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1
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Goodyear MC, Cameron CE. How Proteomics Can Inform Vaccine Design for Sexually Transmitted Infections. Sex Transm Dis 2024; 51:e36-e39. [PMID: 38860670 PMCID: PMC11392604 DOI: 10.1097/olq.0000000000001986] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/12/2024]
Affiliation(s)
- Mara C. Goodyear
- Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada
| | - Caroline E. Cameron
- Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada
- Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA, USA
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2
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Kushwah AS, Dixit H, Upadhyay V, Verma SK, Prasad R. The study of iron- and copper-binding proteome of Fusarium oxysporum and its effector candidates. Proteins 2024; 92:1097-1112. [PMID: 38666709 DOI: 10.1002/prot.26696] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Revised: 03/26/2024] [Accepted: 04/08/2024] [Indexed: 08/07/2024]
Abstract
Fusarium oxysporum f.sp. lycopersici is a phytopathogen which causes vascular wilt disease in tomato plants. The survival tactics of both pathogens and hosts depend on intricate interactions between host plants and pathogenic microbes. Iron-binding proteins (IBPs) and copper-binding proteins (CBPs) play a crucial role in these interactions by participating in enzyme reactions, virulence, metabolism, and transport processes. We employed high-throughput computational tools at the sequence and structural levels to investigate the IBPs and CBPs of F. oxysporum. A total of 124 IBPs and 37 CBPs were identified in the proteome of Fusarium. The ranking of amino acids based on their affinity for binding with iron is Glu > His> Asp > Asn > Cys, and for copper is His > Asp > Cys respectively. The functional annotation, determination of subcellular localization, and Gene Ontology analysis of these putative IBPs and CBPs have unveiled their potential involvement in a diverse array of cellular and biological processes. Three iron-binding glycosyl hydrolase family proteins, along with four CBPs with carbohydrate-binding domains, have been identified as potential effector candidates. These proteins are distinct from the host Solanum lycopersicum proteome. Moreover, they are known to be located extracellularly and function as enzymes that degrade the host cell wall during pathogen-host interactions. The insights gained from this report on the role of metal ions in plant-pathogen interactions can help develop a better understanding of their fundamental biology and control vascular wilt disease in tomato plants.
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Affiliation(s)
- Ankita Singh Kushwah
- Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India
| | - Himisha Dixit
- Centre for Computational Biology & Bioinformatics, Central University of Himachal Pradesh, Kangra, Himachal Pradesh, India
| | - Vipin Upadhyay
- Centre for Computational Biology & Bioinformatics, Central University of Himachal Pradesh, Kangra, Himachal Pradesh, India
| | - Shailender Kumar Verma
- Centre for Computational Biology & Bioinformatics, Central University of Himachal Pradesh, Kangra, Himachal Pradesh, India
- Department of Environmental Studies, University of Delhi, North Campus, Delhi, India
| | - Ramasare Prasad
- Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India
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3
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Turizo MJF, Patell R, Zwicker JI. Identifying novel biomarkers using proteomics to predict cancer-associated thrombosis. BLEEDING, THROMBOSIS AND VASCULAR BIOLOGY 2024; 3:120. [PMID: 38828226 PMCID: PMC11143428 DOI: 10.4081/btvb.2024.120] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 01/22/2024] [Accepted: 04/08/2024] [Indexed: 06/05/2024]
Abstract
Comprehensive protein analyses of plasma are made possible by high-throughput proteomic screens, which may help find new therapeutic targets and diagnostic biomarkers. Patients with cancer are frequently affected by venous thromboembolism (VTE). The limited predictive accuracy of current VTE risk assessment tools highlights the need for new, more targeted biomarkers. Although coagulation biomarkers for the diagnosis, prognosis, and treatment of VTE have been investigated, none of them have the necessary clinical validation or diagnostic accuracy. Proteomics holds the potential to uncover new biomarkers and thrombotic pathways that impact the risk of thrombosis. This review explores the fundamental methods used in proteomics and focuses on particular biomarkers found in VTE and cancer-associated thrombosis.
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Affiliation(s)
- Maria J Fernandez Turizo
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
| | - Rushad Patell
- Division of Medical Oncology and Hematology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
| | - Jeffrey I Zwicker
- Department of Medicine, Hematology Service, Memorial Sloan Kettering Cancer Center, New York, NY
- Weil Cornell Medical College, New York, NY, United States
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4
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Srivastava S, Sandhu N, Liu J, Xie YH. AI-Driven Spectral Decomposition: Predicting the Most Probable Protein Compositions from Surface Enhanced Raman Spectroscopy Spectra of Amino Acids. Bioengineering (Basel) 2024; 11:482. [PMID: 38790349 PMCID: PMC11117800 DOI: 10.3390/bioengineering11050482] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2024] [Revised: 05/05/2024] [Accepted: 05/06/2024] [Indexed: 05/26/2024] Open
Abstract
Surface-enhanced Raman spectroscopy (SERS) is a powerful tool for elucidating the molecular makeup of materials. It possesses the unique characteristics of single-molecule sensitivity and extremely high specificity. However, the true potential of SERS, particularly in capturing the biochemical content of particles, remains underexplored. In this study, we harnessed transformer neural networks to interpret SERS spectra, aiming to discern the amino acid profiles within proteins. By training the network on the SERS profiles of 20 amino acids of human proteins, we explore the feasibility of predicting the predominant proteins within the µL-scale detection volume of SERS. Our results highlight a consistent alignment between the model's predictions and the protein's known amino acid compositions, deepening our understanding of the inherent information contained within SERS spectra. For instance, the model achieved low root mean square error (RMSE) scores and minimal deviation in the prediction of amino acid compositions for proteins such as Bovine Serum Albumin (BSA), ACE2 protein, and CD63 antigen. This novel methodology offers a robust avenue not only for protein analytics but also sets a precedent for the broader realm of spectral analyses across diverse material categories. It represents a solid step forward to establishing SERS-based proteomics.
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Affiliation(s)
| | | | | | - Ya-Hong Xie
- Department of Materials Science and Engineering, University of California, Los Angeles, CA 90095, USA; (S.S.); (N.S.); (J.L.)
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5
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Upadhyay M, Nair D, Moseley GW, Srivastava S, Kondabagil K. Giant Virus Global Proteomics Innovation: Comparative Evaluation of In-Gel and In-Solution Digestion Methods. OMICS : A JOURNAL OF INTEGRATIVE BIOLOGY 2024; 28:170-181. [PMID: 38621149 DOI: 10.1089/omi.2024.0012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/17/2024]
Abstract
With their unusually large genome and particle sizes, giant viruses (GVs) defy the conventional definition of viruses. Although most GVs isolated infect unicellular protozoans, such as amoeba, studies in the last decade have established their much wider prevalence infecting most eukaryotic supergroups and some giant viral families with the potential to be human pathogens. Their complexity, almost autonomous life cycle, and enigmatic evolution necessitate the study of GVs. The accurate assessment of GV proteome is a veritable challenge. We have compared the coverage of global protein identification using different methods for GVs isolated in Mumbai, Mimivirus Bombay (MVB), Powai Lake Megavirus (PLMV), and Kurlavirus (KV), along with two previously studied GVs, Acanthamoeba polyphaga Mimivirus (APMV) and Marseillevirus (MV). Our study shows that the simultaneous use of in-gel and in-solution digestion methods can significantly increase the coverage of protein identification in the global proteome analysis of purified GV particles. Combining the two methods of analyses, we identified an additional 72 proteins in APMV and 114 in MV compared with what have been previously reported. Similarly, proteomes of MVB, PLMV, and KV were analyzed, and a total of 242 proteins in MVB, 287 proteins in PLMV, and 174 proteins in KV were identified. Our results suggest that a combined methodology of in-gel and in-solution methods is more efficient and opens up new avenues for innovation in global proteome analysis of GVs. Future planetary health research on GVs can benefit from consideration of a broader range of proteomics methodologies as illustrated by the present study.
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Affiliation(s)
- Monica Upadhyay
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India
- Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, Australia
| | - Divya Nair
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India
| | - Gregory W Moseley
- Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, Australia
| | - Sanjeeva Srivastava
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India
| | - Kiran Kondabagil
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India
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6
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Li W, Wang Y, Zhang M, Zhao S, Wang M, Zhao R, Chen J, Zhang Y, Xia P. Mass Spectrometry-Based Proteomic Analysis of Potential Host Proteins Interacting with GP5 in PRRSV-Infected PAMs. Int J Mol Sci 2024; 25:2778. [PMID: 38474030 DOI: 10.3390/ijms25052778] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2024] [Revised: 02/25/2024] [Accepted: 02/26/2024] [Indexed: 03/14/2024] Open
Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus causing a large economic impact on the swine industry. The structural protein GP5 of PRRSV plays a pivotal role in its pathogenicity and immune evasion. Virus-host interactions play a crucial part in viral replication and immune escape. Therefore, understanding the interactions between GP5 and host proteins are significant for porcine reproductive and respiratory syndrome (PRRS) control. However, the interaction network between GP5 and host proteins in primary porcine alveolar macrophages (PAMs) has not been reported. In this study, 709 GP5-interacting host proteins were identified in primary PAMs by immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis revealed that these proteins were involved in multiple cellular processes, such as translation, protein transport, and protein stabilization. Subsequently, immunoprecipitation and immunofluorescence assay confirmed that GP5 could interact with antigen processing and presentation pathways related proteins. Finally, we found that GP5 may be a key protein that inhibits the antigen processing and presentation pathway during PRRSV infection. The novel host proteins identified in this study will be the candidates for studying the biological functions of GP5, which will provide new insights into PRRS prevention and vaccine development.
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Affiliation(s)
- Wen Li
- College of Veterinary Medicine, Henan Agricultural University, Longzi Lake 15#, Zhengzhou 450046, China
| | - Yueshuai Wang
- College of Veterinary Medicine, Henan Agricultural University, Longzi Lake 15#, Zhengzhou 450046, China
| | - Mengting Zhang
- College of Veterinary Medicine, Henan Agricultural University, Longzi Lake 15#, Zhengzhou 450046, China
| | - Shijie Zhao
- College of Veterinary Medicine, Henan Agricultural University, Longzi Lake 15#, Zhengzhou 450046, China
| | - Mengxiang Wang
- College of Veterinary Medicine, Henan Agricultural University, Longzi Lake 15#, Zhengzhou 450046, China
| | - Ruijie Zhao
- College of Veterinary Medicine, Henan Agricultural University, Longzi Lake 15#, Zhengzhou 450046, China
| | - Jing Chen
- College of Life Science, Henan Agricultural University, Longzi Lake 15#, Zhengzhou 450046, China
| | - Yina Zhang
- College of Veterinary Medicine, Henan Agricultural University, Longzi Lake 15#, Zhengzhou 450046, China
| | - Pingan Xia
- College of Veterinary Medicine, Henan Agricultural University, Longzi Lake 15#, Zhengzhou 450046, China
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7
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Lu Y, Ran Y, Li H, Wen J, Cui X, Zhang X, Guan X, Cheng M. Micropeptides: origins, identification, and potential role in metabolism-related diseases. J Zhejiang Univ Sci B 2023; 24:1106-1122. [PMID: 38057268 PMCID: PMC10710913 DOI: 10.1631/jzus.b2300128] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2023] [Accepted: 06/06/2023] [Indexed: 12/08/2023]
Abstract
With the development of modern sequencing techniques and bioinformatics, genomes that were once thought to be noncoding have been found to encode abundant functional micropeptides (miPs), a kind of small polypeptides. Although miPs are difficult to analyze and identify, a number of studies have begun to focus on them. More and more miPs have been revealed as essential for energy metabolism homeostasis, immune regulation, and tumor growth and development. Many reports have shown that miPs are especially essential for regulating glucose and lipid metabolism and regulating mitochondrial function. MiPs are also involved in the progression of related diseases. This paper reviews the sources and identification of miPs, as well as the functional significance of miPs for metabolism-related diseases, with the aim of revealing their potential clinical applications.
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Affiliation(s)
| | | | | | | | | | | | | | - Min Cheng
- School of Basic Medicine Sciences, Weifang Medical University, Weifang 261053, China.
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8
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Kushwah AS, Dixit H, Upadhyay V, Yadav S, Verma SK, Prasad R. Elucidating the zinc-binding proteome of Fusarium oxysporum f. sp. lycopersici with particular emphasis on zinc-binding effector proteins. Arch Microbiol 2023; 205:298. [PMID: 37516670 DOI: 10.1007/s00203-023-03638-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Revised: 06/29/2023] [Accepted: 07/14/2023] [Indexed: 07/31/2023]
Abstract
Fusarium oxysporum f. sp. lycopersici is a soil-borne phytopathogenic species which causes vascular wilt disease in the Solanum lycopersicum (tomato). Due to the continuous competition for zinc usage by Fusarium and its host during infection makes zinc-binding proteins a hotspot for focused investigation. Zinc-binding effector proteins are pivotal during the infection process, working in conjunction with other essential proteins crucial for its biological activities. This work aims at identifying and analysing zinc-binding proteins and zinc-binding proteins effector candidates of Fusarium. We have identified three hundred forty-six putative zinc-binding proteins; among these proteins, we got two hundred and thirty zinc-binding proteins effector candidates. The functional annotation, subcellular localization, and Gene Ontology analysis of these putative zinc-binding proteins revealed their probable role in wide range of cellular and biological processes such as metabolism, gene expression, gene expression regulation, protein biosynthesis, protein folding, cell signalling, DNA repair, and RNA processing. Sixteen proteins were found to be putatively secretory in nature. Eleven of these were putative zinc-binding protein effector candidates may be involved in pathogen-host interaction during infection. The information obtained here may enhance our understanding to design, screen, and apply the zinc-metal ion-based antifungal agents to protect the S. lycopersicum and control the vascular wilt caused by F. oxysporum.
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Affiliation(s)
- Ankita Singh Kushwah
- Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, 247667, India
| | - Himisha Dixit
- Centre for Computational Biology & Bioinformatics, Central University of Himachal Pradesh, Kangra, Himachal Pradesh, 176206, India
| | - Vipin Upadhyay
- Centre for Computational Biology & Bioinformatics, Central University of Himachal Pradesh, Kangra, Himachal Pradesh, 176206, India
| | - Siddharth Yadav
- Department of Computer Science and Engineering, Thapar Institute of Engineering & Technology, Patiala, Punjab, 147004, India
| | - Shailender Kumar Verma
- Centre for Computational Biology & Bioinformatics, Central University of Himachal Pradesh, Kangra, Himachal Pradesh, 176206, India
- Department of Environmental Studies, University of Delhi, New Delhi, Delhi, 110007, India
| | - Ramasare Prasad
- Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, 247667, India.
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9
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Abdelbost L, Morel MH, Nascimento TPD, Cameron LC, Bonicel J, Larraz MFS, Mameri H. Sorghum grain germination as a route to improve kafirin digestibility: Biochemical and label free proteomics insights. Food Chem 2023; 424:136407. [PMID: 37224636 DOI: 10.1016/j.foodchem.2023.136407] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2023] [Revised: 04/23/2023] [Accepted: 05/15/2023] [Indexed: 05/26/2023]
Abstract
Kafirin, the sorghum grain storage protein presents lower digestibility compared to its cereals counterparts. Germination has been proposed as an adequate bioprocessing method to improve seed protein digestibility. Here, germination was rationalized so as to evenly sample germinated seeds and the dynamic changes of the proteome and several biochemical markers was connected for the first time with the in vitro protein digestibility of germinated seeds. Free sulfhydryl groups increased during germination and in vitro protein digestibility enhanced. The dynamic in abundance of several enzymes out of which 3 cysteine proteases were found to coincide with appearance of aqueous soluble peptides derived from kafirin at boot time of their degradation. The study provides deep information about the molecular events occurring during sorghum seed germination and reveals potential biomarkers of the kafirin proteolysis. It points a way to improve sorghum nutritional value through controlled germination.
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Affiliation(s)
- Lynda Abdelbost
- UMR 1208 IATE, Univ Montpellier, INRAE, L'Institut-Agro Montpellier, F-34060 Montpellier, France
| | - Marie-Hélène Morel
- UMR 1208 IATE, Univ Montpellier, INRAE, L'Institut-Agro Montpellier, F-34060 Montpellier, France
| | - Talita Pimenta do Nascimento
- Laboratory of Bioactives, Food and Nutrition Graduate Program, Federal University of State of Rio de Janeiro, UNIRIO, Brazil; Center of Innovation in Mass Spectrometry, Laboratory of Protein Biochemistry, UNIRIO, Brazil
| | - Luiz-Claudio Cameron
- Center of Innovation in Mass Spectrometry, Laboratory of Protein Biochemistry, UNIRIO, Brazil
| | - Joëlle Bonicel
- UMR 1208 IATE, Univ Montpellier, INRAE, L'Institut-Agro Montpellier, F-34060 Montpellier, France
| | - Mariana Ferreira Simões Larraz
- Laboratory of Bioactives, Food and Nutrition Graduate Program, Federal University of State of Rio de Janeiro, UNIRIO, Brazil; Center of Innovation in Mass Spectrometry, Laboratory of Protein Biochemistry, UNIRIO, Brazil
| | - Hamza Mameri
- UMR 1208 IATE, Univ Montpellier, INRAE, L'Institut-Agro Montpellier, F-34060 Montpellier, France.
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10
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Popovic Z, Anderson LC, Zhang X, Butcher DS, Blakney GT, Zubarev RA, Marshall AG. Analysis of Isotopically Depleted Proteins Derived from Escherichia coli and Caenorhabditis elegans Cell Lines by Liquid Chromatography 21 T Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2023; 34:137-144. [PMID: 36656140 DOI: 10.1021/jasms.2c00242] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/17/2023]
Abstract
Protein mass measurement by mass spectrometry is complicated by wide isotopic distributions that result from incorporation of heavy isotopes of C, H, N, O, and S, thereby limiting signal-to-noise ratio (SNR) and accurate intact mass determination, particularly for larger proteins [Fenselau et al. Anal. Chem. 1983, 55 (2), 353-356]. Observation of the monoisotopic mass-to-charge ratio (m/z) is the simplest and most accurate way to determine intact protein mass, but as mass increases, the relative abundance of the monoisotopic peak becomes so low that it is often undetectable. Here, we used an isotopically depleted growth medium to culture bacterial cells (Escherichia coli), resulting in isotopically depleted proteins. Isotopically depleted proteins show increased sequence coverage, mass measurement accuracy, and increased S/N of the monoisotopic peak by Fourier transform ion cyclotron resonance mass spectrometry analysis. We then grew Caenorhabditis elegans cells in a medium containing living isotopically depleted E. coli cells, thereby producing the first isotopically depleted eukaryotic proteins. This is the first time isotopic depletion has been implemented for four isotopes (1H, 12C, 14N, and 16O), resulting in the highest degree of depletion ever used for protein analysis and further improving MS analysis.
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Affiliation(s)
- Zeljka Popovic
- National High Magnetic Field Laboratory, Tallahassee, Florida 32310, United States
- Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, United States
| | - Lissa C Anderson
- National High Magnetic Field Laboratory, Tallahassee, Florida 32310, United States
| | - Xuepei Zhang
- Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Solnavagen 1, Solna, 171 77 Stockholm, Sweden
| | - David S Butcher
- National High Magnetic Field Laboratory, Tallahassee, Florida 32310, United States
| | - Greg T Blakney
- National High Magnetic Field Laboratory, Tallahassee, Florida 32310, United States
| | - Roman A Zubarev
- Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Solnavagen 1, Solna, 171 77 Stockholm, Sweden
| | - Alan G Marshall
- National High Magnetic Field Laboratory, Tallahassee, Florida 32310, United States
- Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, United States
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11
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Nasir NSA, Deivasigamani R, Wee MFMR, Hamzah AA, Zaid MHM, Rahim MKA, Kayani AA, Abdulhameed A, Buyong MR. Protein Albumin Manipulation and Electrical Quantification of Molecular Dielectrophoresis Responses for Biomedical Applications. MICROMACHINES 2022; 13:mi13081308. [PMID: 36014230 PMCID: PMC9415755 DOI: 10.3390/mi13081308] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/21/2022] [Revised: 08/06/2022] [Accepted: 08/07/2022] [Indexed: 05/17/2023]
Abstract
Research relating to dielectrophoresis (DEP) has been progressing rapidly through time as it is a strong and controllable technique for manipulation, separation, preconcentration, and partitioning of protein. Extensive studies have been carried out on protein DEP, especially on Bovine Serum Albumin (BSA). However, these studies involve the usage of dye and fluorescent probes to observe DEP responses as the physical properties of protein albumin molecular structure are translucent. The use of dye and the fluorescent probe could later affect the protein's physiology. In this article, we review three methods of electrical quantification of DEP responses: electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and capacitance measurement for protein BSA DEP manipulation. The correlation of these methods with DEP responses is further discussed. Based on the observations on capacitance measurement, it can be deduced that the electrical quantifying method is reliable for identifying DEP responses. Further, the possibility of manipulating the protein and electrically quantifying DEP responses while retaining the original physiology of the protein and without the usage of dye or fluorescent probe is discussed.
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Affiliation(s)
- Nur Shahira Abdul Nasir
- Institute of Microengineering & Nanoelectronics (IMEN), Universiti Kebangsaan Malaysia (UKM), Bangi 43600, Selangor, Malaysia
| | - Revathy Deivasigamani
- Institute of Microengineering & Nanoelectronics (IMEN), Universiti Kebangsaan Malaysia (UKM), Bangi 43600, Selangor, Malaysia
| | - M. F. Mohd Razip Wee
- Institute of Microengineering & Nanoelectronics (IMEN), Universiti Kebangsaan Malaysia (UKM), Bangi 43600, Selangor, Malaysia
| | - Azrul Azlan Hamzah
- Institute of Microengineering & Nanoelectronics (IMEN), Universiti Kebangsaan Malaysia (UKM), Bangi 43600, Selangor, Malaysia
| | - Mohd Hazani Mat Zaid
- Institute of Microengineering & Nanoelectronics (IMEN), Universiti Kebangsaan Malaysia (UKM), Bangi 43600, Selangor, Malaysia
| | | | - Aminuddin Ahmad Kayani
- Functional Materials and Microsystems Research Group and the Micro Nano Research Facility, School of Engineering, RMIT University, Melbourne, VIC 3001, Australia
| | - Abdullah Abdulhameed
- Department of Electronics & Communication Engineering, Faculty of Engineering & Petroleum, Hadhramout University, Al-Mukalla 50512, Hadhramout, Yemen
| | - Muhamad Ramdzan Buyong
- Institute of Microengineering & Nanoelectronics (IMEN), Universiti Kebangsaan Malaysia (UKM), Bangi 43600, Selangor, Malaysia
- Correspondence: ; Tel.: +60-12-385-2713
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12
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Chang L, Wang F, Connolly K, Meng H, Su Z, Cvirkaite-Krupovic V, Krupovic M, Egelman EH, Si D. DeepTracer-ID: De novo protein identification from cryo-EM maps. Biophys J 2022; 121:2840-2848. [PMID: 35769006 PMCID: PMC9388381 DOI: 10.1016/j.bpj.2022.06.025] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Revised: 05/04/2022] [Accepted: 06/23/2022] [Indexed: 11/02/2022] Open
Abstract
The recent revolution in cryo-electron microscopy (cryo-EM) has made it possible to determine macromolecular structures directly from cell extracts. However, identifying the correct protein from the cryo-EM map is still challenging and often needs additional sequence information from other techniques, such as tandem mass spectrometry and/or bioinformatics. Here, we present DeepTracer-ID, a server-based approach to identify the candidate protein in a user-provided organism de novo from a cryo-EM map, without the need for additional information. Our method first uses DeepTracer to generate a protein backbone model that best represents the cryo-EM map, and this model is then searched against the library of AlphaFold2 predictions for all proteins in the given organism. This method is highly accurate and robust for high-resolution cryo-EM maps: in all 13 experimental maps tested blindly, DeepTracer-ID identified the correct proteins as the top candidates. Eight of the maps were of known structures, while the other five unpublished maps were validated by prior protein annotation and careful inspection of the model refined into the map. The program also showed promising results for both homomeric and heteromeric protein complexes. This platform is possible because of the recent breakthroughs in large-scale three-dimensional protein structure prediction.
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Affiliation(s)
- Luca Chang
- Division of Computing and Software Systems, University of Washington Bothell, Bothell, Washington
| | - Fengbin Wang
- Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia.
| | - Kiernan Connolly
- Division of Computing and Software Systems, University of Washington Bothell, Bothell, Washington
| | - Hanze Meng
- Department of Mathematics, University of Washington, Seattle, Washington
| | - Zhangli Su
- Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama
| | | | - Mart Krupovic
- Institut Pasteur, Université Paris Cité, CNRS UMR6047, Archaeal Virology Unit, Paris, France
| | - Edward H Egelman
- Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia.
| | - Dong Si
- Division of Computing and Software Systems, University of Washington Bothell, Bothell, Washington.
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13
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Modifying the pH sensitivity of OmpG nanopore for improved detection at acidic pH. Biophys J 2022; 121:731-741. [PMID: 35131293 PMCID: PMC8943698 DOI: 10.1016/j.bpj.2022.01.023] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2021] [Revised: 01/02/2022] [Accepted: 01/25/2022] [Indexed: 11/22/2022] Open
Abstract
The outer membrane protein G (OmpG) nanopore is a monomeric β-barrel channel consisting of seven flexible extracellular loops. Its most flexible loop, loop 6, can be used to host high-affinity binding ligands for the capture of protein analytes, which induces characteristic current patterns for protein identification. At acidic pH, the ability of OmpG to detect protein analytes is hampered by its tendency toward the closed state, which renders the nanopore unable to reveal current signal changes induced by bound analytes. In this work, critical residues that control the pH-dependent gating of loop 6 were identified, and an OmpG nanopore that can stay predominantly open at a broad range of pHs was created by mutating these pH-sensitive residues. A short single-stranded DNA was chemically tethered to the pH-insensitive OmpG to demonstrate the utility of the OmpG nanopore for sensing complementary DNA and a DNA binding protein at an acidic pH.
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14
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He X, Liu C, Peng J, Li Z, Li F, Wang J, Hu A, Peng M, Huang K, Fan D, Li N, Zhang F, Cai W, Tan X, Hu Z, Deng X, Li Y, Mo X, Li L, Shi Y, Yang L, Zhu Y, Wu Y, Liang H, Liao B, Hong W, He R, Li J, Guo P, Zhuo Y, Zhao L, Hu F, Li W, Zhu W, Zhang Z, Guo Z, Zhang W, Hong X, Cai W, Gu L, Du Z, Zhang Y, Xu J, Zuo T, Deng K, Yan L, Chen X, Chen S, Lei C. COVID-19 induces new-onset insulin resistance and lipid metabolic dysregulation via regulation of secreted metabolic factors. Signal Transduct Target Ther 2021; 6:427. [PMID: 34916489 PMCID: PMC8674414 DOI: 10.1038/s41392-021-00822-x] [Citation(s) in RCA: 67] [Impact Index Per Article: 16.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2021] [Revised: 10/28/2021] [Accepted: 11/02/2021] [Indexed: 12/13/2022] Open
Abstract
Abnormal glucose and lipid metabolism in COVID-19 patients were recently reported with unclear mechanism. In this study, we retrospectively investigated a cohort of COVID-19 patients without pre-existing metabolic-related diseases, and found new-onset insulin resistance, hyperglycemia, and decreased HDL-C in these patients. Mechanistically, SARS-CoV-2 infection increased the expression of RE1-silencing transcription factor (REST), which modulated the expression of secreted metabolic factors including myeloperoxidase, apelin, and myostatin at the transcriptional level, resulting in the perturbation of glucose and lipid metabolism. Furthermore, several lipids, including (±)5-HETE, (±)12-HETE, propionic acid, and isobutyric acid were identified as the potential biomarkers of COVID-19-induced metabolic dysregulation, especially in insulin resistance. Taken together, our study revealed insulin resistance as the direct cause of hyperglycemia upon COVID-19, and further illustrated the underlying mechanisms, providing potential therapeutic targets for COVID-19-induced metabolic complications.
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Affiliation(s)
- Xi He
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Chenshu Liu
- Division of Vascular Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.,National-Guangdong Joint Engineering Laboratory for Diagnosis and Treatment of Vascular Diseases, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Jiangyun Peng
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Guangdong-Hong Kong Joint Laboratory for RNA Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.,Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China
| | - Zilun Li
- Division of Vascular Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.,National-Guangdong Joint Engineering Laboratory for Diagnosis and Treatment of Vascular Diseases, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Fang Li
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China.,Department of Obstetrics and Gynecology, Guangzhou Women and Children Medical Center, Guangzhou Medical University, Guangzhou, China
| | - Jian Wang
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Ao Hu
- Institute of Human Virology, Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China.,Department of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
| | - Meixiu Peng
- National-Guangdong Joint Engineering Laboratory for Diagnosis and Treatment of Vascular Diseases, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Kan Huang
- Division of Vascular Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.,National-Guangdong Joint Engineering Laboratory for Diagnosis and Treatment of Vascular Diseases, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Dongxiao Fan
- Division of Vascular Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.,National-Guangdong Joint Engineering Laboratory for Diagnosis and Treatment of Vascular Diseases, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Na Li
- Division of Vascular Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.,National-Guangdong Joint Engineering Laboratory for Diagnosis and Treatment of Vascular Diseases, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Fuchun Zhang
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Weiping Cai
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Xinghua Tan
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Zhongwei Hu
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Xilong Deng
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Yueping Li
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Xiaoneng Mo
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Linghua Li
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Yaling Shi
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Li Yang
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Yuanyuan Zhu
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Yanrong Wu
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Huichao Liang
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Baolin Liao
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Wenxin Hong
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Ruiying He
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Jiaojiao Li
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Pengle Guo
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Youguang Zhuo
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Lingzhai Zhao
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Fengyu Hu
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Wenxue Li
- Guangzhou Center for Disease Control and Prevention, Guangzhou, China
| | - Wei Zhu
- Guangzhou Center for Disease Control and Prevention, Guangzhou, China
| | - Zefeng Zhang
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Guangdong-Hong Kong Joint Laboratory for RNA Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.,Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China
| | - Zeling Guo
- National-Guangdong Joint Engineering Laboratory for Diagnosis and Treatment of Vascular Diseases, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Wei Zhang
- Wuhan Metware Biotechnology Co., Ltd, Wuhan, China
| | - Xiqiang Hong
- Wuhan Metware Biotechnology Co., Ltd, Wuhan, China
| | - Weikang Cai
- Department of Biomedical Sciences, New York Institute of Technology, College of Osteopathic Medicine, Old Westbury, NY, USA
| | - Lei Gu
- Max Planck Institute for Heart and Lung Research and Cardiopulmonary Institute (CPI), Bad Nauheim, Germany
| | - Ziming Du
- Department of Molecular Diagnostics, Sun Yat-sen Cancer Center, Sun Yat-sen University, Guangzhou, China
| | - Yang Zhang
- School of Public Health (Shenzhen), Sun Yat-sen University, Shenzhen, China
| | - Jin Xu
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China
| | - Tao Zuo
- The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Kai Deng
- Institute of Human Virology, Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China.,Department of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
| | - Li Yan
- Department of Endocrinology, Sun Yat-Sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
| | - Xinwen Chen
- Guangzhou Regenerative Medicine and Health-Guangdong Laboratory (GRMH-GDL), Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China. .,Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
| | - Sifan Chen
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Guangdong-Hong Kong Joint Laboratory for RNA Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China. .,Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
| | - Chunliang Lei
- Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China.
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15
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Eaton AF, Brown D, Merkulova M. The evolutionary conserved TLDc domain defines a new class of (H +)V-ATPase interacting proteins. Sci Rep 2021; 11:22654. [PMID: 34811399 PMCID: PMC8608904 DOI: 10.1038/s41598-021-01809-y] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2021] [Accepted: 11/02/2021] [Indexed: 01/26/2023] Open
Abstract
We recently found that nuclear receptor coactivator 7 (Ncoa7) and Oxr1 interact with the proton-pumping V-ATPase. Ncoa7 and Oxr1 belong to a group of proteins playing a role in the oxidative stress response, that contain the conserved “TLDc” domain. Here we asked if the three other proteins in this family, i.e., Tbc1d24, Tldc1 and Tldc2 also interact with the V-ATPase and if the TLDc domains are involved in all these interactions. By co-immunoprecipitation, endogenous kidney Tbc1d24 (and Ncoa7 and Oxr1) and overexpressed Tldc1 and Tldc2, all interacted with the V-ATPase. In addition, purified TLDc domains of Ncoa7, Oxr1 and Tldc2 (but not Tbc1d24 or Tldc1) interacted with V-ATPase in GST pull-downs. At the amino acid level, point mutations G815A, G845A and G896A in conserved regions of the Ncoa7 TLDc domain abolished interaction with the V-ATPase, and S817A, L926A and E938A mutations resulted in decreased interaction. Furthermore, poly-E motifs upstream of the TLDc domain in Ncoa7 and Tldc2 show a (nonsignificant) trend towards enhancing the interaction with V-ATPase. Our principal finding is that all five members of the TLDc family of proteins interact with the V-ATPase. We conclude that the TLDc motif defines a new class of V-ATPase interacting regulatory proteins.
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Affiliation(s)
- A F Eaton
- Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA
| | - D Brown
- Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA
| | - M Merkulova
- Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA. .,Program in Membrane Biology and Division of Nephrology, Massachusetts General Hospital, Simches Research Center, 128 Cambridge St., Boston, MA, 02114, USA.
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16
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Vandghanooni S, Sanaat Z, Farahzadi R, Eskandani M, Omidian H, Omidi Y. Recent progress in the development of aptasensors for cancer diagnosis: Focusing on aptamers against cancer biomarkers. Microchem J 2021. [DOI: 10.1016/j.microc.2021.106640] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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17
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The brain-derived neurotrophic factor prompts platelet aggregation and secretion. Blood Adv 2021; 5:3568-3580. [PMID: 34546355 DOI: 10.1182/bloodadvances.2020004098] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2020] [Accepted: 06/08/2021] [Indexed: 12/19/2022] Open
Abstract
Brain-derived neurotrophic factor (BDNF) has both autocrine and paracrine roles in neurons, and its release and signaling mechanisms have been extensively studied in the central nervous system. Large quantities of BDNF have been reported in circulation, essentially stored in platelets with concentrations reaching 100- to 1000-fold those of neurons. Despite this abundance, the function of BDNF in platelet biology has not been explored. At low concentrations, BDNF primed platelets, acting synergistically with classical agonists. At high concentrations, BDNF induced complete biphasic platelet aggregation that in part relied on amplification from secondary mediators. Neurotrophin-4, but not nerve growth factor, and an activating antibody against the canonical BDNF receptor tropomyosin-related kinase B (TrkB) induced similar platelet responses to BDNF, suggesting TrkB could be the mediator. Platelets expressed, both at their surface and in their intracellular compartment, a truncated form of TrkB lacking its tyrosine kinase domain. BDNF-induced platelet aggregation was prevented by inhibitors of Ras-related C3 botulinum toxin substrate 1 (Rac1), protein kinase C, and phosphoinositide 3-kinase. BDNF-stimulated platelets secreted a panel of angiogenic and inflammatory cytokines, which may play a role in maintaining vascular homeostasis. Two families with autism spectrum disorder were found to carry rare missense variants in the BDNF gene. Platelet studies revealed defects in platelet aggregation to low concentrations of collagen, as well as reduced adenosine triphosphate secretion in response to adenosine diphosphate. In summary, circulating BDNF levels appear to regulate platelet activation, aggregation, and secretion through activation of a truncated TrkB receptor and downstream kinase-dependent signaling.
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18
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Ruiz Cuevas MV, Hardy MP, Hollý J, Bonneil É, Durette C, Courcelles M, Lanoix J, Côté C, Staudt LM, Lemieux S, Thibault P, Perreault C, Yewdell JW. Most non-canonical proteins uniquely populate the proteome or immunopeptidome. Cell Rep 2021; 34:108815. [PMID: 33691108 PMCID: PMC8040094 DOI: 10.1016/j.celrep.2021.108815] [Citation(s) in RCA: 120] [Impact Index Per Article: 30.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Revised: 01/29/2021] [Accepted: 02/10/2021] [Indexed: 12/16/2022] Open
Abstract
Combining RNA sequencing, ribosome profiling, and mass spectrometry, we elucidate the contribution of non-canonical translation to the proteome and major histocompatibility complex (MHC) class I immunopeptidome. Remarkably, of 14,498 proteins identified in three human B cell lymphomas, 2,503 are non-canonical proteins. Of these, 28% are novel isoforms and 72% are cryptic proteins encoded by ostensibly non-coding regions (60%) or frameshifted canonical genes (12%). Cryptic proteins are translated as efficiently as canonical proteins, have more predicted disordered residues and lower stability, and critically generate MHC-I peptides 5-fold more efficiently per translation event. Translating 5' "untranslated" regions hinders downstream translation of genes involved in transcription, translation, and antiviral responses. Novel protein isoforms show strong enrichment for signaling pathways deregulated in cancer. Only a small fraction of cryptic proteins detected in the proteome contribute to the MHC-I immunopeptidome, demonstrating the high preferential access of cryptic defective ribosomal products to the class I pathway.
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Affiliation(s)
- Maria Virginia Ruiz Cuevas
- Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, QC H3C 3J7, Canada; Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, QC H3C 3J7, Canada
| | - Marie-Pierre Hardy
- Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, QC H3C 3J7, Canada
| | - Jaroslav Hollý
- Cellular Biology Section, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Éric Bonneil
- Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, QC H3C 3J7, Canada
| | - Chantal Durette
- Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, QC H3C 3J7, Canada
| | - Mathieu Courcelles
- Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, QC H3C 3J7, Canada
| | - Joël Lanoix
- Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, QC H3C 3J7, Canada
| | - Caroline Côté
- Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, QC H3C 3J7, Canada
| | - Louis M Staudt
- Lymphoid Malignancies Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Sébastien Lemieux
- Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, QC H3C 3J7, Canada; Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, QC H3C 3J7, Canada
| | - Pierre Thibault
- Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, QC H3C 3J7, Canada; Department of Chemistry, Université de Montréal, Montreal, QC H3C 3J7, Canada
| | - Claude Perreault
- Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, QC H3C 3J7, Canada; Department of Medicine, Université de Montréal, Montreal, QC H3C 3J7, Canada.
| | - Jonathan W Yewdell
- Cellular Biology Section, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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19
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Nikolaou PE, Efentakis P, Abu Qourah F, Femminò S, Makridakis M, Kanaki Z, Varela A, Tsoumani M, Davos CH, Dimitriou CA, Tasouli A, Dimitriadis G, Kostomitsopoulos N, Zuurbier CJ, Vlahou A, Klinakis A, Brizzi MF, Iliodromitis EK, Andreadou I. Chronic Empagliflozin Treatment Reduces Myocardial Infarct Size in Nondiabetic Mice Through STAT-3-Mediated Protection on Microvascular Endothelial Cells and Reduction of Oxidative Stress. Antioxid Redox Signal 2021; 34:551-571. [PMID: 32295413 DOI: 10.1089/ars.2019.7923] [Citation(s) in RCA: 55] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Aims: Empagliflozin (EMPA) demonstrates cardioprotective effects on diabetic myocardium but its infarct-sparing effects in normoglycemia remain unspecified. We investigated the acute and chronic effect of EMPA on infarct size after ischemia-reperfusion (I/R) injury and the mechanisms of cardioprotection in nondiabetic mice. Results: Chronic oral administration of EMPA (6 weeks) reduced myocardial infarct size after 30 min/2 h I/R (26.5% ± 3.9% vs 45.8% ± 3.3% in the control group, p < 0.01). Body weight, blood pressure, glucose levels, and cardiac function remained unchanged between groups. Acute administration of EMPA 24 or 4 h before I/R did not affect infarct size. Chronic EMPA treatment led to a significant reduction of oxidative stress biomarkers. STAT-3 (signal transducer and activator of transcription 3) was activated by Y(705) phosphorylation at the 10th minute of R, but it remained unchanged at 2 h of R and in the acute administration protocols. Proteomic analysis was employed to investigate signaling intermediates and revealed that chronic EMPA treatment regulates several pathways at reperfusion, including oxidative stress and integrin-related proteins that were further evaluated. Superoxide dismutase and vascular endothelial growth factor were increased throughout reperfusion. EMPA pretreatment (24 h) increased the viability of human microvascular endothelial cells in normoxia and on 3 h hypoxia/1 h reoxygenation and reduced reactive oxygen species production. In EMPA-treated murine hearts, CD31-/VEGFR2-positive endothelial cells and the pSTAT-3(Y705) signal derived from endothelial cells were boosted at early reperfusion. Innovation: Chronic EMPA administration reduces infarct size in healthy mice via the STAT-3 pathway and increases the survival of endothelial cells. Conclusion: Chronic but not acute administration of EMPA reduces infarct size through STAT-3 activation independently of diabetes mellitus.
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Affiliation(s)
| | - Panagiotis Efentakis
- Laboratory of Pharmacology, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece
| | - Fairouz Abu Qourah
- Laboratory of Pharmacology, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece
| | - Saveria Femminò
- Department of Medical Sciences, University of Turin, Turin, Italy
| | - Manousos Makridakis
- Biotechnology Laboratory, Centre of Basic Research, Biomedical Research Foundation of the Academy of Athens (BRFAA), Athens, Greece
| | - Zoi Kanaki
- Biomedical Research Foundation Academy of Athens, Athens, Greece
| | - Aimilia Varela
- Cardiovascular Research Laboratory, Biomedical Research Foundation Academy of Athens, Athens, Greece
| | - Maria Tsoumani
- Laboratory of Pharmacology, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece
| | - Constantinos H Davos
- Cardiovascular Research Laboratory, Biomedical Research Foundation Academy of Athens, Athens, Greece
| | - Constantinos A Dimitriou
- Cardiovascular Research Laboratory, Biomedical Research Foundation Academy of Athens, Athens, Greece
| | | | - George Dimitriadis
- 2nd Department of Internal Medicine, Research Institute and Diabetes Center, National and Kapodistrian University of Athens, "Attikon" University Hospital, Athens, Greece
| | - Nikolaos Kostomitsopoulos
- Academy of Athens Biomedical Research Foundation, Centre of Clinical Experimental Surgery and Translational Research, Athens, Greece
| | - Coert J Zuurbier
- Amsterdam UMC, University of Amsterdam, Laboratory of Experimental Intensive Care and Anesthesiology, Department of Anesthesiology, Amsterdam Cardiovascular Sciences, Amsterdam Infection & Immunity, Amsterdam, The Netherlands
| | - Antonia Vlahou
- Biotechnology Laboratory, Centre of Basic Research, Biomedical Research Foundation of the Academy of Athens (BRFAA), Athens, Greece
| | | | - Maria F Brizzi
- Department of Medical Sciences, University of Turin, Turin, Italy
| | - Efstathios K Iliodromitis
- 2nd University Department of Cardiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Ioanna Andreadou
- Laboratory of Pharmacology, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece
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20
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Vinaiphat A, Low JK, Yeoh KW, Chng WJ, Sze SK. Application of Advanced Mass Spectrometry-Based Proteomics to Study Hypoxia Driven Cancer Progression. Front Oncol 2021; 11:559822. [PMID: 33708620 PMCID: PMC7940826 DOI: 10.3389/fonc.2021.559822] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2020] [Accepted: 01/07/2021] [Indexed: 12/24/2022] Open
Abstract
Cancer is one of the largest contributors to the burden of chronic disease in the world and is the second leading cause of death globally. It is associated with episodes of low-oxygen stress (hypoxia or ischemia/reperfusion) that promotes cancer progression and therapeutic resistance. Efforts have been made in the past using traditional proteomic approaches to decipher oxygen deprivation stress-related mechanisms of the disease initiation and progression and to identify key proteins as a therapeutic target for the treatment and prevention. Despite the potential benefits of proteomic in translational research for the discovery of new drugs, the therapeutic outcome with this approach has not met expectations in clinical trials. This is mainly due to the disease complexity which possess a multifaceted molecular pathology. Therefore, novel strategies to identify and characterize clinically important sets of modulators and molecular events for multi-target drug discovery are needed. Here, we review important past and current studies on proteomics in cancer with an emphasis on recent pioneered labeling approaches in mass spectrometry (MS)-based systematic quantitative analysis to improve clinical success. We also discuss the results of the selected innovative publications that integrate advanced proteomic technologies (e.g. MALDI-MSI, pSILAC/SILAC/iTRAQ/TMT-LC-MS/MS, MRM-MS) for comprehensive analysis of proteome dynamics in different biosystems, including cell type, cell species, and subcellular proteome (i.e. secretome and chromatome). Finally, we discuss the future direction and challenges in the application of these technological advancements in mass spectrometry within the context of cancer and hypoxia.
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Affiliation(s)
- Arada Vinaiphat
- School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
| | - Jee Keem Low
- Department of Surgery, Tan Tock Seng Hospital, Singapore, Singapore
| | - Kheng Wei Yeoh
- Department of Radiation Oncology, National Cancer Centre Singapore, Singapore, Singapore
| | - Wee Joo Chng
- Department of Hematology-Oncology, National University Cancer Institute, National University Health System, Singapore, Singapore
| | - Siu Kwan Sze
- School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
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21
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Bansal R, Khan MM, Dasari S, Verma I, Goodlett DR, Manes NP, Nita-Lazar A, Sharma SP, Kumar A, Singh N, Chakraborti A, Gupta V, Dogra MR, Ram J, Gupta A. Proteomic profile of vitreous in patients with tubercular uveitis. Tuberculosis (Edinb) 2021; 126:102036. [PMID: 33359883 PMCID: PMC11005023 DOI: 10.1016/j.tube.2020.102036] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2020] [Revised: 10/31/2020] [Accepted: 11/29/2020] [Indexed: 12/27/2022]
Abstract
OBJECTIVE To elucidate disease-specific host protein profile in vitreous fluid of patients with intraocular inflammation due to tubercular uveitis (TBU). METHODS Vitreous samples from 13 patients with TBU (group A), 7 with non-TBU (group B) and 9 with no uveitis (group C) were analysed by shotgun proteomics using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Differentially expressed proteins (DEPs) were subjected to pathway analysis using WEB-based Gene SeT Analysis Toolkit software. RESULTS Compared to control groups (B + C combined), group A (TBU) displayed 32 (11 upregulated, 21 downregulated) DEPs, which revealed an upregulation of coagulation cascades, complement and classic pathways, and downregulation of metabolism of carbohydrates, gluconeogenesis, glucose metabolism and glycolysis/gluconeogenesis pathways. When compared to group B (non-TBU) alone, TBU displayed 58 DEPs (21 upregulated, 37 downregulated), with an upregulation of apoptosis, KRAS signaling, diabetes pathways, classic pathways, and downregulation of MTORC1 signaling, glycolysis/gluconeogenesis, and glucose metabolism. CONCLUSION This differential protein profile provides novel insights into the molecular mechanisms of TBU and a baseline to explore vitreous biomarkers to differentiate TBU from non-TBU, warranting future studies to identify and validate them as a diagnostic tool in TBU. The enriched pathways generate interesting hypotheses and drive further research.
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Affiliation(s)
- Reema Bansal
- Advanced Eye Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
| | - Mohd M Khan
- University of Maryland, School of Medicine, Baltimore, MD, USA; Laboratory of Immune System Biology (LISB), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, USA.
| | - Surendra Dasari
- Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA.
| | - Indu Verma
- Department of Biochemistry, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
| | | | - Nathan P Manes
- Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
| | - Aleksandra Nita-Lazar
- Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
| | - Surya P Sharma
- Advanced Eye Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
| | - Aman Kumar
- Advanced Eye Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
| | - Nirbhai Singh
- Advanced Eye Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
| | - Anuradha Chakraborti
- Department of Experimental Medicine & Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
| | - Vishali Gupta
- Advanced Eye Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
| | - M R Dogra
- Advanced Eye Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
| | - Jagat Ram
- Advanced Eye Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
| | - Amod Gupta
- Advanced Eye Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
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22
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Rehiman SH, Lim SM, Lim FT, Chin AV, Tan MP, Kamaruzzaman SB, Ramasamy K, Abdul Majeed AB. Fibrinogen isoforms as potential blood-based biomarkers of Alzheimer's disease using a proteomics approach. Int J Neurosci 2020; 132:1014-1025. [PMID: 33280461 DOI: 10.1080/00207454.2020.1860038] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
Abstract
Objective: Alzheimer's disease (AD), the commonest form of dementia which is characterized by progressive decline in cognitive function, can only be definitively diagnosed after death. Although biomarkers may aid diagnosis, currently available AD biomarkers, which are predominantly based on cerebrospinal fluid and neuroimaging facilities, are either invasive or costly. Blood-based biomarkers for AD diagnosis are highly sought after due to its practicality at the clinic. This study was undertaken to determine the differential protein expression in plasma amongst Malaysian AD, mild cognitive impairment (MCI) and non-AD individuals. Methods: A proteomic approach which utilized two-dimensional differential in gel electrophoresis (2 D DIGE) was performed for blood samples from 15 AD, 14 MCI and 15 non-AD individuals. Results: Mass spectrometry (MS)-based protein identification via MALDI ToF/ToF showed that fibrinogen-β-chain (spot 64) and fibrinogen-γ-chain (spot 91) with differential expression ratio >1.5 were significantly upregulated (p < 0.05) in AD patients when compared to non-AD individuals. Further data analysis using Pearson correlation found that the upregulated fibrinogen-γ-chain was weakly but significantly (p < 0.05) and inversely correlated with cognitive decline. Conclusion: Fibrinogen isoforms may play important roles in the vascular pathology of AD as well as neuroinflammation. As such, fibrinogen appears to be a promising blood-based biomarker for AD. Further validation of the present findings in larger population is now warranted.
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Affiliation(s)
- Siti Hajar Rehiman
- Collaborative Drug Discovery Research (CDDR) and Brain Degeneration and Therapeutics Research Group, Faculty of Pharmacy, University Teknologi MARA (UiTM) Cawangan Selangor, Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
| | - Siong Meng Lim
- Collaborative Drug Discovery Research (CDDR) and Brain Degeneration and Therapeutics Research Group, Faculty of Pharmacy, University Teknologi MARA (UiTM) Cawangan Selangor, Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
| | - Fei Tieng Lim
- Collaborative Drug Discovery Research (CDDR) and Brain Degeneration and Therapeutics Research Group, Faculty of Pharmacy, University Teknologi MARA (UiTM) Cawangan Selangor, Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
| | - Ai-Vyrn Chin
- Division of Geriatric Medicine, Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
| | - Maw Pin Tan
- Division of Geriatric Medicine, Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
| | - Shahrul Bahyah Kamaruzzaman
- Division of Geriatric Medicine, Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
| | - Kalavathy Ramasamy
- Collaborative Drug Discovery Research (CDDR) and Brain Degeneration and Therapeutics Research Group, Faculty of Pharmacy, University Teknologi MARA (UiTM) Cawangan Selangor, Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
| | - Abu Bakar Abdul Majeed
- Collaborative Drug Discovery Research (CDDR) and Brain Degeneration and Therapeutics Research Group, Faculty of Pharmacy, University Teknologi MARA (UiTM) Cawangan Selangor, Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
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23
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Wei X, Ma D, Zhang Z, Wang LY, Gray JL, Zhang L, Zhu T, Wang X, Lenhart BJ, Yin Y, Wang Q, Liu C. N-Terminal Derivatization-Assisted Identification of Individual Amino Acids Using a Biological Nanopore Sensor. ACS Sens 2020; 5:1707-1716. [PMID: 32403927 PMCID: PMC7978492 DOI: 10.1021/acssensors.0c00345] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Nanopore technology has been employed as a powerful tool for DNA sequencing and analysis. To extend this method to peptide sequencing, a necessary step is to profile individual amino acids (AAs) through their nanopore stochastic signals, which remains a great challenge because of the low signal-to-noise ratio and unpredictable conformational changes of AAs during their translocation through nanopores. We showed that the combination of an N-terminal derivatization strategy of AAs with nanopore technology could lead to effective in situ differentiation of AAs. Four different derivatization reactions have been tested with five selected AAs: Ala, Phe, Tyr, His, and Asp. Using an α-hemolysin nanopore, we demonstrated the feasibility of derivatization-assisted identification of AAs regardless of their charge composition and polarity. The method was further applied to discriminate each individual AA in testing data sets using their established nanopore profiles from training data sets. We envision that this proof-of-concept study will not only pave a way for identification of individual AAs but also lead to future applications in protein/peptide sequencing using the nanopore technology.
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Affiliation(s)
- Xiaojun Wei
- Biomedical Engineering Program, University of South Carolina, Columbia, South Carolina 29208, United States
- Department of Chemical Engineering, University of South Carolina, Columbia, South Carolina 29208, United States
| | - Dumei Ma
- Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, United States
- Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 Fujian, China
| | - Zehui Zhang
- Biomedical Engineering Program, University of South Carolina, Columbia, South Carolina 29208, United States
| | - Leon Y Wang
- Department of Chemical Engineering, University of South Carolina, Columbia, South Carolina 29208, United States
| | - Jonathan L Gray
- Biomedical Engineering Program, University of South Carolina, Columbia, South Carolina 29208, United States
| | - Libo Zhang
- Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, United States
| | - Tianyu Zhu
- Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, United States
| | - Xiaoqin Wang
- Department of Chemical Engineering, University of South Carolina, Columbia, South Carolina 29208, United States
| | - Brian J Lenhart
- Department of Chemical Engineering, University of South Carolina, Columbia, South Carolina 29208, United States
| | - Yingwu Yin
- Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 Fujian, China
| | - Qian Wang
- Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, United States
| | - Chang Liu
- Biomedical Engineering Program, University of South Carolina, Columbia, South Carolina 29208, United States
- Department of Chemical Engineering, University of South Carolina, Columbia, South Carolina 29208, United States
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24
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Suvarna K, Honda K, Muroi M, Kondoh Y, Watanabe N, Osada H. Identification of Target Protein for Bio-active Small Molecule Using Photo-cross Linked Beads and MALDI-TOF Mass Spectrometry. Bio Protoc 2020; 10:e3517. [PMID: 33654742 DOI: 10.21769/bioprotoc.3517] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2019] [Revised: 12/23/2019] [Accepted: 12/24/2019] [Indexed: 11/02/2022] Open
Abstract
Development of methods for protein identification is one of the important aspects of proteomics. Here, we report a protocol for the preparation of compound conjugated beads by photo-crosslinking, affinity purification, gel electrophoresis, and highly sensitive mass spectrometric assay for drug-target identification. Although there are several other methods used for drug-target identification, such as biochemical fractionation or radioactive ligand binding assay, affinity purification is widely used for its straight-forward and easy approach. To identify the target protein of an inhibitor of cancer cell-accelerated fibroblast migration, we prepared the inhibitor-conjugated beads by photo-crosslinking. Proteins were pulled down from cell lysates by the compound beads and separated by SDS-PAGE, and a specifically pulled down protein was cut out, trypsin-digested, analyzed using matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF-MS) and identified by peptide mass fingerprinting (PMF) method. Since the photo-crosslinking enables the immobilization of ligands on an affinity matrix in a functional group-independent manner, we do not have to determine the functional group of the compound to conjugate the matrix. In addition, as compared to other MS techniques such as electrospray ionization, MALDI offers a less complex sample preparation procedure and higher sensitivity, and thus is better suited for the rapid identification of proteins isolated by gel electrophoresis.
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Affiliation(s)
- Kruthi Suvarna
- Bio-Active Compounds Discovery Research Unit, RIKEN CSRS, Saitama, 351-0198, Japan.,Tokyo Medical Dental University, Yushima, Tokyo, 113-8510, Japan
| | - Kaori Honda
- Bio-Active Compounds Discovery Research Unit, RIKEN CSRS, Saitama, 351-0198, Japan.,Chemical Biology Research Group, RIKEN CSRS, Saitama, 351-0198, Japan
| | - Makoto Muroi
- Chemical Biology Research Group, RIKEN CSRS, Saitama, 351-0198, Japan
| | - Yasumitsu Kondoh
- Chemical Biology Research Group, RIKEN CSRS, Saitama, 351-0198, Japan
| | - Nobumoto Watanabe
- Bio-Active Compounds Discovery Research Unit, RIKEN CSRS, Saitama, 351-0198, Japan.,Tokyo Medical Dental University, Yushima, Tokyo, 113-8510, Japan.,RIKEN-Max Planck Joint Research Division, RIKEN CSRS, Saitama, 351-0198, Japan
| | - Hiroyuki Osada
- Chemical Biology Research Group, RIKEN CSRS, Saitama, 351-0198, Japan.,RIKEN-Max Planck Joint Research Division, RIKEN CSRS, Saitama, 351-0198, Japan
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25
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Ouwendijk WJD, Dekker LJM, van den Ham HJ, Lenac Rovis T, Haefner ES, Jonjic S, Haas J, Luider TM, Verjans GMGM. Analysis of Virus and Host Proteomes During Productive HSV-1 and VZV Infection in Human Epithelial Cells. Front Microbiol 2020; 11:1179. [PMID: 32547533 PMCID: PMC7273502 DOI: 10.3389/fmicb.2020.01179] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2020] [Accepted: 05/08/2020] [Indexed: 12/21/2022] Open
Abstract
Herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) are two closely related human alphaherpesviruses that persistently infect most adults worldwide and cause a variety of clinically important diseases. Herpesviruses are extremely well adapted to their hosts and interact broadly with cellular proteins to regulate virus replication and spread. However, it is incompletely understood how HSV-1 and VZV interact with the host proteome during productive infection. This study determined the temporal changes in virus and host protein expression during productive HSV-1 and VZV infection in the same cell type. Results demonstrated the temporally coordinated expression of HSV-1 and VZV proteins in infected cells. Analysis of the host proteomes showed that both viruses affected extracellular matrix composition, transcription, RNA processing and cell division. Moreover, the prominent role of epidermal growth factor receptor (EGFR) signaling during productive HSV-1 and VZV infection was identified. Stimulation and inhibition of EGFR leads to increased and decreased virus replication, respectively. Collectively, the comparative temporal analysis of viral and host proteomes in productively HSV-1 and VZV-infected cells provides a valuable resource for future studies aimed to identify target(s) for antiviral therapy development.
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Affiliation(s)
- Werner J. D. Ouwendijk
- Department of Viroscience, Erasmus MC, Rotterdam, Netherlands
- *Correspondence: Werner J. D. Ouwendijk,
| | | | - Henk-Jan van den Ham
- Department of Viroscience, Erasmus MC, Rotterdam, Netherlands
- Enpicom B.V., ‘s-Hertogenbosch, Netherlands
| | - Tihana Lenac Rovis
- Center for Proteomics and Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
| | - Erik S. Haefner
- Experimental and Translational Oncology, University Medical Center Mainz, Mainz, Germany
| | - Stipan Jonjic
- Center for Proteomics and Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
| | - Jürgen Haas
- Division of Infection and Pathway Medicine, University of Edinburgh, Edinburgh, United Kingdom
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26
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Ten Cate V, Koeck T, Panova-Noeva M, Rapp S, Prochaska JH, Lenz M, Schulz A, Eggebrecht L, Hermanns MI, Heitmeier S, Krahn T, Laux V, Münzel T, Leineweber K, Konstantinides SV, Wild PS. A prospective cohort study to identify and evaluate endotypes of venous thromboembolism: Rationale and design of the Genotyping and Molecular Phenotyping in Venous ThromboEmbolism project (GMP-VTE). Thromb Res 2019; 181:84-91. [PMID: 31374513 DOI: 10.1016/j.thromres.2019.07.019] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2019] [Revised: 07/17/2019] [Accepted: 07/21/2019] [Indexed: 01/02/2023]
Abstract
Several clinical, genetic and acquired risk factors for venous thromboembolism (VTE) have been identified. However, the molecular pathophysiology and mechanisms of disease progression remain poorly understood. This is reflected by uncertainties regarding the primary and secondary prevention of VTE and the optimal duration of antithrombotic therapy. A growing body of literature points to clinically relevant differences between VTE phenotypes (e.g. deep vein thrombosis (DVT) versus pulmonary embolism (PE), unprovoked versus provoked VTE). Extensive links to cardiovascular, inflammatory and immune-related morbidities are testament to the complexity of the disease. The GMP-VTE project is a prospective, multi-center cohort study on individuals with objectively confirmed VTE. Sequential data sampling was performed at the time of the acute event and during serial follow-up investigations. Various data levels (e.g. clinical, genetic, proteomic and platelet data) are available for multi-dimensional data analyses by means of advanced statistical, bioinformatic and machine learning methods. The GMP-VTE project comprises n = 663 individuals with acute VTE (mean age: 60.3 ± 15.9 years; female sex: 42.8%). In detail, 28.4% individuals (n = 188) had acute isolated DVT, whereas 71.6% subjects (n = 475) had PE with or without concomitant DVT. In the study sample, 28.9% (n = 129) of individuals with PE and 30.1% (n = 55) of individuals with isolated DVT had a recurrent VTE event at the time of study enrolment. The systems-oriented approach for the comprehensive dataset of the GMP-VTE project may generate new biological insights into the pathophysiology of VTE and refine our current understanding and management of VTE.
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Affiliation(s)
- V Ten Cate
- Preventive Cardiology and Preventive Medicine, Center for Cardiology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany; Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - T Koeck
- Preventive Cardiology and Preventive Medicine, Center for Cardiology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - M Panova-Noeva
- Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany; Preventive Cardiology and Preventive Medicine, Center for Cardiology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Rhine Main, University Medical Center of the Johannes Gutenberg University Mainz, Germany
| | - S Rapp
- Preventive Cardiology and Preventive Medicine, Center for Cardiology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - J H Prochaska
- Preventive Cardiology and Preventive Medicine, Center for Cardiology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany; Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Rhine Main, University Medical Center of the Johannes Gutenberg University Mainz, Germany
| | - M Lenz
- Preventive Cardiology and Preventive Medicine, Center for Cardiology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany; Institute of Organismic and Molecular Evolution, Johannes Gutenberg University Mainz, Mainz, Germany
| | - A Schulz
- Preventive Cardiology and Preventive Medicine, Center for Cardiology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - L Eggebrecht
- Preventive Cardiology and Preventive Medicine, Center for Cardiology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany; Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - M I Hermanns
- Preventive Cardiology and Preventive Medicine, Center for Cardiology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany; School of Chemistry, Biology and Pharmacy, Fresenius University of Applied Sciences, Idstein, Germany
| | | | - T Krahn
- Bayer AG, Wuppertal, Germany
| | - V Laux
- Bayer AG, Wuppertal, Germany
| | - T Münzel
- Center for Cardiology - Cardiology I, University Medical Center of the Johannes Gutenberg University Mainz, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Rhine Main, University Medical Center of the Johannes Gutenberg University Mainz, Germany; Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | | | - S V Konstantinides
- Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany; Department of Cardiology, Democritus University of Thrace, University General Hospital, Greece
| | - P S Wild
- Preventive Cardiology and Preventive Medicine, Center for Cardiology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany; Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Rhine Main, University Medical Center of the Johannes Gutenberg University Mainz, Germany.
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27
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Beneke T, Demay F, Hookway E, Ashman N, Jeffery H, Smith J, Valli J, Becvar T, Myskova J, Lestinova T, Shafiq S, Sadlova J, Volf P, Wheeler RJ, Gluenz E. Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections. PLoS Pathog 2019; 15:e1007828. [PMID: 31242261 PMCID: PMC6615630 DOI: 10.1371/journal.ppat.1007828] [Citation(s) in RCA: 72] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2018] [Revised: 07/09/2019] [Accepted: 05/08/2019] [Indexed: 11/29/2022] Open
Abstract
The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite’s life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single paralysed mutant lacking the central pair protein PF16 or an uncoordinated swimmer lacking the axonemal protein MBO2 showed that these promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies. Leishmania are protozoan parasites, transmitted between mammals by the bite of phlebotomine sand flies. Promastigote forms in the sand fly have a long flagellum, which is motile and used for anchoring the parasites to prevent clearance with the digested blood meal remnants. To dissect flagellar functions and their importance in life cycle progression, we generated here a comprehensive list of >300 flagellar proteins and produced a CRISPR-Cas9 gene knockout library of 100 mutant Leishmania. We studied their behaviour in vitro before examining their fate in the sand fly Lutzomyia longipalpis. Measuring mutant swimming speeds showed that about half behaved differently compared to the wild type: a few swam faster, many slower and some were completely paralysed. We also found a group of uncoordinated swimmers. To test whether flagellar motility is required for parasite migration from the fly midgut to the foregut from where they reach the next host, we infected sand flies with a mixed mutant population. Each mutant carried a unique tag and tracking these tags up to nine days after infection showed that paralysed and uncoordinated Leishmania were rapidly lost from flies. These data indicate that directional swimming is important for successful colonisation of sand flies.
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Affiliation(s)
- Tom Beneke
- Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
| | - François Demay
- University of Lille 1, Cité Scientifique, Villeneuve d’Ascq, France
| | - Edward Hookway
- Research Department of Pathology, University College London, London, United Kingdom
| | - Nicole Ashman
- Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
| | - Heather Jeffery
- Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
| | - James Smith
- Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
| | - Jessica Valli
- Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
| | - Tomas Becvar
- Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic
| | - Jitka Myskova
- Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic
| | - Tereza Lestinova
- Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic
| | - Shahaan Shafiq
- Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
- Department of Biological and Medical Sciences, Oxford Brookes University, Gipsy Lane, Oxford, United Kingdom
| | - Jovana Sadlova
- Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic
| | - Petr Volf
- Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic
| | - Richard John Wheeler
- Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
- Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Eva Gluenz
- Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
- * E-mail:
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28
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Sharma A, Sharma D, Verma SK. In silico Study of Iron, Zinc and Copper Binding Proteins of Pseudomonas syringae pv. lapsa: Emphasis on Secreted Metalloproteins. Front Microbiol 2018; 9:1838. [PMID: 30186242 PMCID: PMC6110883 DOI: 10.3389/fmicb.2018.01838] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2018] [Accepted: 07/23/2018] [Indexed: 11/17/2022] Open
Abstract
The phytopathogenic bacteria, Pseudomonas syringae pv. lapsa (P. syringae pv. lapsa) infects the staple food crop wheat. Metalloproteins play important roles in plant-pathogen interactions. Hence, the present work is aimed to predict and analyze the iron (Fe), zinc (Zn), and copper (Cu) binding proteins of P. syringae pv. lapsa which help in its growth, adaptation, survival and pathogenicity. A total of 232 Fe, 307 Zn, and 38 Cu-binding proteins have been identified. The functional annotation, subcellular localization and gene ontology enriched network analysis revealed their role in wide range of biological activities of the phytopathogen. Among the identified metalloproteins, a total of 29 Fe-binding, 31 Zn-binding, and 5 Cu-binding proteins were found to be secreted in nature. These putative secreted metalloproteins may perform diverse cellular and biological functions ranging from transport, response to oxidative stress, proteolysis, antimicrobial resistance, metabolic processes, protein folding and DNA repair. The observations obtained here may provide initial information required to draft new schemes to control microbial infections of staple food crops and will further help in developing sustainable agriculture.
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Affiliation(s)
- Ankita Sharma
- Centre for Computational Biology and Bioinformatics, School of Life Sciences, Central University of Himachal Pradesh, Kangra, India
| | - Dixit Sharma
- Centre for Computational Biology and Bioinformatics, School of Life Sciences, Central University of Himachal Pradesh, Kangra, India
| | - Shailender K Verma
- Centre for Computational Biology and Bioinformatics, School of Life Sciences, Central University of Himachal Pradesh, Kangra, India
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29
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Han X, Xie Y, Wu Q, Wu S. A novel protein digestion method with the assistance of alternating current denaturation for high efficient protein digestion and mass spectrometry analysis. Talanta 2018; 184:382-387. [PMID: 29674058 DOI: 10.1016/j.talanta.2018.03.017] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2018] [Revised: 03/01/2018] [Accepted: 03/07/2018] [Indexed: 12/24/2022]
Abstract
Protein denaturation has always displayed a huge necessity for mass spectrometry (MS)-based protein identification methods in proteomics. In this research, a novel protein digestion method with the assistance of alternating current (AC) denaturation has been proposed and evaluated. In this method, merely, 200 mM ammonium bicarbonate buffer solution (pH, 8.2) was used to dissolve proteins and act as the electrolyte, and protein denaturation could be achieved in several seconds. For apo-transferrin, ovalbumin and bovine serum albumin that are resistant to digestion in their native states, confident amino acid sequence coverage by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis were obtained after 200 v AC denaturation. The applicability of this method was further investigated via analyzing a rat liver proteome sample using nano reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoRPLC-ESI-MS/MS). As a result, 458 proteins were identified which is comparable to the in-solution digestion via 8 M urea denaturation (375 proteins). All these results demonstrated that AC denaturation could offer an efficient assistance for a clean and high-throughput digestion in the individual level and proteome level.
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Affiliation(s)
- Xiaoxun Han
- Hubei Collaborative Innovation Center for Rare Metal Chemistry, Hubei Key Laboratory of Pollutant Analysis & Reuse Technology, College of Chemistry and Chemical Engineering, Hubei Normal University, Cihu Road No. 11, Huangshi 435002, China
| | - Yiming Xie
- Hubei Collaborative Innovation Center for Rare Metal Chemistry, Hubei Key Laboratory of Pollutant Analysis & Reuse Technology, College of Chemistry and Chemical Engineering, Hubei Normal University, Cihu Road No. 11, Huangshi 435002, China
| | - Qin Wu
- Hubei Collaborative Innovation Center for Rare Metal Chemistry, Hubei Key Laboratory of Pollutant Analysis & Reuse Technology, College of Chemistry and Chemical Engineering, Hubei Normal University, Cihu Road No. 11, Huangshi 435002, China
| | - Shuaibin Wu
- Hubei Collaborative Innovation Center for Rare Metal Chemistry, Hubei Key Laboratory of Pollutant Analysis & Reuse Technology, College of Chemistry and Chemical Engineering, Hubei Normal University, Cihu Road No. 11, Huangshi 435002, China.
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Lewis NH, Hitchcock DB, Dryden IL, Rose JR. Peptide refinement by using a stochastic search. J R Stat Soc Ser C Appl Stat 2018. [DOI: 10.1111/rssc.12280] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
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Laumont CM, Perreault C. Exploiting non-canonical translation to identify new targets for T cell-based cancer immunotherapy. Cell Mol Life Sci 2018; 75:607-621. [PMID: 28823056 PMCID: PMC11105255 DOI: 10.1007/s00018-017-2628-4] [Citation(s) in RCA: 56] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2017] [Revised: 08/03/2017] [Accepted: 08/16/2017] [Indexed: 01/11/2023]
Abstract
Cryptic MHC I-associated peptides (MAPs) are produced via two mechanisms: translation of protein-coding genes in non-canonical reading frames and translation of allegedly non-coding sequences. In general, cryptic MAPs are coded by relatively short open reading frames whose translation can be regulated at the level of initiation, elongation or termination. In contrast to conventional MAPs, the processing of cryptic MAPs is frequently proteasome independent. The existence of cryptic MAPs derived from allegedly non-coding regions enlarges the scope of CD8 T cell immunosurveillance from a mere ~2% to as much as ~75% of the human genome. Considering that 99% of cancer-specific mutations are located in those allegedly non-coding regions, cryptic MAPs could furthermore represent a particularly rich source of tumor-specific antigens. However, extensive proteogenomic analyses will be required to determine the breath as well as the temporal and spatial plasticity of the cryptic MAP repertoire in normal and neoplastic cells.
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Affiliation(s)
- Céline M Laumont
- Institute for Research in Immunology and Cancer, Université de Montréal, Station Centre-Ville, PO Box 6128, Montreal, QC, H3C 3J7, Canada
- Department of Medicine, Faculty of Medicine, Université de Montréal, Station Centre-Ville, PO Box 6128, Montreal, QC, H3C 3J7, Canada
| | - Claude Perreault
- Institute for Research in Immunology and Cancer, Université de Montréal, Station Centre-Ville, PO Box 6128, Montreal, QC, H3C 3J7, Canada.
- Department of Medicine, Faculty of Medicine, Université de Montréal, Station Centre-Ville, PO Box 6128, Montreal, QC, H3C 3J7, Canada.
- Division of Hematology, Hôpital Maisonneuve-Rosemont, 5415 de l'Assomption Boulevard, Montreal, QC, H1T 2M4, Canada.
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Wang H, Yang Z, Du S, Ma L, Liao Y, Wang Y, Toth I, Fan J. Characterization of Pectobacterium carotovorum proteins differentially expressed during infection of Zantedeschia elliotiana in vivo and in vitro which are essential for virulence. MOLECULAR PLANT PATHOLOGY 2018; 19:35-48. [PMID: 27671364 PMCID: PMC6638092 DOI: 10.1111/mpp.12493] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/09/2016] [Revised: 09/05/2016] [Accepted: 09/19/2016] [Indexed: 05/22/2023]
Abstract
The identification of phytopathogen proteins that are differentially expressed during the course of the establishment of an infection is important to better understand the infection process. In vitro approaches, using plant extracts added to culture medium, have been used to identify such proteins, but the biological relevance of these findings for in planta infection are often uncertain until confirmed by in vivo studies. Here, we compared the proteins of Pectobacterium carotovorum ssp. carotovorum strain PccS1 differentially expressed in Luria-Bertani medium supplemented with extracts of the ornamental plant Zantedeschia elliotiana cultivar 'Black Magic' (in vitro) and in plant tissues (in vivo) by two-dimensional electrophoresis coupled with mass spectrometry. A total of 53 differentially expressed proteins (>1.5-fold) were identified (up-regulated or down-regulated in vitro, in vivo or both). Proteins that exhibited increased expression in vivo but not in vitro, or in both conditions, were identified, and deletions were made in a number of genes encoding these proteins, four of which (clpP, mreB, flgK and eda) led to a loss of virulence on Z. elliotiana, although clpP and mreB were later also shown to be reduced in growth in rich and minimal media. Although clpP, flgK and mreB have previously been reported as playing a role in virulence in plants, this is the first report of such a role for eda, which encodes 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, a key enzyme in Entner-Doudoroff metabolism. The results highlight the value of undertaking in vivo as well as in vitro approaches for the identification of new bacterial virulence factors.
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Affiliation(s)
- Huan Wang
- College of Plant ProtectionNanjing Agricultural UniversityNanjing210095China
| | - Zhongling Yang
- College of Plant ProtectionNanjing Agricultural UniversityNanjing210095China
| | - Shuo Du
- College of Plant ProtectionNanjing Agricultural UniversityNanjing210095China
| | - Lin Ma
- College of Plant ProtectionNanjing Agricultural UniversityNanjing210095China
| | - Yao Liao
- College of Plant ProtectionNanjing Agricultural UniversityNanjing210095China
| | - Yujie Wang
- College of Plant ProtectionNanjing Agricultural UniversityNanjing210095China
| | - Ian Toth
- Cell and Molecular SciencesJames Hutton InstituteDundeeDD2 5DAUK
| | - Jiaqin Fan
- College of Plant ProtectionNanjing Agricultural UniversityNanjing210095China
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DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions. Cell Rep 2017; 17:303-315. [PMID: 27681439 DOI: 10.1016/j.celrep.2016.08.095] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2016] [Revised: 05/06/2016] [Accepted: 08/29/2016] [Indexed: 01/06/2023] Open
Abstract
We adapted the yeast 2-hybrid assay to simultaneously uncover multiple transient protein interactions within a single screen by using a strategy termed DEEPN (dynamic enrichment for evaluation of protein networks). This approach incorporates high-throughput DNA sequencing and computation to follow competition among a plasmid population encoding interacting partners. To demonstrate the capacity of DEEPN, we identify a wide range of ubiquitin-binding proteins, including interactors that we verify biochemically. To demonstrate the specificity of DEEPN, we show that DEEPN allows simultaneous comparison of candidate interactors across multiple bait proteins, allowing differential interactions to be identified. This feature was used to identify interactors that distinguish between GTP- and GDP-bound conformations of Rab5.
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Adeola HA, Van Wyk JC, Arowolo A, Ngwanya RM, Mkentane K, Khumalo NP. Emerging Diagnostic and Therapeutic Potentials of Human Hair Proteomics. Proteomics Clin Appl 2017; 12. [PMID: 28960873 DOI: 10.1002/prca.201700048] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2017] [Revised: 06/09/2017] [Indexed: 01/22/2023]
Abstract
The use of noninvasive human substrates to interrogate pathophysiological conditions has become essential in the post- Human Genome Project era. Due to its high turnover rate, and its long term capability to incorporate exogenous and endogenous substances from the circulation, hair testing is emerging as a key player in monitoring long term drug compliance, chronic alcohol abuse, forensic toxicology, and biomarker discovery, among other things. Novel high-throughput 'omics based approaches like proteomics have been underutilized globally in comprehending human hair morphology and its evolving use as a diagnostic testing substrate in the era of precision medicine. There is paucity of scientific evidence that evaluates the difference in drug incorporation into hair based on lipid content, and very few studies have addressed hair growth rates, hair forms, and the biological consequences of hair grooming or bleaching. It is apparent that protein-based identification using the human hair proteome would play a major role in understanding these parameters akin to DNA single nucleotide polymorphism profiling, up to single amino acid polymorphism resolution. Hence, this work seeks to identify and discuss the progress made thus far in the field of molecular hair testing using proteomic approaches, and identify ways in which proteomics would improve the field of hair research, considering that the human hair is mostly composed of proteins. Gaps in hair proteomics research are identified and the potential of hair proteomics in establishing a historic medical repository of normal and disease-specific proteome is also discussed.
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Affiliation(s)
- Henry A Adeola
- Division of Dermatology, Department of Medicine, Faculty of Health Sciences and Groote Schuur Hospital, University of Cape Town, Cape Town, South Africa.,Hair and Skin Research Laboratory, Groote Schuur Hospital, Cape Town, South Africa
| | - Jennifer C Van Wyk
- Division of Dermatology, Department of Medicine, Faculty of Health Sciences and Groote Schuur Hospital, University of Cape Town, Cape Town, South Africa.,Hair and Skin Research Laboratory, Groote Schuur Hospital, Cape Town, South Africa
| | - Afolake Arowolo
- Division of Dermatology, Department of Medicine, Faculty of Health Sciences and Groote Schuur Hospital, University of Cape Town, Cape Town, South Africa.,Hair and Skin Research Laboratory, Groote Schuur Hospital, Cape Town, South Africa
| | - Reginald M Ngwanya
- Division of Dermatology, Department of Medicine, Faculty of Health Sciences and Groote Schuur Hospital, University of Cape Town, Cape Town, South Africa
| | - Khwezikazi Mkentane
- Division of Dermatology, Department of Medicine, Faculty of Health Sciences and Groote Schuur Hospital, University of Cape Town, Cape Town, South Africa.,Hair and Skin Research Laboratory, Groote Schuur Hospital, Cape Town, South Africa
| | - Nonhlanhla P Khumalo
- Division of Dermatology, Department of Medicine, Faculty of Health Sciences and Groote Schuur Hospital, University of Cape Town, Cape Town, South Africa.,Hair and Skin Research Laboratory, Groote Schuur Hospital, Cape Town, South Africa
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Gianazza E, Miller I, Guerrini U, Palazzolo L, Parravicini C, Eberini I. Gender proteomics I. Which proteins in non-sexual organs. J Proteomics 2017; 178:7-17. [PMID: 28988882 DOI: 10.1016/j.jprot.2017.10.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2017] [Revised: 09/26/2017] [Accepted: 10/04/2017] [Indexed: 12/15/2022]
Abstract
Differences related to gender have long been neglected but recent investigations show that they are widespread and may be recognized with all types of omics approaches, both in tissues and in biological fluids. Our review compiles evidence collected with proteomics techniques in our species, mainly focusing on baseline parameters in non-sexual organs in healthy men and women. Data from human specimens had to be replaced with information from other mammals every time invasive procedures of sample procurement were involved. SIGNIFICANCE As our knowledge, and the methods to build it, get refined, gender differences need to receive more and more attention, as they influence the outcome of all aspects in lifestyle, including diet, exercise and environmental factors. In turn this background modulates a differential susceptibility to some disease, or a different pathogenetic mechanism, depending on gender, and a different response to pharmacological therapy. Preparing this review we meant to raise awareness about the gender issue. We anticipate that more and more often, in the future, separate evaluations will be carried out on male and female subjects as an alternative - and an upgrade - to the current approach of reference and test groups being 'matched for age and sex'.
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Affiliation(s)
- Elisabetta Gianazza
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, I-20133 Milano, Italy.
| | - Ingrid Miller
- Institut für Medizinische Biochemie, Veterinärmedizinische Universität Wien, Veterinärplatz 1, A-1210 Wien, Austria
| | - Uliano Guerrini
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, I-20133 Milano, Italy
| | - Luca Palazzolo
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, I-20133 Milano, Italy
| | - Chiara Parravicini
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, I-20133 Milano, Italy
| | - Ivano Eberini
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, I-20133 Milano, Italy
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Subhadarshanee B, Mohanty A, Jagdev MK, Vasudevan D, Behera RK. Surface charge dependent separation of modified and hybrid ferritin in native PAGE: Impact of lysine 104. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2017; 1865:1267-1273. [DOI: 10.1016/j.bbapap.2017.07.012] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/21/2017] [Revised: 07/05/2017] [Accepted: 07/20/2017] [Indexed: 01/06/2023]
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Kennel PJ, Saha A, Maldonado DA, Givens R, Brunjes DL, Castillero E, Zhang X, Ji R, Yahi A, George I, Mancini DM, Koller A, Fine B, Zorn E, Colombo PC, Tatonetti N, Chen EI, Schulze PC. Serum exosomal protein profiling for the non-invasive detection of cardiac allograft rejection. J Heart Lung Transplant 2017; 37:409-417. [PMID: 28789823 DOI: 10.1016/j.healun.2017.07.012] [Citation(s) in RCA: 65] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2017] [Revised: 06/28/2017] [Accepted: 07/16/2017] [Indexed: 12/30/2022] Open
Abstract
BACKGROUND Exosomes are cell-derived circulating vesicles that play an important role in cell-cell communication. Exosomes are actively assembled and carry messenger RNAs, microRNAs and proteins. The "gold standard" for cardiac allograft surveillance is endomyocardial biopsy (EMB), an invasive technique with a distinct complication profile. The development of novel, non-invasive methods for the early diagnosis of allograft rejection is warranted. We hypothesized that the exosomal proteome is altered in acute rejection, allowing for a distinction between non-rejection and rejection episodes. METHODS Serum samples were collected from heart transplant (HTx) recipients with no rejection, acute cellular rejection (ACR) and antibody-mediated rejection (AMR). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of serum exosome was performed using a mass spectrometer (Orbitrap Fusion Tribrid). RESULTS Principal component analysis (PCA) revealed a clustering of 3 groups: (1) control and heart failure (HF); (2) HTx without rejection; and (3) ACR and AMR. A total of 45 proteins were identified that could distinguish between groups (q < 0.05). Comparison of serum exosomal proteins from control, HF and non-rejection HTx revealed 17 differentially expressed proteins in at least 1 group (q < 0.05). Finally, comparisons of non-rejection HTx, ACR and AMR serum exosomes revealed 15 differentially expressed proteins in at least 1 group (q < 0.05). Of these 15 proteins, 8 proteins are known to play a role in the immune response. Of note, the majority of proteins identified were associated with complement activation, adaptive immunity such as immunoglobulin components and coagulation. CONCLUSIONS Characterizing of circulating exosomal proteome in different cardiac disease states reveals unique protein expression patterns indicative of the respective pathologies. Our data suggest that HTx and allograft rejection alter the circulating exosomal protein content. Exosomal protein analysis could be a novel approach to detect and monitor acute transplant rejection and lead to the development of predictive and prognostic biomarkers.
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Affiliation(s)
- Peter J Kennel
- Division of Cardiology, Department of Medicine, Columbia University Medical Center, New York, New York, USA; Department of Medicine, Weill-Cornell Medical College, New York, New York, USA; Department of Internal Medicine I, Division of Cardiology, University Hospital Jena, Friedrich Schiller University Jena, Jena, Germany
| | - Amit Saha
- Division of Cardiology, Department of Medicine, Columbia University Medical Center, New York, New York, USA
| | - Dawn A Maldonado
- Division of Cardiology, Department of Medicine, Columbia University Medical Center, New York, New York, USA
| | - Raymond Givens
- Division of Cardiology, Department of Medicine, Columbia University Medical Center, New York, New York, USA
| | - Danielle L Brunjes
- Division of Cardiology, Department of Medicine, Columbia University Medical Center, New York, New York, USA
| | - Estibaliz Castillero
- Division of Cardiothoracic Surgery, Department of Surgery, Columbia University Medical Center, New York, New York, USA
| | - Xiaokan Zhang
- Division of Cardiology, Department of Medicine, Columbia University Medical Center, New York, New York, USA
| | - Ruiping Ji
- Division of Cardiology, Department of Medicine, Columbia University Medical Center, New York, New York, USA
| | - Alexandre Yahi
- Department of Biomedical Informatics, Columbia University, New York, New York, USA
| | - Isaac George
- Division of Cardiothoracic Surgery, Department of Surgery, Columbia University Medical Center, New York, New York, USA
| | - Donna M Mancini
- Division of Cardiology, Department of Medicine, Columbia University Medical Center, New York, New York, USA; Mount Sinai Heart, New York, New York, USA
| | - Antonius Koller
- The Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York, USA
| | - Barry Fine
- Division of Cardiology, Department of Medicine, Columbia University Medical Center, New York, New York, USA
| | - Emmanuel Zorn
- Columbia Center for Translational Immunology, Columbia University Medical Center, New York, New York, USA
| | - Paolo C Colombo
- Division of Cardiology, Department of Medicine, Columbia University Medical Center, New York, New York, USA
| | - Nicholas Tatonetti
- Department of Biomedical Informatics, Columbia University, New York, New York, USA
| | - Emily I Chen
- Columbia Center for Translational Immunology, Columbia University Medical Center, New York, New York, USA; Department of Pharmacology, Columbia University Medical Center, New York, New York, USA
| | - P Christian Schulze
- Division of Cardiology, Department of Medicine, Columbia University Medical Center, New York, New York, USA; Department of Internal Medicine I, Division of Cardiology, University Hospital Jena, Friedrich Schiller University Jena, Jena, Germany.
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ITIM receptors: more than just inhibitors of platelet activation. Blood 2017; 129:3407-3418. [PMID: 28465343 DOI: 10.1182/blood-2016-12-720185] [Citation(s) in RCA: 61] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2016] [Accepted: 04/24/2017] [Indexed: 12/12/2022] Open
Abstract
Since their discovery, immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptors have been shown to inhibit signaling from immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors in almost all hematopoietic cells, including platelets. However, a growing body of evidence has emerged demonstrating that this is an oversimplification, and that ITIM-containing receptors are versatile regulators of platelet signal transduction, with functions beyond inhibiting ITAM-mediated platelet activation. PECAM-1 was the first ITIM-containing receptor identified in platelets and appeared to conform to the established model of ITIM-mediated attenuation of ITAM-driven activation. PECAM-1 was therefore widely accepted as a major negative regulator of platelet activation and thrombosis for many years, but more recent findings suggest a more complex role for this receptor, including the facilitation of αIIbβ3-mediated platelet functions. Since the identification of PECAM-1, several other ITIM-containing platelet receptors have been discovered. These include G6b-B, a critical regulator of platelet reactivity and production, and the noncanonical ITIM-containing receptor TREM-like transcript-1, which is localized to α-granules in resting platelets, binds fibrinogen, and acts as a positive regulator of platelet activation. Despite structural similarities and shared binding partners, including the Src homology 2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2, knockout and transgenic mouse models have revealed distinct phenotypes and nonredundant functions for each ITIM-containing receptor in the context of platelet homeostasis. These roles are likely influenced by receptor density, compartmentalization, and as-yet unknown binding partners. In this review, we discuss the diverse repertoire of ITIM-containing receptors in platelets, highlighting intriguing new functions, controversies, and future areas of investigation.
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Proteome scale identification, classification and structural analysis of iron-binding proteins in bread wheat. J Inorg Biochem 2017; 170:63-74. [DOI: 10.1016/j.jinorgbio.2017.02.012] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2016] [Revised: 01/23/2017] [Accepted: 02/10/2017] [Indexed: 12/26/2022]
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Sharma A, Sharma D, Verma SK. Proteome wide identification of iron binding proteins of Xanthomonas translucens pv. undulosa: focus on secretory virulent proteins. Biometals 2017; 30:127-141. [DOI: 10.1007/s10534-017-9991-3] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2016] [Accepted: 01/08/2017] [Indexed: 12/19/2022]
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Ujang JA, Kwan SH, Ismail MN, Lim BH, Noordin R, Othman N. Proteome analysis of excretory-secretory proteins of Entamoeba histolytica HM1:IMSS via LC-ESI-MS/MS and LC-MALDI-TOF/TOF. Clin Proteomics 2016; 13:33. [PMID: 27895543 PMCID: PMC5120466 DOI: 10.1186/s12014-016-9135-8] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2016] [Accepted: 11/16/2016] [Indexed: 11/23/2022] Open
Abstract
BACKGROUND Excretory-secretory (ES) proteins of E. histolytica are thought to play important roles in the host invasion, metabolism, and defence. Elucidation of the types and functions of E. histolytica ES proteins can further our understanding of the disease pathogenesis. Thus, the aim of this study is to use proteomics approach to better understand the complex ES proteins of the protozoa. METHODS E. histolytica ES proteins were prepared by culturing the trophozoites in protein-free medium. The ES proteins were identified using two mass spectrometry tools, namely, LC-ESI-MS/MS and LC-MALDI-TOF/TOF. The identified proteins were then classified according to their biological processes, molecular functions, and cellular components using the Panther classification system (PantherDB). RESULTS A complementary list of 219 proteins was identified; this comprised 201 proteins detected by LC-ESI-MS/MS and 107 proteins by LC-MALDI-TOF/TOF. Of the 219 proteins, 89 were identified by both mass-spectrometry systems, while 112 and 18 proteins were detected exclusively by LC-ESI-MS/MS and LC-MALDI-TOF/TOF respectively. Biological protein functional analysis using PantherDB showed that 27% of the proteins were involved in metabolic processes. Using molecular functional and cellular component analyses, 35% of the proteins were found to be involved in catalytic activity, and 21% were associated with the cell parts. CONCLUSION This study showed that complementary use of LC-ESI-MS/MS and LC-MALDI-TOF/TOF has improved the identification of ES proteins. The results have increased our understanding of the types of proteins excreted/secreted by the amoeba and provided further evidence of the involvement of ES proteins in intestinal colonisation and evasion of the host immune system, as well as in encystation and excystation of the parasite.
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Affiliation(s)
- Jorim Anak Ujang
- Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Gelugor, Penang Malaysia
| | - Soon Hong Kwan
- Analytical Biochemistry Research Centre, Universiti Sains Malaysia, 11800 Gelugor, Penang Malaysia
| | - Mohd Nazri Ismail
- Analytical Biochemistry Research Centre, Universiti Sains Malaysia, 11800 Gelugor, Penang Malaysia
| | - Boon Huat Lim
- School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan Malaysia
| | - Rahmah Noordin
- Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Gelugor, Penang Malaysia
| | - Nurulhasanah Othman
- Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Gelugor, Penang Malaysia
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Jones J, Mirzaei M, Ravishankar P, Xavier D, Lim DS, Shin DH, Bianucci R, Haynes PA. Identification of proteins from 4200-year-old skin and muscle tissue biopsies from ancient Egyptian mummies of the first intermediate period shows evidence of acute inflammation and severe immune response. PHILOSOPHICAL TRANSACTIONS. SERIES A, MATHEMATICAL, PHYSICAL, AND ENGINEERING SCIENCES 2016; 374:rsta.2015.0373. [PMID: 27644972 PMCID: PMC5031639 DOI: 10.1098/rsta.2015.0373] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 06/28/2016] [Indexed: 05/18/2023]
Abstract
We performed proteomics analysis on four skin and one muscle tissue samples taken from three ancient Egyptian mummies of the first intermediate period, approximately 4200 years old. The mummies were first dated by radiocarbon dating of the accompany-\break ing textiles, and morphologically examined by scanning electron microscopy of additional skin samples. Proteins were extracted, separated on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels, and in-gel digested with trypsin. The resulting peptides were analysed using nanoflow high-performance liquid chromatography-mass spectrometry. We identified a total of 230 unique proteins from the five samples, which consisted of 132 unique protein identifications. We found a large number of collagens, which was confirmed by our microscopy data, and is in agreement with previous studies showing that collagens are very long-lived. As expected, we also found a large number of keratins. We identified numerous proteins that provide evidence of activation of the innate immunity system in two of the mummies, one of which also contained proteins indicating severe tissue inflammation, possibly indicative of an infection that we can speculate may have been related to the cause of death.This article is part of the themed issue 'Quantitative mass spectrometry'.
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Affiliation(s)
- Jana Jones
- Department of Ancient History, Macquarie University, North Ryde, NSW 2109, Australia
| | - Mehdi Mirzaei
- Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109, Australia
| | - Prathiba Ravishankar
- Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109, Australia
| | - Dylan Xavier
- Australian Proteome Analysis Facility, Macquarie University, North Ryde, NSW 2109, Australia
| | - Do Seon Lim
- Department of Dental Hygiene, College of Health Sciences, Eulji University, Sungnam, South Korea
| | - Dong Hoon Shin
- Department of Anatomy, Seoul National University, College of Medicine, Seoul, South Korea
| | - Raffaella Bianucci
- Department of Public Health and Paediatric Sciences, Legal Medicine Section, University of Turin, 10126 Turin, Italy UMR 7268, Laboratoire d'Anthropologie bio-culturelle, Droit, Étique and Santé (ADÉS), Faculté de Médecine de Marseille, 13344 Marseille, France
| | - Paul A Haynes
- Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109, Australia
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Kang C, Lee Y, Lee JE. Recent advances in mass spectrometry-based proteomics of gastric cancer. World J Gastroenterol 2016; 22:8283-8293. [PMID: 27729735 PMCID: PMC5055859 DOI: 10.3748/wjg.v22.i37.8283] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/28/2016] [Revised: 07/28/2016] [Accepted: 08/10/2016] [Indexed: 02/06/2023] Open
Abstract
The last decade has witnessed remarkable technological advances in mass spectrometry-based proteomics. The development of proteomics techniques has enabled the reliable analysis of complex proteomes, leading to the identification and quantification of thousands of proteins in gastric cancer cells, tissues, and sera. This quantitative information has been used to profile the anomalies in gastric cancer and provide insights into the pathogenic mechanism of the disease. In this review, we mainly focus on the advances in mass spectrometry and quantitative proteomics that were achieved in the last five years and how these up-and-coming technologies are employed to track biochemical changes in gastric cancer cells. We conclude by presenting a perspective on quantitative proteomics and its future applications in the clinic and translational gastric cancer research.
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Thulasi Raman SN, Zhou Y. Networks of Host Factors that Interact with NS1 Protein of Influenza A Virus. Front Microbiol 2016; 7:654. [PMID: 27199973 PMCID: PMC4855030 DOI: 10.3389/fmicb.2016.00654] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2016] [Accepted: 04/19/2016] [Indexed: 11/13/2022] Open
Abstract
Pigs are an important host of influenza A viruses due to their ability to generate reassortant viruses with pandemic potential. NS1 protein of influenza A viruses is a key virulence factor and a major antagonist of innate immune responses. It is also involved in enhancing viral mRNA translation and regulation of virus replication. Being a protein with pleiotropic functions, NS1 has a variety of cellular interaction partners. Hence, studies on swine influenza viruses (SIV) and identification of swine influenza NS1-interacting host proteins is of great interest. Here, we constructed a recombinant SIV carrying a Strep-tag in the NS1 protein and infected primary swine respiratory epithelial cells (SRECs) with this virus. The Strep-tag sequence in the NS1 protein enabled us to purify intact, the NS1 protein and its interacting protein complex specifically. We identified cellular proteins present in the purified complex by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and generated a dataset of these proteins. 445 proteins were identified by LC-MS/MS and among them 192 proteins were selected by setting up a threshold based on MS parameters. The selected proteins were analyzed by bioinformatics and were categorized as belonging to different functional groups including translation, RNA processing, cytoskeleton, innate immunity, and apoptosis. Protein interaction networks were derived using these data and the NS1 interactions with some of the specific host factors were verified by immunoprecipitation. The novel proteins and the networks revealed in our study will be the potential candidates for targeted study of the molecular interaction of NS1 with host proteins, which will provide insights into the identification of new therapeutic targets to control influenza infection and disease pathogenesis.
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Affiliation(s)
- Sathya N Thulasi Raman
- Vaccine and Infectious Disease Organization - International Vaccine Centre, University of Saskatchewan, SaskatoonSK, Canada; Vaccinology and Immunotherapeutics Program, School of Public Health, University of Saskatchewan, SaskatoonSK, Canada
| | - Yan Zhou
- Vaccine and Infectious Disease Organization - International Vaccine Centre, University of Saskatchewan, SaskatoonSK, Canada; Vaccinology and Immunotherapeutics Program, School of Public Health, University of Saskatchewan, SaskatoonSK, Canada
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Starck SR, Tsai JC, Chen K, Shodiya M, Wang L, Yahiro K, Martins-Green M, Shastri N, Walter P. Translation from the 5' untranslated region shapes the integrated stress response. Science 2016; 351:aad3867. [PMID: 26823435 DOI: 10.1126/science.aad3867] [Citation(s) in RCA: 274] [Impact Index Per Article: 30.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Translated regions distinct from annotated coding sequences have emerged as essential elements of the proteome. This includes upstream open reading frames (uORFs) present in mRNAs controlled by the integrated stress response (ISR) that show "privileged" translation despite inhibited eukaryotic initiation factor 2-guanosine triphosphate-initiator methionyl transfer RNA (eIF2·GTP·Met-tRNA(i )(Met)). We developed tracing translation by T cells to directly measure the translation products of uORFs during the ISR. We identified signature translation events from uORFs in the 5' untranslated region of binding immunoglobulin protein (BiP) mRNA (also called heat shock 70-kilodalton protein 5 mRNA) that were not initiated at the start codon AUG. BiP expression during the ISR required both the alternative initiation factor eIF2A and non-AUG-initiated uORFs. We propose that persistent uORF translation, for a variety of chaperones, shelters select mRNAs from the ISR, while simultaneously generating peptides that could serve as major histocompatibility complex class I ligands, marking cells for recognition by the adaptive immune system.
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Affiliation(s)
- Shelley R Starck
- Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, CA 94143, USA. Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
| | - Jordan C Tsai
- Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, CA 94143, USA
| | - Keling Chen
- Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
| | - Michael Shodiya
- Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
| | - Lei Wang
- Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA
| | - Kinnosuke Yahiro
- Departments of Molecular Infectiology, Graduate School of Medicine, Chiba University, Chiba, Japan
| | - Manuela Martins-Green
- Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA
| | - Nilabh Shastri
- Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
| | - Peter Walter
- Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, CA 94143, USA.
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Ghaheri S, Masoum S, Gholami A. Resolving of challenging gas chromatography–mass spectrometry peak clusters in fragrance samples using multicomponent factorization approaches based on polygon inflation algorithm. J Chromatogr A 2016; 1429:317-28. [DOI: 10.1016/j.chroma.2015.12.003] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2015] [Revised: 11/19/2015] [Accepted: 12/02/2015] [Indexed: 10/22/2022]
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Narayan M, Seeley KW, Jinwal UK. Identification and quantitative analysis of cellular proteins affected by treatment with withaferin a using a SILAC-based proteomics approach. JOURNAL OF ETHNOPHARMACOLOGY 2015; 175:86-92. [PMID: 26392330 DOI: 10.1016/j.jep.2015.09.024] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/01/2015] [Revised: 08/24/2015] [Accepted: 09/17/2015] [Indexed: 06/05/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE Withaferin A (WA) is a major bioactive compound isolated from the medicinal plant Withania somnifera Dunal, also known as "Ashwagandha". A number of published reports suggest various uses for WA including its function as an anti-inflammatory and anti-angiogenic drug molecule. The effects of WA at the molecular level in a cellular environment are not well understood. Knowledge of the molecular mechanism of action of WA could enhance its therapeutic value and may reveal novel pathways it may modulate. MATERIALS AND METHODS In order to identify and characterize proteins affected by treatment with WA, we used SILAC- based proteomics analysis on a mouse microglial cell line (N9), which replicates phenotypic characteristics of primary microglial cells. RESULTS Using stable isotope labeling of amino acids in cell culture (SILAC) and mass spectrometry (MS), a total of 2300 unique protein groups were identified from three biological replicates, with significant expression changes in 32 non-redundant proteins. The top biological functions associated with these differentially expressed proteins include cell death and survival, free radical scavenging, and carbohydrate metabolism. Specifically, several heat shock proteins (Hsps) were found to be upregulated, which suggests that the chaperonic machinery might be regulated by WA. Furthermore, our study revealed several novel protein molecules that were not previously reported to be affected by WA. Among them, annexin A1, a key anti-inflammatory molecule in microglial cells was found to be downregulated. Hsc70, Hsp90α and Hsp105 were found to be upregulated. We also found sequestosome1/p62 (p62) to be upregulated. We performed Ingenuity Pathway Analysis (IPA) and found a number of pathways that were affected by WA treatment. CONCLUSIONS SILAC-based proteomics analysis of a microglial cell model revealed several novel proteins whose expression is regulated by WA and probable pathways regulated by WA.
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Affiliation(s)
- Malathi Narayan
- Department of Pharmaceutical Sciences, College of Pharmacy, Byrd Alzheimer's Institute, University of South Florida-Health, 4001 E. Fletcher Ave, MDC36, Tampa, FL 33613, United States
| | - Kent W Seeley
- Florida Center of Excellence for Drug Discovery & Innovation at the University of South Florida, 3720 Spectrum Blvd., Suite 303, IDR Building, Tampa, FL 33612, United States
| | - Umesh K Jinwal
- Department of Pharmaceutical Sciences, College of Pharmacy, Byrd Alzheimer's Institute, University of South Florida-Health, 4001 E. Fletcher Ave, MDC36, Tampa, FL 33613, United States.
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Fischer JL, Lutomski CA, El-Baba TJ, Siriwardena-Mahanama BN, Weidner SM, Falkenhagen J, Allen MJ, Trimpin S. Matrix-Assisted Ionization-Ion Mobility Spectrometry-Mass Spectrometry: Selective Analysis of a Europium-PEG Complex in a Crude Mixture. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2015; 26:2086-2095. [PMID: 26453417 DOI: 10.1007/s13361-015-1233-8] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/01/2014] [Revised: 07/09/2015] [Accepted: 07/17/2015] [Indexed: 06/05/2023]
Abstract
The analytical utility of a new and simple to use ionization method, matrix-assisted ionization (MAI), coupled with ion mobility spectrometry (IMS) and mass spectrometry (MS) is used to characterize a 2-armed europium(III)-containing poly(ethylene glycol) (Eu-PEG) complex directly from a crude sample. MAI was used with the matrix 1,2-dicyanobenzene, which affords low chemical background relative to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). MAI provides high ion abundance of desired products in comparison to ESI and MALDI. Inductively coupled plasma-MS measurements were used to estimate a maximum of 10% of the crude sample by mass was the 2-arm Eu-PEG complex, supporting evidence of selective ionization of Eu-PEG complexes using the new MAI matrix, 1,2-dicyanobenzene. Multiply charged ions formed in MAI enhance the IMS gas-phase separation, especially relative to the singly charged ions observed with MALDI. Individual components are cleanly separated and readily identified, allowing characterization of the 2-arm Eu-PEG conjugate from a mixture of the 1-arm Eu-PEG complex and unreacted starting materials. Size-exclusion chromatography, liquid chromatography at critical conditions, MALDI-MS, ESI-MS, and ESI-IMS-MS had difficulties with this analysis, or failed. Graphical Abstract ᅟ.
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Affiliation(s)
- Joshua L Fischer
- Department of Chemistry, Wayne State University, Detroit, MI, 48202, USA
| | - Corinne A Lutomski
- Department of Chemistry, Wayne State University, Detroit, MI, 48202, USA
| | - Tarick J El-Baba
- Department of Chemistry, Wayne State University, Detroit, MI, 48202, USA
| | | | - Steffen M Weidner
- BAM Federal Institute for Materials Research and Testing, D-12489, Berlin, Germany
| | - Jana Falkenhagen
- BAM Federal Institute for Materials Research and Testing, D-12489, Berlin, Germany
| | - Matthew J Allen
- Department of Chemistry, Wayne State University, Detroit, MI, 48202, USA
| | - Sarah Trimpin
- Department of Chemistry, Wayne State University, Detroit, MI, 48202, USA.
- MSTM, LLC, Newark, DE, 19711, USA.
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Ji Y, Liu M, Bachschmid MM, Costello CE, Lin C. Surfactant-Induced Artifacts during Proteomic Sample Preparation. Anal Chem 2015; 87:5500-4. [PMID: 25945600 DOI: 10.1021/acs.analchem.5b00249] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Bottom-up proteomics is a powerful tool for characterization of protein post-translational modifications (PTMs), where PTMs are identified at the peptide level by mass spectrometry (MS) following protein digestion. However, enzymatic digestion is associated with additional sample processing steps that may potentially introduce artifactual modifications. Here, during an MS study of the PTMs of the regulator of G-protein signaling 4, we discovered that the use of ProteaseMAX, which is an acid-labile surfactant commonly used to improve protein solubilization and digestion efficiency, can lead to in vitro modifications on cysteine residues. These hydrophobic modifications resemble S-palmitoylation and hydroxyfarnesylation, thus discouraging the use of ProteaseMAX in studies of lipid modifications of proteins. Furthermore, since they target the cysteine thiol group, the presence of these artifacts will inevitably lead to inaccuracies in quantitative analysis of cysteine modifications.
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Goodswen SJ, Barratt JLN, Kennedy PJ, Ellis JT. Improving the gene structure annotation of the apicomplexan parasite Neospora caninum fulfils a vital requirement towards an in silico-derived vaccine. Int J Parasitol 2015; 45:305-18. [PMID: 25747726 DOI: 10.1016/j.ijpara.2015.01.006] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2014] [Revised: 01/12/2015] [Accepted: 01/12/2015] [Indexed: 12/16/2022]
Abstract
Neospora caninum is an apicomplexan parasite which can cause abortion in cattle, instigating major economic burden. Vaccination has been proposed as the most cost-effective control measure to alleviate this burden. Consequently the overriding aspiration for N. caninum research is the identification and subsequent evaluation of vaccine candidates in animal models. To save time, cost and effort, it is now feasible to use an in silico approach for vaccine candidate prediction. Precise protein sequences, derived from the correct open reading frame, are paramount and arguably the most important factor determining the success or failure of this approach. The challenge is that publicly available N. caninum sequences are mostly derived from gene predictions. Annotated inaccuracies can lead to erroneously predicted vaccine candidates by bioinformatics programs. This study evaluates the current N. caninum annotation for potential inaccuracies. Comparisons with annotation from a closely related pathogen, Toxoplasma gondii, are also made to distinguish patterns of inconsistency. More importantly, a mRNA sequencing (RNA-Seq) experiment is used to validate the annotation. Potential discrepancies originating from a questionable start codon context and exon boundaries were identified in 1943 protein coding sequences. We conclude, where experimental data were available, that the majority of N. caninum gene sequences were reliably predicted. Nevertheless, almost 28% of genes were identified as questionable. Given the limitations of RNA-Seq, the intention of this study was not to replace the existing annotation but to support or oppose particular aspects of it. Ideally, many studies aimed at improving the annotation are required to build a consensus. We believe this study, in providing a new resource on gene structure and annotation, is a worthy contributor to this endeavour.
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Affiliation(s)
- Stephen J Goodswen
- School of Medical and Molecular Sciences, University of Technology Sydney (UTS), 15 Broadway, Ultimo, NSW 2007, Australia.
| | - Joel L N Barratt
- School of Medical and Molecular Sciences, University of Technology Sydney (UTS), 15 Broadway, Ultimo, NSW 2007, Australia
| | - Paul J Kennedy
- School of Software, Faculty of Engineering and Information Technology and the Centre for Quantum Computation and Intelligent Systems at the University of Technology Sydney (UTS), 15 Broadway, Ultimo, NSW 2007, Australia
| | - John T Ellis
- School of Medical and Molecular Sciences, University of Technology Sydney (UTS), 15 Broadway, Ultimo, NSW 2007, Australia
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