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1
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Downs M, Zaia J, Sethi MK. Mass spectrometry methods for analysis of extracellular matrix components in neurological diseases. MASS SPECTROMETRY REVIEWS 2023; 42:1848-1875. [PMID: 35719114 PMCID: PMC9763553 DOI: 10.1002/mas.21792] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/08/2022] [Revised: 04/12/2022] [Accepted: 05/24/2022] [Indexed: 06/15/2023]
Abstract
The brain extracellular matrix (ECM) is a highly glycosylated environment and plays important roles in many processes including cell communication, growth factor binding, and scaffolding. The formation of structures such as perineuronal nets (PNNs) is critical in neuroprotection and neural plasticity, and the formation of molecular networks is dependent in part on glycans. The ECM is also implicated in the neuropathophysiology of disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and Schizophrenia (SZ). As such, it is of interest to understand both the proteomic and glycomic makeup of healthy and diseased brain ECM. Further, there is a growing need for site-specific glycoproteomic information. Over the past decade, sample preparation, mass spectrometry, and bioinformatic methods have been developed and refined to provide comprehensive information about the glycoproteome. Core ECM molecules including versican, hyaluronan and proteoglycan link proteins, and tenascin are dysregulated in AD, PD, and SZ. Glycomic changes such as differential sialylation, sulfation, and branching are also associated with neurodegeneration. A more thorough understanding of the ECM and its proteomic, glycomic, and glycoproteomic changes in brain diseases may provide pathways to new therapeutic options.
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Affiliation(s)
- Margaret Downs
- Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University, Boston, Massachusetts, USA
| | - Joseph Zaia
- Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University, Boston, Massachusetts, USA
- Bioinformatics Program, Boston University, Boston, Massachusetts, USA
| | - Manveen K Sethi
- Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University, Boston, Massachusetts, USA
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2
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Kumar S. Strategies to Enrich, Identify, and Characterize Glycoproteome in Blood Plasma Using Liquid Chromatography High-Resolution Mass Spectrometry. Methods Mol Biol 2023; 2628:155-172. [PMID: 36781784 DOI: 10.1007/978-1-0716-2978-9_10] [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: 02/15/2023]
Abstract
Blood and blood-derived components such as plasma and serum are considered as excellent resources that can be utilized to understand the biology of higher eukaryotic organisms including human beings. In research and clinical studies, blood plasma and serum are used to monitor health conditions, progression, and severity of diseases. Many of the disease-related studies utilize plasma and serum due to their disease relevance and accessibility as they can be collected from patients and healthy donors through minimally invasive approaches. Despite its significance, the unique proteome composition, complexity, wide dynamic range, and heterogeneity of the plasma proteins highlight critical factors that challenge the existing analytical technologies. Depletion of high abundant proteins is one among the accepted methods that can simplify the plasma proteome complexity; however, collateral loss of critical proteins should be anticipated. Selective protein enrichment seems to be a better alternative to depletion. Glycosylation of proteins is a dominant posttranslational modification known for its biological as well as diagnostic and therapeutic potential. Most of the reported therapeutic targets for diagnosis and monitoring are found to be glycosylated. In this chapter, a protocol for selective and reproducible enrichment of glycoproteins from blood plasma followed by identification through liquid chromatography high-resolution mass spectrometry has been documented.
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Affiliation(s)
- Saravanan Kumar
- Proteomics Lab, Thermo Fisher Scientific India, First Technology Place, Bangalore, Karnataka, India
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3
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Šimunović J, Gašperšič J, Černigoj U, Vidič J, Štrancar A, Novokmet M, Razdorov G, Pezer M, Lauc G, Trbojević-Akmačić I. High-throughput immunoaffinity enrichment and N-glycan analysis of human plasma haptoglobin. Biotechnol Bioeng 2023; 120:491-502. [PMID: 36324280 DOI: 10.1002/bit.28280] [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: 08/24/2022] [Revised: 10/24/2022] [Accepted: 10/30/2022] [Indexed: 11/06/2022]
Abstract
Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC-UHPLC-FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.
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Affiliation(s)
| | | | - Urh Černigoj
- Sartorius BIA Separations d.o.o., Ajdovščina, Slovenia
| | - Jana Vidič
- Sartorius BIA Separations d.o.o., Ajdovščina, Slovenia
| | - Aleš Štrancar
- Sartorius BIA Separations d.o.o., Ajdovščina, Slovenia
| | | | | | - Marija Pezer
- Genos Glycoscience Research Laboratory, Zagreb, Croatia
| | - Gordan Lauc
- Genos Glycoscience Research Laboratory, Zagreb, Croatia.,Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia
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4
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Lectin-Based Affinity Enrichment and Characterization of N-Glycoproteins from Human Tear Film by Mass Spectrometry. MOLECULES (BASEL, SWITZERLAND) 2023; 28:molecules28020648. [PMID: 36677706 PMCID: PMC9864693 DOI: 10.3390/molecules28020648] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/30/2022] [Revised: 01/03/2023] [Accepted: 01/06/2023] [Indexed: 01/11/2023]
Abstract
The glycosylation of proteins is one of the most common post-translational modifications (PTMs) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome, maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of tear film might promote the development of chronic eye diseases, indicating glycoproteins as a valuable source for biomarker discovery or drug target identification. Therefore, the present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and to characterize their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we first accumulated native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA, and UEA I, termed 4L) was applied to glycopeptide enrichment from human tear sample digests, followed by MS-based detection and localization of their specific N-glycosylation sites. As the main result, our study identified a total of 26 N glycosylation sites of 11 N-glycoproteins in the tear sample pools of healthy individuals (n = 3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP), and extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, already associated with the pathogenesis of various chronic eye diseases such as dry eye syndrome (DES). In conclusion, the results of the present study will serve as an important tear film N-glycoprotein catalog for future studies focusing on human tear film and ocular surface-related inflammatory diseases.
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5
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Li J, Zhang J, Xu M, Yang Z, Yue S, Zhou W, Gui C, Zhang H, Li S, Wang PG, Yang S. Advances in glycopeptide enrichment methods for the analysis of protein glycosylation over the past decade. J Sep Sci 2022; 45:3169-3186. [PMID: 35816156 DOI: 10.1002/jssc.202200292] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2022] [Revised: 06/16/2022] [Accepted: 07/01/2022] [Indexed: 11/12/2022]
Abstract
Advances in bioanalytical technology have accelerated the analysis of complex protein glycosylation, which is beneficial to understanding glycosylation in drug discovery and disease diagnosis. Due to its biological uniqueness in the course of disease occurrence and development, disease-specific glycosylation requires quantitative characterization of protein glycosylation. We provide a comprehensive review of recent advances in glycosylation analysis, including workflows for glycoprotein digestion, glycopeptide separation and enrichment, and mass-spectrometry sequencing. We specifically focus on different strategies for glycopeptide enrichment through physical interaction, chemical oxidation, or metabolic labeling of intact glycopeptides. The recent advances and challenges of O-glycosylation analysis are presented, and the development of improved enrichment methods combining different proteases to analyze O-glycosylation is also proposed. This article is protected by copyright. All rights reserved.
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Affiliation(s)
- Jiajia Li
- Center for Clinical Mass Spectrometry, College of Pharmaceutical Sciences, Soochow University, Jiangsu, 215123, China
| | - Jie Zhang
- Center for Clinical Mass Spectrometry, College of Pharmaceutical Sciences, Soochow University, Jiangsu, 215123, China
| | - Mingming Xu
- Center for Clinical Mass Spectrometry, College of Pharmaceutical Sciences, Soochow University, Jiangsu, 215123, China
| | - Zeren Yang
- AstraZeneca, Medimmune Ct, Frederick, MD, 21703, USA
| | - Shuang Yue
- Center for Clinical Mass Spectrometry, College of Pharmaceutical Sciences, Soochow University, Jiangsu, 215123, China
| | - Wanlong Zhou
- U.S. Food and Drug Administration, Forensic Chemistry Center, Cincinnati, OH, 45237, USA
| | - Chunshan Gui
- Department of Pharmaceutical Analysis, College of Pharmaceutical Sciences, Soochow University, Jiangsu, 215123, China
| | - Haiyang Zhang
- Department of Pharmaceutical Analysis, College of Pharmaceutical Sciences, Soochow University, Jiangsu, 215123, China
| | - Shuwei Li
- Nanjing Apollomics Biotech, Inc., Nanjing, Jiangsu, 210033, China
| | - Perry G Wang
- U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, College Park, MD, 20740, USA
| | - Shuang Yang
- Center for Clinical Mass Spectrometry, College of Pharmaceutical Sciences, Soochow University, Jiangsu, 215123, China.,Department of Pharmaceutical Analysis, College of Pharmaceutical Sciences, Soochow University, Jiangsu, 215123, China
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6
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Identification and Relative Quantification of hFSH Glycoforms in Women's Sera via MS-PRM-Based Approach. Pharmaceutics 2021; 13:pharmaceutics13060798. [PMID: 34071747 PMCID: PMC8226871 DOI: 10.3390/pharmaceutics13060798] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2021] [Revised: 04/23/2021] [Accepted: 04/28/2021] [Indexed: 11/17/2022] Open
Abstract
Follicle-stimulating hormone (FSH) is a glycohormone synthesized by adenohypophysis, and it stimulates ovulation in women and spermatogenesis in men by binding to its receptor (FSHR). FSHR is involved in several mechanisms to transduce intracellular signals in response to the FSH stimulus. Exogenous FSH is currently used in the clinic for ovarian hyperstimulation during in vitro fertilization in women, and for treatment of infertility caused by gonadotropin deficiency in men. The glycosylation of FSH strongly affects the binding affinity to its receptor, hence significantly influencing the biological activity of the hormone. Therefore, the accurate measurement and characterization of serum hFSH glycoforms will contribute to elucidating the complex mechanism of action by which different glycoforms elicit distinct biological activity. Nowadays ELISA is the official method with which to monitor serum hFSH, but the test is unable to distinguish between the different FSH glycovariants and is therefore unsuitable to study the biological activity of this hormone. This study presents a preliminary alternative strategy for identifying and quantifying serum hFSH glycoforms based on immunopurification assay and mass spectrometry (MS), and parallel reaction monitoring (PRM) analysis. In this study, we provide an MS–PRM data acquisition method for hFSH glycopeptides identification with high specificity and their quantification by extracting the chromatographic traces of selected fragments of glycopeptides. Once set up for all its features, the proposed method could be transferred to the clinic to improve fertility treatments and follow-ups in men and women.
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7
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di Masi A, De Simone G, Ciaccio C, D'Orso S, Coletta M, Ascenzi P. Haptoglobin: From hemoglobin scavenging to human health. Mol Aspects Med 2020; 73:100851. [PMID: 32660714 DOI: 10.1016/j.mam.2020.100851] [Citation(s) in RCA: 83] [Impact Index Per Article: 16.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2020] [Revised: 03/23/2020] [Accepted: 03/25/2020] [Indexed: 02/07/2023]
Abstract
Haptoglobin (Hp) belongs to the family of acute-phase plasma proteins and represents the most important plasma detoxifier of hemoglobin (Hb). The basic Hp molecule is a tetrameric protein built by two α/β dimers. Each Hp α/β dimer is encoded by a single gene and is synthesized as a single polypeptide. Following post-translational protease-dependent cleavage of the Hp polypeptide, the α and β chains are linked by disulfide bridge(s) to generate the mature Hp protein. As human Hp gene is characterized by two common Hp1 and Hp2 alleles, three major genotypes can result (i.e., Hp1-1, Hp2-1, and Hp2-2). Hp regulates Hb clearance from circulation by the macrophage-specific receptor CD163, thus preventing Hb-mediated severe consequences for health. Indeed, the antioxidant and Hb binding properties of Hp as well as its ability to stimulate cells of the monocyte/macrophage lineage and to modulate the helper T-cell type 1 and type 2 balance significantly associate with a variety of pathogenic disorders (e.g., infectious diseases, diabetes, cardiovascular diseases, and cancer). Alternative functions of the variants Hp1 and Hp2 have been reported, particularly in the susceptibility and protection against infectious (e.g., pulmonary tuberculosis, HIV, and malaria) and non-infectious (e.g., diabetes, cardiovascular diseases and obesity) diseases. Both high and low levels of Hp are indicative of clinical conditions: Hp plasma levels increase during infections, inflammation, and various malignant diseases, and decrease during malnutrition, hemolysis, hepatic disease, allergic reactions, and seizure disorders. Of note, the Hp:Hb complexes display heme-based reactivity; in fact, they bind several ferrous and ferric ligands, including O2, CO, and NO, and display (pseudo-)enzymatic properties (e.g., NO and peroxynitrite detoxification). Here, genetic, biochemical, biomedical, and biotechnological aspects of Hp are reviewed.
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Affiliation(s)
- Alessandra di Masi
- Department of Sciences, Roma Tre University, Viale Guglielmo Marconi 446, I-00146 Roma, Italy
| | - Giovanna De Simone
- Department of Sciences, Roma Tre University, Viale Guglielmo Marconi 446, I-00146 Roma, Italy
| | - Chiara Ciaccio
- Department of Clinical Sciences and Translational Medicine, University of Roma "Tor Vergata", Via Montpellier 1, I-00133, Roma, Italy; Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Via Celso Ulpiani 27, I-70126, Bari, Italy
| | - Silvia D'Orso
- Department of Sciences, Roma Tre University, Viale Guglielmo Marconi 446, I-00146 Roma, Italy
| | - Massimo Coletta
- Department of Clinical Sciences and Translational Medicine, University of Roma "Tor Vergata", Via Montpellier 1, I-00133, Roma, Italy; Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Via Celso Ulpiani 27, I-70126, Bari, Italy
| | - Paolo Ascenzi
- Interdepartmental Laboratory for Electron Microscopy, Roma Tre University, Via della Vasca Navale 79, I-00146, Roma, Italy.
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8
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Cao Q, Yu Q, Liu Y, Chen Z, Li L. Signature-Ion-Triggered Mass Spectrometry Approach Enabled Discovery of N- and O-Linked Glycosylated Neuropeptides in the Crustacean Nervous System. J Proteome Res 2020; 19:634-643. [PMID: 31875397 DOI: 10.1021/acs.jproteome.9b00525] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Crustaceans are commonly used model organisms to study neuromodulation. Despite numerous reported crustacean neuropeptide families and their functions, there has been no report on neuropeptide glycosylation. This is in part due to a lack of sensitive methods that enable deciphering this intricate low-abundance post-translational modification, even though glycosylation has been shown to play an important role in neuromodulation. Here, we describe the discovery of glycosylated neuropeptides with an enrichment-free approach, taking advantage of signature oxonium ions produced in higher-energy collision dissociation (HCD) MS/MS spectra. The detection of the oxonium ions in the HCD scans suggests glycan attachment to peptides, allowing electron-transfer/higher-energy collision dissociation (EThcD) to be performed to selectively elucidate structural information of glycosylated neuropeptides that are buried in nonglycosylated peptides. Overall, 4 N-linked and 14 O-linked glycosylated neuropeptides have been identified for the first time in the crustacean nervous system. In addition, 91 novel putative neuropeptides have been discovered based on the collected HCD scans. This hybrid approach, coupling a shotgun method for neuropeptide discovery and targeted strategy for glycosylation characterization, enables the first report on glycosylated neuropeptides in crustaceans and the discovery of additional neuropeptides simultaneously. The elucidation of novel glycosylated neuropeptides sheds light on the crustacean peptidome and offers novel insights into future neuropeptide functional studies.
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Affiliation(s)
- Qinjingwen Cao
- Department of Chemistry , University of Wisconsin-Madison , 1101 University Avenue , Madison , Wisconsin 53706 , United States
| | - Qing Yu
- School of Pharmacy , University of Wisconsin-Madison , 777 Highland Avenue , Madison , Wisconsin 53705 , United States
| | - Yang Liu
- Department of Chemistry , University of Wisconsin-Madison , 1101 University Avenue , Madison , Wisconsin 53706 , United States
| | - Zhengwei Chen
- Department of Chemistry , University of Wisconsin-Madison , 1101 University Avenue , Madison , Wisconsin 53706 , United States
| | - Lingjun Li
- Department of Chemistry , University of Wisconsin-Madison , 1101 University Avenue , Madison , Wisconsin 53706 , United States.,School of Pharmacy , University of Wisconsin-Madison , 777 Highland Avenue , Madison , Wisconsin 53705 , United States
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9
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Chen Z, Huang J, Li L. Recent advances in mass spectrometry (MS)-based glycoproteomics in complex biological samples. Trends Analyt Chem 2019; 118:880-892. [PMID: 31579312 PMCID: PMC6774629 DOI: 10.1016/j.trac.2018.10.009] [Citation(s) in RCA: 63] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Protein glycosylation plays a key role in various biological processes and disease-related pathological progression. Mass spectrometry (MS)-based glycoproteomics is a powerful approach that provides a system-wide profiling of the glycoproteome in a high-throughput manner. There have been numerous significant technological advances in this field, including improved glycopeptide enrichment, hybrid fragmentation techniques, emerging specialized software packages, and effective quantitation strategies, as well as more dedicated workflows. With increasingly sophisticated glycoproteomics tools on hand, researchers have extensively adapted this approach to explore different biological systems both in terms of in-depth glycoproteome profiling and comparative glycoproteome analysis. Quantitative glycoproteomics enables researchers to discover novel glycosylation-based biomarkers in various diseases with potential to offer better sensitivity and specificity for disease diagnosis. In this review, we present recent methodological developments in MS-based glycoproteomics and highlight its utility and applications in answering various questions in complex biological systems.
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Affiliation(s)
- Zhengwei Chen
- Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53705, USA
| | - Junfeng Huang
- School of Pharmacy, University of Wisconsin-Madison, Madison, WI, 53705, USA
| | - Lingjun Li
- Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53705, USA
- School of Pharmacy, University of Wisconsin-Madison, Madison, WI, 53705, USA
- School of Life Sciences, Tianjin University, Tianjin, 300072, China
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10
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Measurement of the total angiotensinogen and its reduced and oxidised forms in human plasma using targeted LC-MS/MS. Anal Bioanal Chem 2019; 411:427-437. [PMID: 30465161 PMCID: PMC6336742 DOI: 10.1007/s00216-018-1455-2] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2018] [Revised: 10/17/2018] [Accepted: 10/25/2018] [Indexed: 11/28/2022]
Abstract
Angiotensinogen (AGT) is a critical protein in the renin-angiotensin-aldosterone system and may have an important role in the pathogenesis of pre-eclampsia. The disulphide linkage between cysteines 18 and 138 has a key role in the redox switch of AGT which modulates the release of angiotensin I with consequential effects on blood pressure. In this paper, we report a quantitative targeted LC-MS/MS method for the reliable measurement of the total AGT and its reduced and oxidised forms in human plasma. AGT was selectively enriched from human plasma using two-dimensional chromatography employing concanavalin A lectin affinity and reversed phase steps and then deglycosylated using PNGase F. A differential alkylation approach was coupled with targeted LC-MS/MS method to identify the two AGT forms in the plasma chymotryptic digest. An additional AGT proteolytic marker peptide was identified and used to measure total AGT levels. The developed MS workflow enabled the reproducible detection of total AGT and its two distinct forms in human plasma with analytical precision of ≤ 15%. The LC-MS/MS assay for total AGT in plasma showed a linear response (R2 = 0.992) with a limit of quantification in the low nanomolar range. The method gave suitable validation characteristics for biomedical application to the quantification of the oxidation level and the total level of AGT in plasma samples collected from normal and pre-eclamptic patients.
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11
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Ma Y, Li X, Li W, Liu Z. Glycan-Imprinted Magnetic Nanoparticle-Based SELEX for Efficient Screening of Glycoprotein-Binding Aptamers. ACS APPLIED MATERIALS & INTERFACES 2018; 10:40918-40926. [PMID: 30379519 DOI: 10.1021/acsami.8b14441] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/08/2023]
Abstract
Nucleic acid aptamers, as useful alternatives of antibodies, have found a large range of promising applications such as affinity separation and bioassays. The screening of aptamers is critical for their applications. Aptamers are often screened by an in vitro methodology called SELEX (systematic evolution of ligands by exponential enrichment). Although numerous SELEX methods have been established to facilitate the selection, new efficient selection methods are still much needed. Molecularly imprinted polymers, which are antibody alternatives at the material level and competitors of aptamers, have not been used as a platform for aptamer selection yet so far. In this study, a glycan-imprinted magnetic nanoparticles (MNPs)-based SELEX was developed to efficiently screen aptamers against glycoproteins. Glycan-imprinted MNPs were used as an affinity interface to bind target glycoprotein, and then the target glycoprotein-bound MNPs were used as an affinity substrate for aptamer selection. The glycan-imprinted MNPs were synthesized by a state-of-the-art imprinting approach called boronate affinity controllable oriented surface imprinting. The glycan-imprinted MNPs exhibited high affinity and specificity and therefore allowed preferential binding toward target glycoproteins while excluding unwanted species. Two representative glycoproteins, including RNase B and transferrin, were employed as target glycoproteins, and aptamers with high affinity and specificity toward the two target glycoproteins were screened out in 3 rounds. This method exhibited some merits, such as high affinity, fast speed, and avoiding negative screening. Therefore, the glycan-imprinted MNP-based SELEX approach holds great values for the efficient screening of high-performance aptamers.
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Affiliation(s)
- Yanyan Ma
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering , Nanjing University , Nanjing 210023 , China
| | - Xinglin Li
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering , Nanjing University , Nanjing 210023 , China
| | - Wei Li
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering , Nanjing University , Nanjing 210023 , China
| | - Zhen Liu
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering , Nanjing University , Nanjing 210023 , China
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12
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Waniwan JT, Chen YJ, Capangpangan R, Weng SH, Chen YJ. Glycoproteomic Alterations in Drug-Resistant Nonsmall Cell Lung Cancer Cells Revealed by Lectin Magnetic Nanoprobe-Based Mass Spectrometry. J Proteome Res 2018; 17:3761-3773. [PMID: 30261726 DOI: 10.1021/acs.jproteome.8b00433] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Understanding the functional role of glycosylation-mediated pathogenesis requires deep characterization of glycoproteome, which remains extremely challenging due to the inherently complex nature of glycoproteins. We demonstrate the utility of lectin-magnetic nanoprobe (MNP@lectin) coupled to Orbitrap HCD-CID-MS/MS for complementary glycotope-specific enrichment and site-specific glycosylation analysis of the glycoproteome. By three nanoprobes, MNP@ConA, MNP@AAL, and MNP@SNA, our results revealed the first large-scale glycoproteome of nonsmall cell lung cancer (NSCLC) with 2290 and 2767 nonredundant glycopeptides confidently identified (Byonic score ≥100) in EGFR-TKI-sensitive PC9 and -resistant PC9-IR cells, respectively, especially with more fucosylated and sialylated glycopeptides in PC9-IR cells. The complementary enrichment was demonstrated with only five glycopeptides commonly enriched in three MNP@lectins. Glycotope specificity of 79 and 62% for enrichment was achieved using MNP@AAL and MNP@SNA, respectively. Label-free quantitation revealed predominant fucosylation in PC9-IR cells, suggesting its potential role associated with NSCLC resistance. Moreover, without immunoprecipitation, this multilectin nanoprobe allows the sensitive identification of 51 glycopeptides from 10 of 12 reported sites from onco-protein EGFR. Our results not only demonstrated a sensitive approach to study the vastly under-represented N-glycoprotome but also may pave the way for a glycoproteomic atlas to further explore the site-specific function of glycoproteins associated with drug resistance in NSCLC.
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Affiliation(s)
- Juanilita T Waniwan
- Department of Chemistry , National Taiwan University , Taipei 115 , Taiwan.,Chemical Biology and Molecular Biophysics , Taiwan International Graduate Program , Taipei 115 , Taiwan.,Institute of Chemistry , Academia Sinica , Taipei 115 , Taiwan
| | - Yi-Ju Chen
- Institute of Chemistry , Academia Sinica , Taipei 115 , Taiwan
| | - Rey Capangpangan
- Caraga State University , Butuan City 8600 , Philippines.,Institute of Chemistry , Academia Sinica , Taipei 115 , Taiwan
| | - Shao-Hsing Weng
- Institute of Chemistry , Academia Sinica , Taipei 115 , Taiwan.,Genome and Systems Biology Degree Program , National Taiwan University , Taipei 115 , Taiwan
| | - Yu-Ju Chen
- Department of Chemistry , National Taiwan University , Taipei 115 , Taiwan.,Institute of Chemistry , Academia Sinica , Taipei 115 , Taiwan
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13
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Frost DC, Li L. Recent advances in mass spectrometry-based glycoproteomics. ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY 2018; 95:71-123. [PMID: 24985770 DOI: 10.1016/b978-0-12-800453-1.00003-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Protein glycosylation plays fundamental roles in many biological processes as one of the most common, and the most complex, posttranslational modification. Alterations in glycosylation profile are now known to be associated with many diseases. As a result, the discovery and detailed characterization of glycoprotein disease biomarkers is a primary interest of biomedical research. Advances in mass spectrometry (MS)-based glycoproteomics and glycomics are increasingly enabling qualitative and quantitative approaches for site-specific structural analysis of protein glycosylation. While the complexity presented by glycan heterogeneity and the wide dynamic range of clinically relevant samples like plasma, serum, cerebrospinal fluid, and tissue make comprehensive analyses of the glycoproteome a challenging task, the ongoing efforts into the development of glycoprotein enrichment, enzymatic digestion, and separation strategies combined with novel quantitative MS methodologies have greatly improved analytical sensitivity, specificity, and throughput. This review summarizes current MS-based glycoproteomics approaches and highlights recent advances in its application to cancer biomarker and neurodegenerative disease research.
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Affiliation(s)
- Dustin C Frost
- School of Pharmacy, University of Wisconsin, Madison, Wisconsin, USA
| | - Lingjun Li
- School of Pharmacy, University of Wisconsin, Madison, Wisconsin, USA; Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA.
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14
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Ribeiro AC, Ferreira R, Freitas R. Plant Lectins: Bioactivities and Bioapplications. STUDIES IN NATURAL PRODUCTS CHEMISTRY 2018. [DOI: 10.1016/b978-0-444-64056-7.00001-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/03/2022]
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15
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Jung HJ. Chemical Proteomic Approaches Targeting Cancer Stem Cells: A Review of Current Literature. Cancer Genomics Proteomics 2017; 14:315-327. [PMID: 28870999 PMCID: PMC5611518 DOI: 10.21873/cgp.20042] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2017] [Revised: 07/18/2017] [Accepted: 07/20/2017] [Indexed: 12/24/2022] Open
Abstract
Cancer stem cells (CSCs) have been proposed as central drivers of tumor initiation, progression, recurrence, and therapeutic resistance. Therefore, identifying stem-like cells within cancers and understanding their properties is crucial for the development of effective anticancer therapies. Recently, chemical proteomics has become a powerful tool to efficiently determine protein networks responsible for CSC pathophysiology and comprehensively elucidate molecular mechanisms of drug action against CSCs. This review provides an overview of major methodologies utilized in chemical proteomic approaches. In addition, recent successful chemical proteomic applications targeting CSCs are highlighted. Future direction of potential CSC research by integrating chemical genomic and proteomic data obtained from a single biological sample of CSCs are also suggested in this review.
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Affiliation(s)
- Hye Jin Jung
- Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University, Asan, Republic of Korea
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16
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ADAM Metalloprotease-Released Cancer Biomarkers. Trends Cancer 2017; 3:482-490. [DOI: 10.1016/j.trecan.2017.05.001] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2017] [Revised: 04/28/2017] [Accepted: 05/03/2017] [Indexed: 12/14/2022]
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17
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Pan L, Aguilar HA, Wang L, Iliuk A, Tao WA. Three-Dimensionally Functionalized Reverse Phase Glycoprotein Array for Cancer Biomarker Discovery and Validation. J Am Chem Soc 2016; 138:15311-15314. [PMID: 27933927 DOI: 10.1021/jacs.6b10239] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Glycoproteins have vast structural diversity that plays an important role in many biological processes and have great potential as disease biomarkers. Here, we report a novel functionalized reverse phase protein array (RPPA), termed polymer-based reverse phase glycoprotein array (polyGPA), to capture and profile glycoproteomes specifically, and validate glycoproteins. Nitrocellulose membrane functionalized with globular hydroxyaminodendrimers was used to covalently capture preoxidized glycans on glycoproteins from complex protein samples such as biofluids. The captured glycoproteins were subsequently detected using the same validated antibodies as in RPPA. We demonstrated the outstanding specificity, sensitivity, and quantitative capabilities of polyGPA by capturing and detecting purified as well as endogenous α-1-acid glycoprotein (AGP) in human plasma. We further applied quantitative N-glycoproteomics and the strategy to validate a panel of glycoproteins identified as potential biomarkers for bladder cancer by analyzing urine glycoproteins from bladder cancer patients or matched healthy individuals.
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Affiliation(s)
| | | | | | - Anton Iliuk
- Tymora Analytical Operations , West Lafayette, Indiana 47906, United States
| | - W Andy Tao
- Tymora Analytical Operations , West Lafayette, Indiana 47906, United States.,Center for Cancer Research, Purdue University , West Lafayette, Indiana 47907, United States
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18
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Pan S, Brentnall TA, Chen R. Glycoproteins and glycoproteomics in pancreatic cancer. World J Gastroenterol 2016; 22:9288-9299. [PMID: 27895417 PMCID: PMC5107693 DOI: 10.3748/wjg.v22.i42.9288] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2016] [Revised: 08/23/2016] [Accepted: 09/14/2016] [Indexed: 02/06/2023] Open
Abstract
Aberrations in protein glycosylation and polysaccharides play a pivotal role in pancreatic tumorigenesis, influencing cancer progression, metastasis, immuno-response and chemoresistance. Abnormal expression in sugar moieties can impact the function of various glycoproteins, including mucins, surface receptors, adhesive proteins, proteoglycans, as well as their effectors and binding ligands, resulting in an increase in pancreatic cancer invasiveness and a cancer-favored microenvironment. Recent advance in glycoproteomics, glycomics and other chemical biology techniques have been employed to better understand the complex mechanism of glycosylation events and how they orchestrate molecular activities in genomics, proteomics and metabolomics implicated in pancreatic adenocarcinoma. A variety of strategies have been demonstrated targeting protein glycosylation and polysaccharides for diagnostic and therapeutic development.
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19
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Zhang S, Shang S, Li W, Qin X, Liu Y. Insights on N-glycosylation of human haptoglobin and its association with cancers. Glycobiology 2016; 26:684-692. [PMID: 26873173 DOI: 10.1093/glycob/cww016] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2015] [Accepted: 02/08/2016] [Indexed: 12/11/2022] Open
Abstract
Protein glycosylation is one of the most significant post-translation modifications and plays a critical role in various biological functions. Haptoglobin (Hp) is one of the acute-phase response proteins secreted by liver. Its glycosylation could be analyzed by many analytical techniques qualitatively and quantitatively. The glycosylation alterations of Hp are reported to be associated with different kinds of diseases. The main glycosylation alterations of Hp in cancer appear to be the presence of aberrantly fucosylated and sialylated structures as well as increased branching. In this mini review, we provided a brief overview of Hp structure and biological function, discussed its glycosylation alterations in different cancers, and described the existing technologies for analyzing glycosylation site and glycan of Hp. Given the importance of Hp glycosylation, its unknown and unclear biological complexity and significances, Hp glycosylation has become a major target in cancer research. Development of sensitive and specific detection of Hp glycosylation including large-scale validation may be significant steps forward to its clinical application.
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Affiliation(s)
- Shu Zhang
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai 200032, China
| | - Shuxin Shang
- Department of Clinical Laboratory, First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Wei Li
- Cancer Research Center, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China
| | - Xue Qin
- Department of Clinical Laboratory, First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi, China
| | - Yinkun Liu
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai 200032, China.,Cancer Research Center, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China
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20
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Clinical diagnostics and therapy monitoring in the congenital disorders of glycosylation. Glycoconj J 2016; 33:345-58. [PMID: 26739145 PMCID: PMC4891361 DOI: 10.1007/s10719-015-9639-x] [Citation(s) in RCA: 48] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2015] [Revised: 11/03/2015] [Accepted: 11/18/2015] [Indexed: 12/20/2022]
Abstract
Abnormal protein glycosylation is observed in many common disorders like cancer, inflammation, Alzheimer’s disease and diabetes. However, the actual use of this information in clinical diagnostics is still very limited. Information is usually derived from analysis of total serum N-glycan profiling methods, whereas the current use of glycoprotein biomarkers in the clinical setting is commonly based on protein levels. It can be envisioned that combining protein levels and their glycan isoforms would increase specificity for early diagnosis and therapy monitoring. To establish diagnostic assays, based on the mass spectrometric analysis of protein-specific glycosylation abnormalities, still many technical improvements have to be made. In addition, clinical validation is equally important as well as an understanding of the genetic and environmental factors that determine the protein-specific glycosylation abnormalities. Important lessons can be learned from the group of monogenic disorders in the glycosylation pathway, the Congenital Disorders of Glycosylation (CDG). Now that more and more genetic defects are being unraveled, we start to learn how genetic factors influence glycomics profiles of individual and total serum proteins. Although only in its initial stages, such studies suggest the importance to establish diagnostic assays for protein-specific glycosylation profiling, and the need to look beyond the single glycoprotein diagnostic test. Here, we review progress in and lessons from genetic disease, and review the increasing opportunities of mass spectrometry to analyze protein glycosylation in the clinical diagnostic setting. Furthermore, we will discuss the possibilities to expand current CDG diagnostics and how this can be used to approach glycoprotein biomarkers for more common diseases.
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21
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22
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Xue X, Zhao Y, Zhang X, Zhang C, Kumar A, Zhang X, Zou G, Wang PC, Zhang J, Liang XJ. Phenylboronic acid-functionalized magnetic nanoparticles for one-step saccharides enrichment and mass spectrometry analysis. BIOPHYSICS REPORTS 2015; 1:61-70. [PMID: 26942220 PMCID: PMC4762129 DOI: 10.1007/s41048-015-0002-3] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2015] [Accepted: 04/01/2015] [Indexed: 11/16/2022] Open
Abstract
Abstract In this work, 2-(2-aminoethoxy) ethanol-blocked phenylboronic acid-functionalized magnetic nanoparticles (blocked PMNPs) were fabricated for selective enrichment of different types of saccharides. The phenylboronic acid was designed for capturing the cis-diols moieties on saccharides molecules, and the 2-(2-aminoethoxy) ethanol can deplete the nonspecific absorption of peptides and proteins which always coexisted with saccharides. For mass spectrometry analysis, the PMNPs bound saccharides can be directly applied onto the MALDI plate with matrix without removing the PMNPs. By PMNPs, the simple saccharide (glucose) could be detected in pmol level. The complex saccharides can also be reliably purified and analyzed; 16 different N-glycans were successfully identified from ovalbumin, and the high-abundance N-glycans can be detected even when the ovalbumin was in very low concentration (2 μg). In human milk, ten different oligosaccharides were identified, and the lactose can still be detected when the human milk concentration was low to 0.01 μL. Graphical Abstract ![]()
Electronic supplementary material The online version of this article (doi:10.1007/s41048-015-0002-3) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Xiangdong Xue
- CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190 China
| | - Yuanyuan Zhao
- CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190 China
| | - Xu Zhang
- CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190 China
| | - Chunqiu Zhang
- CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190 China
| | - Anil Kumar
- CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190 China
| | - Xiaoning Zhang
- Laboratory of Pharmaceutics, School of Medicine, Tsinghua University, Beijing, 100084 China
| | - Guozhang Zou
- CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190 China
| | - Paul C Wang
- Fu Jen Catholic University, Taipei, 24205 China
| | - Jinchao Zhang
- College of Chemistry & Environmental Science, Chemical Biology Key Laboratory of Hebei Province, Hebei University, Baoding, 071002 China
| | - Xing-Jie Liang
- CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190 China
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23
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Pouw RB, Vredevoogd DW, Kuijpers TW, Wouters D. Of mice and men: The factor H protein family and complement regulation. Mol Immunol 2015; 67:12-20. [PMID: 25824240 DOI: 10.1016/j.molimm.2015.03.011] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2015] [Revised: 03/09/2015] [Accepted: 03/10/2015] [Indexed: 10/23/2022]
Abstract
For decades immunological research has relied, with variable success, on mouse models to investigate diseases and possible therapeutic interventions. With the approval of the first therapeutic antibody targeting complement, called eculizumab, as therapy in paroxysmal nocturnal hemoglobinuria (PNH) and more recently atypical hemolytic uremic syndrome (aHUS), the viability of targeting the complement system was demonstrated. The potent, endogenous complement regulators have become of increasing interest as templates for designing and developing new therapeutics. Recently, complement inhibitors based on (parts of) the human complement regulator factor H (FH) are being examined for therapeutic intervention in inflammatory conditions. The first step to evaluate the potency of a new drug is often testing it in a mouse model for the target disease. However, translating results to human conditions requires a good understanding of similarities and, more importantly, differences between the human and mouse complement system and particularly regulation. This review will provide a comprehensive overview of the complement regulator FH and its closely related proteins and current views on their role in mice and men.
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Affiliation(s)
- R B Pouw
- Department of Immunopathology, Sanquin Blood Supply, Division Research and Landsteiner laboratory of the Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands; Department of Pediatric Hematology, Immunology & Infectious Diseases, Emma Children's Hospital, Academic Medical Centre, Amsterdam, the Netherlands.
| | - D W Vredevoogd
- Department of Immunopathology, Sanquin Blood Supply, Division Research and Landsteiner laboratory of the Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands
| | - T W Kuijpers
- Department of Pediatric Hematology, Immunology & Infectious Diseases, Emma Children's Hospital, Academic Medical Centre, Amsterdam, the Netherlands; Department of Blood Cell Research, Sanquin Blood Supply, Division Research and Landsteiner laboratory of the Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands
| | - D Wouters
- Department of Immunopathology, Sanquin Blood Supply, Division Research and Landsteiner laboratory of the Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands
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24
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Citartan M, Gopinath SC, Chen Y, Lakshmipriya T, Tang TH. Monitoring recombinant human erythropoietin abuse among athletes. Biosens Bioelectron 2015; 63:86-98. [DOI: 10.1016/j.bios.2014.06.068] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2014] [Revised: 06/02/2014] [Accepted: 06/27/2014] [Indexed: 11/16/2022]
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25
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Baycin Hizal D, Wolozny D, Colao J, Jacobson E, Tian Y, Krag SS, Betenbaugh MJ, Zhang H. Glycoproteomic and glycomic databases. Clin Proteomics 2014; 11:15. [PMID: 24725457 PMCID: PMC3996109 DOI: 10.1186/1559-0275-11-15] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2013] [Accepted: 02/20/2014] [Indexed: 11/17/2022] Open
Abstract
Protein glycosylation serves critical roles in the cellular and biological processes of many organisms. Aberrant glycosylation has been associated with many illnesses such as hereditary and chronic diseases like cancer, cardiovascular diseases, neurological disorders, and immunological disorders. Emerging mass spectrometry (MS) technologies that enable the high-throughput identification of glycoproteins and glycans have accelerated the analysis and made possible the creation of dynamic and expanding databases. Although glycosylation-related databases have been established by many laboratories and institutions, they are not yet widely known in the community. Our study reviews 15 different publicly available databases and identifies their key elements so that users can identify the most applicable platform for their analytical needs. These databases include biological information on the experimentally identified glycans and glycopeptides from various cells and organisms such as human, rat, mouse, fly and zebrafish. The features of these databases - 7 for glycoproteomic data, 6 for glycomic data, and 2 for glycan binding proteins are summarized including the enrichment techniques that are used for glycoproteome and glycan identification. Furthermore databases such as Unipep, GlycoFly, GlycoFish recently established by our group are introduced. The unique features of each database, such as the analytical methods used and bioinformatical tools available are summarized. This information will be a valuable resource for the glycobiology community as it presents the analytical methods and glycosylation related databases together in one compendium. It will also represent a step towards the desired long term goal of integrating the different databases of glycosylation in order to characterize and categorize glycoproteins and glycans better for biomedical research.
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Affiliation(s)
- Deniz Baycin Hizal
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA
| | - Daniel Wolozny
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA
| | - Joseph Colao
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA
| | - Elena Jacobson
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA
| | - Yuan Tian
- Department of Pathology, Johns Hopkins University, Baltimore, MD, USA
| | - Sharon S Krag
- Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Michael J Betenbaugh
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA
| | - Hui Zhang
- Department of Pathology, Johns Hopkins University, Baltimore, MD, USA
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26
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Chen W, Smeekens JM, Wu R. A universal chemical enrichment method for mapping the yeast N-glycoproteome by mass spectrometry (MS). Mol Cell Proteomics 2014; 13:1563-72. [PMID: 24692641 DOI: 10.1074/mcp.m113.036251] [Citation(s) in RCA: 78] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Glycosylation is one of the most common and important protein modifications in biological systems. Many glycoproteins naturally occur at low abundances, which makes comprehensive analysis extremely difficult. Additionally, glycans are highly heterogeneous, which further complicates analysis in complex samples. Lectin enrichment has been commonly used, but each lectin is inherently specific to one or several carbohydrates, and thus no single or collection of lectin(s) can bind to all glycans. Here we have employed a boronic acid-based chemical method to universally enrich glycopeptides. The reaction between boronic acids and sugars has been extensively investigated, and it is well known that the interaction between boronic acid and diols is one of the strongest reversible covalent bond interactions in an aqueous environment. This strong covalent interaction provides a great opportunity to catch glycopeptides and glycoproteins by boronic acid, whereas the reversible property allows their release without side effects. More importantly, the boronic acid-diol recognition is universal, which provides great capability and potential for comprehensively mapping glycosylation sites in complex biological samples. By combining boronic acid enrichment with PNGase F treatment in heavy-oxygen water and MS, we have identified 816 N-glycosylation sites in 332 yeast proteins, among which 675 sites were well-localized with greater than 99% confidence. The results demonstrated that the boronic acid-based chemical method can effectively enrich glycopeptides for comprehensive analysis of protein glycosylation. A general trend seen within the large data set was that there were fewer glycosylation sites toward the C termini of proteins. Of the 332 glycoproteins identified in yeast, 194 were membrane proteins. Many proteins get glycosylated in the high-mannose N-glycan biosynthetic and GPI anchor biosynthetic pathways. Compared with lectin enrichment, the current method is more cost-efficient, generic, and effective. This method can be extensively applied to different complex samples for the comprehensive analysis of protein glycosylation.
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Affiliation(s)
- Weixuan Chen
- From the ‡School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA
| | - Johanna M Smeekens
- From the ‡School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA
| | - Ronghu Wu
- From the ‡School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA
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27
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Chen W, Smeekens JM, Wu R. Comprehensive Analysis of Protein N-Glycosylation Sites by Combining Chemical Deglycosylation with LC–MS. J Proteome Res 2014; 13:1466-73. [DOI: 10.1021/pr401000c] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Affiliation(s)
- Weixuan Chen
- School
of Chemistry and Biochemistry
and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, United States
| | - Johanna M. Smeekens
- School
of Chemistry and Biochemistry
and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, United States
| | - Ronghu Wu
- School
of Chemistry and Biochemistry
and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, United States
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28
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Wang Y, Liu M, Xie L, Fang C, Xiong H, Lu H. Highly efficient enrichment method for glycopeptide analyses: using specific and nonspecific nanoparticles synergistically. Anal Chem 2014; 86:2057-64. [PMID: 24471740 DOI: 10.1021/ac403236q] [Citation(s) in RCA: 82] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
We invented a new method for highly efficient and specific enrichment of glycopeptides using two different nanomaterials synergistically. One is boronic-acid-functionalized Fe3O4 nanoparticles, enriching glycopeptides through formation of cyclic boronate esters between the boronic acid groups and the cis-diol groups on glycopeptides. The other nanomaterial is conventional poly(methyl methacrylate) nanobeads, which have strong adsorption toward nonglycopeptides. By optimizing the proportion of these two materials, extremely high sensitivity and selectivity are achieved in analyzing the standard glycopeptides/nonglycopeptides mixture solutions. Since the washing step is not necessary for these conditions, the enrichment process is simplified and the recovery efficiency of target glycopeptides reaches 90%. Finally, this approach is successfully applied to analyze human serum with the sample volume as little as 1 μL, in which 147 different N-glycosylation peptides within 66 unique glycoproteins are identified. All these performances by the synergistic enrichment are much better than employing one specific enrichment agent alone.
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Affiliation(s)
- Yali Wang
- Department of Chemistry, Fudan University , Shanghai 200433, P. R. China
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Abstract
Glycoproteins play an important role in cell signaling and cell-cell interaction. The alterations of glycoproteins are often relevant to progression of diseases, and these changed glycoproteins can be important biomarkers. The lectin-based glycoproteomic technology has extensively been used for high-throughput screening of potential glycoprotein biomarkers. Here we describe a multi-lectin affinity chromatography and label-free quantitative glycoproteomic approach for discovery of glycoprotein biomarkers relevant to differentiation of glioblastoma stem cells.
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Affiliation(s)
- Jintang He
- Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI, USA
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30
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Li QK, Gabrielson E, Askin F, Chan DW, Zhang H. Glycoproteomics using fluid-based specimens in the discovery of lung cancer protein biomarkers: promise and challenge. Proteomics Clin Appl 2014; 7:55-69. [PMID: 23112109 DOI: 10.1002/prca.201200105] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2012] [Revised: 10/01/2012] [Accepted: 10/05/2012] [Indexed: 12/29/2022]
Abstract
Lung cancer is the leading cancer in the United States and worldwide. In spite of the rapid progression in personalized treatments, the overall survival rate of lung cancer patients is still suboptimal. Over the past decade, tremendous efforts have been focused on the discovery of protein biomarkers to facilitate the early detection and monitoring of lung cancer progression during treatment. In addition to tumor tissues and cancer cell lines, a variety of biological material has been studied. Particularly in recent years, studies using fluid-based specimen or so-called "fluid-biopsy" specimens have progressed rapidly. Fluid specimens are relatively easier to collect than tumor tissue, and they can be repeatedly sampled during the disease progression. Glycoproteins are the major content of fluid specimens and have long been recognized to play fundamental roles in many physiological and pathological processes. In this review, we focus the discussion on recent advances of glycoproteomics, particularly in the identification of potential glyco protein biomarkers using fluid-based specimens in lung cancer. The purpose of this review is to summarize current strategies, achievements, and perspectives in the field. This insight will highlight the discovery of tumor-associated glycoprotein biomarkers in lung cancer and their potential clinical applications.
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Affiliation(s)
- Qing Kay Li
- Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD 21224, USA.
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31
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N-glycan occupancy of Arabidopsis N-glycoproteins. J Proteomics 2013; 93:343-55. [PMID: 23994444 DOI: 10.1016/j.jprot.2013.07.032] [Citation(s) in RCA: 62] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2013] [Revised: 07/08/2013] [Accepted: 07/27/2013] [Indexed: 11/24/2022]
Abstract
UNLABELLED Most secreted proteins in eukaryotes are modified on the amino acid consensus sequence NxS/T by an N-glycan through the process of N-glycosylation. The N-glycans on glycoproteins are processed in the endoplasmic reticulum (ER) to different mannose-type N-glycans or, when the protein passes through the Golgi apparatus, to different complex glycan forms. Here we describe the capturing of N-glycopeptides from a trypsin digest of total protein extracts of Arabidopsis plants and release of these captured peptides following Peptide N-glycosidase (PNGase) treatment for analysis of N-glycan site-occupancy. The mixture of peptides released as a consequence of the PNGase treatment was analyzed by two dimensional nano-LC-MS. As the PNGase treatment of glycopeptides results in the deamidation of the asparagine (N) in the NxS/T site of the released peptide, this asparagine (N) to aspartic acid (D) conversion is used as a glycosylation 'signature'. The efficiency of PNGase F and PNGase A in peptide release is discussed. The identification of proteins with a single glycopeptide was limited by the used search algorithm but could be improved using a reference database including deamidated peptide sequences. Additional stringency settings were used for filtering results to minimize false discovery. This resulted in identification of 330 glycopeptides on 173 glycoproteins from Arabidopsis, of which 28 putative glycoproteins, that were previously not annotated as secreted protein in The Arabidopsis Information Resource database (TAIR). Furthermore, the identified glycosylation site occupancy helped to determine the correct topology for membrane proteins. A quantitative comparison of peptide signal was made between wild type and complex-glycan-less (cgl) mutant Arabidopsis from three replicate leaf samples using a label-free MS peak comparison. As an example, the identified membrane protein SKU5 (AT4G12420) showed differential glycopeptide intensity ratios between WT and cgl indicating heterogeneous glycan modification on single protein. BIOLOGICAL SIGNIFICANCE Proteins that enter the secretory pathway are mostly modified by N-glycans. The function of N-glycosylation has been well studied in mammals. However, in plants the function of N-glycosylation is still unclear, because glycosylation mutants in plants often do not have a clear phenotype. Here we analyzed which proteins are modified by N-glycans in plants by developing a glycopeptide enrichment method for plant proteins. Subsequently, label free comparative proteomics was employed using protein fractions from wild type and from a mutant which is blocked in modification of the N-glycan into complex glycans. The results provide new information on N-glycosylation sites on numerous secreted proteins. Results allow for specific mapping of multiple glycosylation site occupancy on proteins, which provides information on which glycosylation sites are protected or non-used from downstream processing and thus presumably are buried into the protein structure. Glycoproteomics can therefore contribute to protein structure analysis. Indeed, mapping the glycosylation sites on membrane proteins gives information on the topology of protein folds over the membrane. We thus were able to correct the topology prediction of three membrane proteins. Besides, these studies also identified limitations in the software that is used to identify single modified peptide per protein. This article is part of a Special Issue entitled: Translational Plant Proteomics.
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Cao L, Yu L, Guo Z, Li X, Xue X, Liang X. Application of a strong anion exchange material in electrostatic repulsion–hydrophilic interaction chromatography for selective enrichment of glycopeptides. J Chromatogr A 2013; 1299:18-24. [DOI: 10.1016/j.chroma.2013.05.037] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2013] [Revised: 05/15/2013] [Accepted: 05/15/2013] [Indexed: 12/13/2022]
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Wu J, Xie X, Nie S, Buckanovich RJ, Lubman DM. Altered expression of sialylated glycoproteins in ovarian cancer sera using lectin-based ELISA assay and quantitative glycoproteomics analysis. J Proteome Res 2013; 12:3342-52. [PMID: 23731285 DOI: 10.1021/pr400169n] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Herein, we identify and confirm differentially expressed sialoglycoproteins in the serum of patients with ovarian cancer. On the basis of Sambucus nigra (SNA) lectin enrichment and on an isobaric chemical labeling quantitative strategy, clusterin (CLUS), leucine-rich alpha-2-glycoprotein (LRG1), hemopexin (HEMO), vitamin D-binding protein (VDB), and complement factor H (CFH) were found to be differentially expressed in the serum of patients with ovarian cancer compared to benign diseases. The abnormal sialylation levels of CLUS, CFH, and HEMO in serum of ovarian cancer patients were verified by a lectin-based ELISA assay. ELISA assays were further applied to measure total protein level changes of these glycoproteins. Protein levels of CLUS were found to be down-regulated in the serum of ovarian cancer patients, while protein levels of LRG1 were increased. The combination of CLUS and LRG1 (AUC = 0.837) showed improved performance for distinguishing stage III ovarian cancer from benign diseases compared to CA125 alone (AUC = 0.811). In differentiating early stage ovarian cancer from benign diseases or healthy controls, LRG1 showed comparable performance to CA125. An independent sample set was further used to confirm the ability of these candidate markers to detect patients with ovarian cancer. Our study provides a comprehensive strategy for the identification of candidate biomarkers that show the potential for diagnosis of ovarian cancer. Further studies using a large number of samples are necessary to validate the utility of this panel of proteins.
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Affiliation(s)
- Jing Wu
- Department of Surgery, University of Michigan, Ann Arbor, Michigan 48109, USA
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Abstract
Human tear fluid is a complex biological fluid that is actually the extracellular fluid of the surface of the eye and can be considered a dilute protein solution. The quality of the tears are critical for vision as they provide the mirror smooth reflex for the surface of the eye and hence focusing of images on the retina. Changes in tear composition may reflect the health of the epithelial cells lining the ocular surface and they have been shown to be useful to discover biomarkers for eye diseases. Glycoproteins are potentially important biomarkers of disease and therapeutic targets and can also be found in human tears. In this chapter, we concentrated on technical details in quantitative proteomic analysis of N-linked glycoproteins in human tears by combining hydrazide chemistry enrichment of N-linked glycoproteins and iTRAQ technology.
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Fanayan S, Smith JT, Lee LY, Yan F, Snyder M, Hancock WS, Nice E. Proteogenomic analysis of human colon carcinoma cell lines LIM1215, LIM1899, and LIM2405. J Proteome Res 2013; 12:1732-42. [PMID: 23458625 DOI: 10.1021/pr3010869] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
As part of the genome-wide and chromosome-centric human proteomic project (C-HPP), we have integrated shotgun proteomics approach and a genome-wide transcriptomic approach (RNA-Seq) of a set of human colon cancer cell lines (LIM1215, LIM1899 and LIM2405) that were selected to represent a wide range of pathological states of colorectal cancer. The combination of a standard proteomics approach (1D-gel electrophoresis coupled to LC/ion trap mass spectrometry) and RNA-Seq allowed us to exploit the greater depth of the transcriptomics measurement (∼ 9800 transcripts per cell line) versus the protein observations (∼ 1900 protein identifications per cell line). Conversely, the proteomics data were helpful in identifying both cancer associated proteins with differential expression patterns as well as protein networks and pathways which appear to be deregulated in these cell lines. Examples of potential markers include mortalin, nucleophosmin, ezrin, LASP1, alpha and beta forms of spectrin, exportin, the carcinoembryonic antigen family, EGFR and MET. Interaction analyses identified the large intermediate filament family, the protein folding network and adapter proteins in focal adhesion networks, which included the CDC42 and RHOA signaling pathways that may have potential for identifying phenotypic states representing poorly and moderately differentiated states of CRC, with or without metastases.
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Affiliation(s)
- Susan Fanayan
- Department of Chemistry and Biomolecular Sciences, Macquarie University , Sydney, NSW 2109, Australia
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Tummala S, Titus M, Wilson L, Wang C, Ciatto C, Foster D, Szabo Z, Guttman A, Li C, Bettencourt B, Jayaraman M, Deroot J, Thill G, Kocisko D, Pollard S, Charisse K, Kuchimanchi S, Hinkle G, Milstein S, Myers R, Wu SL, Karger B, Rossomando A. Evaluation of exogenous siRNA addition as a metabolic engineering tool for modifying biopharmaceuticals. Biotechnol Prog 2013; 29:415-24. [PMID: 23172735 PMCID: PMC4226405 DOI: 10.1002/btpr.1667] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2012] [Revised: 11/08/2012] [Indexed: 12/19/2022]
Abstract
Traditional metabolic engineering approaches, including homologous recombination, zinc-finger nucleases, and short hairpin RNA, have previously been used to generate biologics with specific characteristics that improve efficacy, potency, and safety. An alternative approach is to exogenously add soluble small interfering RNA (siRNA) duplexes, formulated with a cationic lipid, directly to cells grown in shake flasks or bioreactors. This approach has the following potential advantages: no cell line development required, ability to tailor mRNA silencing by adjusting siRNA concentration, simultaneous silencing of multiple target genes, and potential temporal control of down regulation of target gene expression. In this study, we demonstrate proof of concept of the siRNA feeding approach as a metabolic engineering tool in the context of increasing monoclonal antibody (MAb) afucosylation. First, potent siRNA duplexes targeting fut8 and gmds were dosed into shake flasks with cells that express an anti-CD20 MAb. Dose response studies demonstrated the ability to titrate the silencing effect. Furthermore, siRNA addition resulted in no deleterious effects on cell growth, final protein titer, or specific productivity. In bioreactors, antibodies produced by cells following siRNA treatment exhibited improved functional characteristics compared to antibodies from untreated cells, including increased levels of afucosylation (63%), a 17-fold improvement in FCgRIIIa binding, and an increase in specific cell lysis by up to 30%, as determined in an Antibody-Dependent Cellular Cytoxicity (ADCC) assay. In addition, standard purification procedures effectively cleared the exogenously added siRNA and transfection agent. Moreover, no differences were observed when other key product quality structural attributes were compared to untreated controls. These results establish that exogenous addition of siRNA represents a potentially novel metabolic engineering tool to improve biopharmaceutical function and quality that can complement existing metabolic engineering methods.
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Affiliation(s)
- Seshu Tummala
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | - Michael Titus
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | - Lee Wilson
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | - Chunhua Wang
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | - Carlo Ciatto
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | - Donald Foster
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | - Zoltan Szabo
- Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA 02115
| | - Andras Guttman
- Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA 02115
| | - Chen Li
- Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA 02115
| | | | | | - Jack Deroot
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | - Greg Thill
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | - David Kocisko
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | - Stuart Pollard
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | - Klaus Charisse
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | | | - Greg Hinkle
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | - Stuart Milstein
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | - Rachel Myers
- Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142
| | - Shiaw-Lin Wu
- Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA 02115
| | - Barry Karger
- Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA 02115
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Ma D, Li L. Searching for reliable premortem protein biomarkers for prion diseases: progress and challenges to date. Expert Rev Proteomics 2013; 9:267-80. [PMID: 22809206 DOI: 10.1586/epr.12.20] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Prion diseases are a unique family of fatal neurodegenerative diseases caused by abnormal folding of normal cellular prion proteins in the brain. Due to the high risk of prion disease transmission and the lack of effective treatment to cure or delay the disease progression, prion diseases pose a serious threat to public health. To control and prevent prion diseases, an early diagnosis is urgently needed. Proteomic analysis has emerged as a powerful technology to decipher biological and pathophysiological processes and identify protein biomarkers indicative of disease. In this article, the authors review the use of the latest proteomic technologies for the identification of promising prion disease biomarkers, the challenges that exist in biomarker development pipelines and the new directions for utilizing proteomics for future biomarker discovery in the context of prion disease diagnostics.
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Affiliation(s)
- Di Ma
- School of Pharmacy, University of Wisconsin at Madison, 777 Highland Avenue, Madison, WI 53705, USA
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Fanayan S, Smith JT, Sethi MK, Cantor D, Goode R, Simpson RJ, Baker MS, Hancock WS, Nice E. Chromosome 7-Centric Analysis of Proteomics Data from a Panel of Human Colon Carcinoma Cell Lines. J Proteome Res 2012; 12:89-96. [DOI: 10.1021/pr300906y] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Affiliation(s)
- Susan Fanayan
- Department of Chemistry and
Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia
| | - Joshua T. Smith
- Barnett Institute and Department
of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States
| | - Manveen K. Sethi
- Department of Chemistry and
Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia
| | - David Cantor
- Department of Chemistry and
Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia
| | - Robert Goode
- Department of Biochemistry & Molecular Biology, Monash University, Clayton, VIC 3800, Australia
| | - Richard J. Simpson
- La
Trobe Institute for Molecular
Science, La Trobe University, Bundoora,
Victoria 3086, Australia
| | - Mark S. Baker
- Department of Chemistry and
Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia
| | - William S. Hancock
- Department of Chemistry and
Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia
- Barnett Institute and Department
of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States
| | - Edouard Nice
- Department of Biochemistry & Molecular Biology, Monash University, Clayton, VIC 3800, Australia
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Lu Y, Bie Z, Liu Y, Liu Z. Fine-tuning the specificity of boronate affinity monoliths toward glycoproteins through pH manipulation. Analyst 2012; 138:290-8. [PMID: 23139927 DOI: 10.1039/c2an36048a] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Boronate affinity functionalized materials have recently drawn increasing attention due to their capability to selectively isolate and enrich glycoproteins and glycopeptides. As cheaper and more stable competitors to lectins, boronic acids are generally believed to yield a relatively wider spectrum specificity to glycoproteins. For better understanding and effective utilization of boronate affinity, it is necessary to establish if boronic acids exhibit lectin-like narrow specificity towards individual or a sub-class of glycoproteins. Here we report a pH manipulation strategy for fine-tuning the specificity of boronate affinity monoliths towards two sub-classes of glycoproteins, sialylated and nonsialylated glycoproteins. When the binding pH > the pK(a) of the boronic acid by one pH unit or more, the boronate affinity monolith preferentially binds to glycoproteins containing neutral sugars and excludes sialic acid containing glycoproteins due to electrostatic repulsion. When the binding pH < the pK(a) by one pH unit or more, the boronate affinity monolith binds to sialylated glycoproteins due to the exceptional binding affinity of the boronic acid towards sialic acid residues. The alternative specificity towards sialic acid and neutral sugar was first verified using an off-line combination of boronate affinity extraction with nano-ESI-Orbitrap MS/MS detection. The alternative specificity towards sialylated and nonsialylated glycoproteins was then demonstrated by means of off-line combination of boronate affinity extraction with MALDI-TOF MS. Finally, the developed approach was applied to the alternative extraction of intact sialylated and nonsialylated glycoproteins spiked in human serum.
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Affiliation(s)
- Yue Lu
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
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40
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Pan S, Tamura Y, Chen R, May D, McIntosh MW, Brentnall TA. Large-scale quantitative glycoproteomics analysis of site-specific glycosylation occupancy. MOLECULAR BIOSYSTEMS 2012; 8:2850-6. [PMID: 22892896 PMCID: PMC3463725 DOI: 10.1039/c2mb25268f] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Disease-associated aberrant glycosylation may be protein specific and glycosylation site specific. Quantitative assessment of glycosylation changes at a site-specific molecular level may represent one of the initial steps for systematically revealing the glycosylation abnormalities associated with a disease or biological state. Comparative quantitative profiling of glycoproteome to provide accurate quantification of site-specific glycosylation occupancy has been a challenging task, requiring a concerted approach drawing from a variety of techniques. In this report, we present a quantitative glycoproteomics method that allows global scale identification and comparative quantification of glycosylation site occupancy using mass spectrometry. We further demonstrated this approach by quantitatively characterizing the N-glycoproteome of human pancreas.
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Affiliation(s)
- Sheng Pan
- Department of Medicine, University of Washington, Seattle, WA 98195, USA.
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41
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Li QK, Gabrielson E, Zhang H. Application of glycoproteomics for the discovery of biomarkers in lung cancer. Proteomics Clin Appl 2012; 6:244-56. [PMID: 22641610 DOI: 10.1002/prca.201100042] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Lung cancer is the leading cause of cancer-related deaths in the United States. Approximately 40-60% of lung cancer patients present with locally advanced or metastatic disease at the time of diagnosis. Lung cancer development and progression are a multistep process that is characterized by abnormal gene and protein expressions ultimately leading to phenotypic change. Glycoproteins have long been recognized to play fundamental roles in many physiological and pathological processes, particularly in cancer genesis and progression. In order to improve the survival rate of lung cancer patients, the discovery of early diagnostic and prognostic biomarkers is urgently needed. Herein, we reviewed the recent technological developments of glycoproteomics and published data in the field of glycoprotein biomarkers in lung cancer, and discussed their utility and limitations for the discovery of potential biomarkers in lung cancer. Although numerous papers have already acknowledged the importance of the discovery of cancer biomarkers, the systemic study of glycoproteins in lung cancer using glycoproteomic approaches is still suboptimal. Recent development in the glycoproteomics will provide new platforms for identification of potential protein biomarkers in lung cancers.
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Affiliation(s)
- Qing Kay Li
- Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD 21287, USA.
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Barone R, Sturiale L, Palmigiano A, Zappia M, Garozzo D. Glycomics of pediatric and adulthood diseases of the central nervous system. J Proteomics 2012; 75:5123-39. [DOI: 10.1016/j.jprot.2012.07.007] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2012] [Revised: 06/05/2012] [Accepted: 07/04/2012] [Indexed: 12/14/2022]
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Nilsson J, Halim A, Grahn A, Larson G. Targeting the glycoproteome. Glycoconj J 2012; 30:119-36. [PMID: 22886069 PMCID: PMC3552370 DOI: 10.1007/s10719-012-9438-6] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2012] [Revised: 07/06/2012] [Accepted: 07/26/2012] [Indexed: 12/12/2022]
Abstract
Despite numerous original publications describing the structural complexity of N- and O-linked glycans on glycoproteins, only very few answer the basic question of which particular glycans are linked to which amino acid residues along the polypeptide chain. Such structural information is of fundamental importance for understanding the biological roles of complex glycosylations as well as deciphering their non-template driven biosynthesis. This review focuses on presenting and commenting on recent strategies, specifically aimed at identifying the glycoproteome of cultured cells and biological samples, using targeted and global enrichment procedures and utilizing the high resolution power, high through-put capacity and complementary fragmentation techniques of tandem mass spectrometry. The goal is to give an update of this emerging field of protein and glyco-sciences and suggest routes to bridge the data gap between the two aspects of glycoprotein characteristics, i.e. glycan structures and their attachment sites.
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Affiliation(s)
- Jonas Nilsson
- Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, The Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital, Gothenburg 413 45, Sweden
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Shetty V, Hafner J, Shah P, Nickens Z, Philip R. Investigation of ovarian cancer associated sialylation changes in N-linked glycopeptides by quantitative proteomics. Clin Proteomics 2012; 9:10. [PMID: 22856521 PMCID: PMC3488482 DOI: 10.1186/1559-0275-9-10] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2012] [Accepted: 06/26/2012] [Indexed: 11/29/2022] Open
Abstract
Background In approximately 80% of patients, ovarian cancer is diagnosed when the patient is already in the advanced stages of the disease. CA125 is currently used as the marker for ovarian cancer; however, it lacks specificity and sensitivity for detecting early stage disease. There is a critical unmet need for sensitive and specific routine screening tests for early diagnosis that can reduce ovarian cancer lethality by reliably detecting the disease at its earliest and treatable stages. Results In this study, we investigated the N-linked sialylated glycopeptides in serum samples from healthy and ovarian cancer patients using Lectin-directed Tandem Labeling (LTL) and iTRAQ quantitative proteomics methods. We identified 45 N-linked sialylated glycopeptides containing 46 glycosylation sites. Among those, ten sialylated glycopeptides were significantly up-regulated in ovarian cancer patients’ serum samples. LC-MS/MS analysis of the non-glycosylated peptides from the same samples, western blot data using lectin enriched glycoproteins of various ovarian cancer type samples, and PNGase F (+/−) treatment confirmed the sialylation changes in the ovarian cancer samples. Conclusion Herein, we demonstrated that several proteins are aberrantly sialylated in N-linked glycopeptides in ovarian cancer and detection of glycopeptides with abnormal sialylation changes may have the potential to serve as biomarkers for ovarian cancer.
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45
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Liu J, Ren L, Liu Y, Li H, Liu Z. Weak anion exchange chromatographic profiling of glycoprotein isoforms on a polymer monolithic capillary. J Chromatogr A 2012; 1228:276-82. [DOI: 10.1016/j.chroma.2011.08.079] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2011] [Revised: 08/13/2011] [Accepted: 08/21/2011] [Indexed: 10/17/2022]
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46
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A general approach for the purification and quantitative glycomic analysis of human plasma. Anal Bioanal Chem 2012; 402:2687-700. [DOI: 10.1007/s00216-012-5712-5] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2011] [Revised: 12/28/2011] [Accepted: 01/04/2012] [Indexed: 11/26/2022]
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47
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Lee S, Valentine SJ, Reilly JP, Clemmer DE. Analyzing a Mixture of Disaccharides by IMS-VUVPD-MS. INTERNATIONAL JOURNAL OF MASS SPECTROMETRY 2012; 309:161-167. [PMID: 22518093 PMCID: PMC3327510 DOI: 10.1016/j.ijms.2011.09.013] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/08/2023]
Abstract
Comparative analyses utilizing collision induced dissociation (CID) and vacuum ultraviolet photodissociation (VUVPD) for seven isobaric disaccharides have been performed in order to differentiate the linkage type and anomeric configuration of the isomers. Although an individual CID spectrum of a disaccharide ion provides information related to its structure, CID does not sufficiently differentiate mixture components due to the identical mass-to-charge values of most of the intense fragments. In contrast to the ambiguity of the CID analyses for the disaccharide mixture, VUVPD (157 nm) generates unique fragments for each disaccharide ion that are useful for distinguishing individual components from the mixture. When combined with a gas-phase ion mobility separation of the ions, the identification of each component from the mixture can be obtained.
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48
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Ma WF, Li LL, Zhang Y, An Q, You LJ, Li JM, Zhang YT, Xu S, Yu M, Guo J, Lu HJ, Wang CC. Ligand-free strategy for ultrafast and highly selective enrichment of glycopeptides using Ag-coated magnetic nanoarchitectures. ACTA ACUST UNITED AC 2012. [DOI: 10.1039/c2jm35196j] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
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49
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Safina G. Application of surface plasmon resonance for the detection of carbohydrates, glycoconjugates, and measurement of the carbohydrate-specific interactions: A comparison with conventional analytical techniques. A critical review. Anal Chim Acta 2012; 712:9-29. [DOI: 10.1016/j.aca.2011.11.016] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2011] [Revised: 10/07/2011] [Accepted: 11/04/2011] [Indexed: 12/16/2022]
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50
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Lin Z, Lo A, Simeone DM, Ruffin MT, Lubman DM. An N-glycosylation Analysis of Human Alpha-2-Macroglobulin Using an Integrated Approach. ACTA ACUST UNITED AC 2012; 5:127-134. [PMID: 23028207 DOI: 10.4172/jpb.1000224] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Assignment of glycosylation sites and site microheterogeneity is of both biological and clinical significance. Herein, the detailed N-glycosylation pattern of human serum alpha-2-macroglobulin was studied using an integrative approach, including permethylation of N-glycans, collision induced dissociation (CID) and electron transfer dissociation (ETD) of chymotryptic N-glycopeptides, and partial deglycosylation of chymotryptic N-glycopeptides with endo-β-N-acetylglucosaminidase F3 (Endo F3). Three N-glycosylation sites were found to be occupied by four biantennary complex type N-glycans using N-glycan analysis and the ETD/CID method. Endo F3 assisted mass spectrometric analysis yielded five N-glycosylation sites with and without core fucosylation. In total, six out of eight potential N-glycosylation sites were identified using this approach. This integrative approach was performed using only 10 μL of human serum for both N-glycosylation site assignment and site microheterogeneity determination.
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Affiliation(s)
- Zhenxin Lin
- Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109, USA
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