Glycans, Glycosite, and Intact Glycopeptide Analysis of N-Linked Glycoproteins Using Liquid Handling Systems

Overview

Journal Anal Chem

Specialty Chemistry

Date 2019 Dec 21

PMID 31859482

Citations 18

Authors

Shao-Yung Chen

Mingming Dong

Ganglong Yang

Yangying Zhou

David J Clark

T Mamie Lih

Michael Schnaubelt

Zichen Liu

Hui Zhang

Affiliations

Soon will be listed here.

Abstract

Aberrant glycosylation has been shown to associate with disease progression, and with glycoproteins representing the major protein component of biological fluids this makes them attractive targets for disease monitoring. Leveraging glycoproteomic analysis via mass spectrometry (MS) could provide the insight into the altered glycosylation patterns that relate to disease progression. However, investigation of large sample cohorts requires rapid, efficient, and highly reproducible sample preparation. To address the limitation, we developed a high-throughput method for characterizing glycans, glycosites, and intact glycopeptides (IGPs) derived from N-linked glycoproteins. We combined disparate peptide enrichment strategies (i.e., hydrophilic and hydrophobic) and a liquid handling platform allowing for a high throughput and rapid enrichment of IGP in a 96-well plate format. The C18/MAX-Tip workflow reduced sample processing time and facilitated the selective enrichment of IGPs from complex samples. Furthermore, our approach enabled the analysis of deglycosylated peptides and glycans from enriched IGPs following PNGase F digest. Following development and optimization of the C18/MAX-Tip methodology using the standard glycoprotein, fetuin, we investigated normal urine samples to obtain N-linked glycoprotein information. Together, our method enables a high-throughput enrichment of glycan, glycosites, and IGPs from biological samples.

Citing Articles

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References

Berven F, Ahmad R, Clauser K, Carr S . Optimizing performance of glycopeptide capture for plasma proteomics. J Proteome Res. 2010; 9(4):1706-15. PMC: 2855884. DOI: 10.1021/pr900845m. View

Guo Z, Liu X, Li M, Shao C, Tao J, Sun W . Differential urinary glycoproteome analysis of type 2 diabetic nephropathy using 2D-LC-MS/MS and iTRAQ quantification. J Transl Med. 2015; 13:371. PMC: 4660682. DOI: 10.1186/s12967-015-0712-9. View

Palmisano G, Lendal S, Engholm-Keller K, Leth-Larsen R, Parker B, Larsen M . Selective enrichment of sialic acid-containing glycopeptides using titanium dioxide chromatography with analysis by HILIC and mass spectrometry. Nat Protoc. 2010; 5(12):1974-82. DOI: 10.1038/nprot.2010.167. View

Nagai-Okatani C, Minamino N . Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure. PLoS One. 2016; 11(6):e0150210. PMC: 4900630. DOI: 10.1371/journal.pone.0150210. View

Chen J, Shah P, Zhang H . Solid phase extraction of N-linked glycopeptides using hydrazide tip. Anal Chem. 2013; 85(22):10670-4. PMC: 4082910. DOI: 10.1021/ac401812b. View

Toghi Eshghi S, Shah P, Yang W, Li X, Zhang H . GPQuest: A Spectral Library Matching Algorithm for Site-Specific Assignment of Tandem Mass Spectra to Intact N-glycopeptides. Anal Chem. 2015; 87(10):5181-8. PMC: 4721644. DOI: 10.1021/acs.analchem.5b00024. View

Tabb D, Vega-Montoto L, Rudnick P, Variyath A, Ham A, Bunk D . Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry. J Proteome Res. 2009; 9(2):761-76. PMC: 2818771. DOI: 10.1021/pr9006365. View

Ercan A, Cui J, Chatterton D, Deane K, Hazen M, Brintnell W . Aberrant IgG galactosylation precedes disease onset, correlates with disease activity, and is prevalent in autoantibodies in rheumatoid arthritis. Arthritis Rheum. 2010; 62(8):2239-48. PMC: 4118465. DOI: 10.1002/art.27533. View

Nilsson J, Ruetschi U, Halim A, Hesse C, Carlsohn E, Brinkmalm G . Enrichment of glycopeptides for glycan structure and attachment site identification. Nat Methods. 2009; 6(11):809-11. DOI: 10.1038/nmeth.1392. View

10.

Khoury G, Baliban R, Floudas C . Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database. Sci Rep. 2011; 1. PMC: 3201773. DOI: 10.1038/srep00090. View

11.

Yang S, Hu Y, Sokoll L, Zhang H . Simultaneous quantification of N- and O-glycans using a solid-phase method. Nat Protoc. 2017; 12(6):1229-1244. PMC: 5877797. DOI: 10.1038/nprot.2017.034. View

12.

Kawahara R, Ortega F, Rosa-Fernandes L, Guimaraes V, Quina D, Nahas W . Distinct urinary glycoprotein signatures in prostate cancer patients. Oncotarget. 2018; 9(69):33077-33097. PMC: 6145689. DOI: 10.18632/oncotarget.26005. View

13.

Sun S, Shah P, Toghi Eshghi S, Yang W, Trikannad N, Yang S . Comprehensive analysis of protein glycosylation by solid-phase extraction of N-linked glycans and glycosite-containing peptides. Nat Biotechnol. 2015; 34(1):84-8. PMC: 4872599. DOI: 10.1038/nbt.3403. View

14.

Nie H, Li Y, Sun X . Recent advances in sialic acid-focused glycomics. J Proteomics. 2012; 75(11):3098-112. PMC: 3367064. DOI: 10.1016/j.jprot.2012.03.050. View

15.

Zou Z, Ibisate M, Zhou Y, Aebersold R, Xia Y, Zhang H . Synthesis and evaluation of superparamagnetic silica particles for extraction of glycopeptides in the microtiter plate format. Anal Chem. 2008; 80(4):1228-34. DOI: 10.1021/ac701950h. View

16.

Zhou J, Yang W, Hu Y, Hoti N, Liu Y, Shah P . Site-Specific Fucosylation Analysis Identifying Glycoproteins Associated with Aggressive Prostate Cancer Cell Lines Using Tandem Affinity Enrichments of Intact Glycopeptides Followed by Mass Spectrometry. Anal Chem. 2017; 89(14):7623-7630. PMC: 5599242. DOI: 10.1021/acs.analchem.7b01493. View

17.

Ceroni A, Maass K, Geyer H, Geyer R, Dell A, Haslam S . GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J Proteome Res. 2008; 7(4):1650-9. DOI: 10.1021/pr7008252. View

18.

Hu Y, Shah P, Clark D, Ao M, Zhang H . Reanalysis of Global Proteomic and Phosphoproteomic Data Identified a Large Number of Glycopeptides. Anal Chem. 2018; 90(13):8065-8071. PMC: 6440470. DOI: 10.1021/acs.analchem.8b01137. View

19.

Clark D, Hu Y, Schnaubelt M, Fu Y, Ponce S, Chen S . Simple Tip-Based Sample Processing Method for Urinary Proteomic Analysis. Anal Chem. 2019; 91(9):5517-5522. PMC: 6512789. DOI: 10.1021/acs.analchem.8b05234. View

20.

An H, Miyamoto S, Lancaster K, Kirmiz C, Li B, Lam K . Profiling of glycans in serum for the discovery of potential biomarkers for ovarian cancer. J Proteome Res. 2006; 5(7):1626-35. DOI: 10.1021/pr060010k. View