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. 2021 Mar;1(3):e84.
doi: 10.1002/cpz1.84.

Glycoproteomic Sample Processing, LC-MS, and Data Analysis Using GlycReSoft

Meizhe Wang  1 , Asif Shajahan  2 , Lauren E Pepi  2 , Parastoo Azadi  2 , Joseph Zaia  1   3
Affiliations

Glycoproteomic Sample Processing, LC-MS, and Data Analysis Using GlycReSoft

Meizhe Wang et al. Curr Protoc. 2021 Mar.

Erratum in

Abstract

Identification of N- and O-glycosylation on specific sites of proteins, along with glycan structural information, is necessary to determine the roles glycoproteins play in normal and pathologic cellular functions. Because such glycosylation is macro- and micro-heterogeneous and alters the dissociation behavior of glycopeptides, specific sample preparation, mass spectrometry, and data analysis techniques are required. Advanced tandem mass spectrometry-based glycoproteomics coupled with powerful data mining algorithms are key elements for characterization of protein glycosylation. This article includes the detailed, streamlined sample preparation method for liquid chromatography-mass spectrometry data acquisition and subsequent bioinformatics-based data annotation using the publicly available GlycReSoft program for highly efficient identification and quantification of glycoprotein glycosylation. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Characterization of glycans and site occupancy on purified glycoprotein Support Protocol 1: In-gel digestion of glycoproteins Support Protocol 2: Detection of glycoproteins from cells/tissue through glycopeptide enrichment Basic Protocol 2: Acquisition of glycopeptides through high-resolution nano-LC-MS/MS Basic Protocol 3: Identification and quantification of glycopeptides using GlycReSoft.

Keywords: GlycReSoft; bioinformatics; glycomics; glycoproteomics; mass spectrometry.

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Figures

Figure 1.
Figure 1.
Workflow of glycopeptide analysis with GlycReSoft.
Figure 2.
Figure 2.
Glycoproteomics sample processing. (A). Extract proteins from cells/tissues and enrich membrane proteins by differential centrifugation; (B) Reduce, alkylate, and precipitate the membrane protein lysates. Digest the proteins by protease and enrich the glycopeptides by iHILIC solid phase extraction (SPE) cartridges. Filter the sample and inject into LC-MS/MS system. Process the data and identify the glycosylation and site occupancy by GlycReSoft; (C) Reduce, alkylate and buffer exchange the glycoprotein solution. Digest the proteins by protease, filter the sample and inject into nano-LCMS/MS system. Process the data and identify the glycosylation and site occupancy by GlycReSoft.
Figure 3.
Figure 3.
Add sample to GlycReSoft workspace.
Figure 4.
Figure 4.
Build glycan search space.
Figure 5.
Figure 5.
Build glycopeptide search space.
Figure 6.
Figure 6.
Search glycopeptide sequence.
Figure 7.
Figure 7.
Glycopeptide search results. (A) The assigned glycopeptides are shown for each protein in the sample using pileup diagrams. (B) A table of glycopeptides assigned for each protein is given. Clicking on a table entry opens a window that shows the annotated tandem mass spectrum and extracted ion chromatogram. (C) Bar plots show the distribution of glycopeptide glycoforms identified for each glycosite.
See this image and copyright information in PMC

References

    1. Alagesan K, Khilji SK, & Kolarich D. (2017). It is all about the solvent: on the importance of the mobile phase for ZIC-HILIC glycopeptide enrichment. Anal Bioanal Chem, 409(2), 529–538. doi:10.1007/s00216-016-0051-6 - DOI - PMC - PubMed
    1. An HJ, Froehlich JW, & Lebrilla CB (2009). Determination of glycosylation sites and site-specific heterogeneity in glycoproteins. Curr Opin Chem Biol, 13(4), 421–426. doi:10.1016/j.cbpa.2009.07.022 - DOI - PMC - PubMed
    1. Brockhausen I, & Stanley P. (2015). O-GalNAc Glycans. In rd A. Varki, Cummings RD, Esko JD, Stanley P, Hart GW, Aebi M, Darvill AG, Kinoshita T, Packer NH, Prestegard JH, Schnaar RL, & Seeberger PH (Eds.), Essentials of Glycobiology (pp. 113–123). Cold Spring; Harbor (NY).
    1. Cao L, Qu Y, Zhang Z, Wang Z, Prytkova I, & Wu S. (2016). Intact glycopeptide characterization using mass spectrometry. Expert Rev Proteomics, 13(5), 513–522. doi:10.1586/14789450.2016.1172965 - DOI - PMC - PubMed
    1. Cao W, Huang J, Jiang B, Gao X, & Yang P. (2016). Highly Selective Enrichment of Glycopeptides Based on Zwitterionically Functionalized Soluble Nanopolymers. Sci Rep, 6, 29776. doi:10.1038/srep29776 - DOI - PMC - PubMed

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