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Review
. 2018 Sep 12;118(17):7886-7930.
doi: 10.1021/acs.chemrev.7b00732. Epub 2018 Mar 19.

Mass Spectrometry Approaches to Glycomic and Glycoproteomic Analyses

L Renee Ruhaak  1 , Gege Xu  2 , Qiongyu Li  2 , Elisha Goonatilleke  2 , Carlito B Lebrilla  2   3   4
Affiliations
Review

Mass Spectrometry Approaches to Glycomic and Glycoproteomic Analyses

L Renee Ruhaak et al. Chem Rev. .

Abstract

Glycomic and glycoproteomic analyses involve the characterization of oligosaccharides (glycans) conjugated to proteins. Glycans are produced through a complicated nontemplate driven process involving the competition of enzymes that extend the nascent chain. The large diversity of structures, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies of glycans all conspire to make the analysis arguably much more difficult than any other biopolymer. Furthermore, the large number of glycoforms associated with a specific protein site makes it more difficult to characterize than any post-translational modification. Nonetheless, there have been significant progress, and advanced separation and mass spectrometry methods have been at its center and the main reason for the progress. While glycomic and glycoproteomic analyses are still typically available only through highly specialized laboratories, new software and workflow is making it more accessible. This review focuses on the role of mass spectrometry and separation methods in advancing glycomic and glycoproteomic analyses. It describes the current state of the field and progress toward making it more available to the larger scientific community.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
An illustration of the several levels of complexity in glycoprotein analysis.
Figure 2.
Figure 2.
Site-specific glycosylation with occupancy information on secretory IgA from human milk. An example of the complexity of glycosylation even on a single protein. Reprinted with permission from ref . Copyright 2015 American Chemical Society.
Figure 3.
Figure 3.
Mechanistic pathway for the formation of N-glycans.
Figure 4.
Figure 4.
Variation in the N- and O-glycosylation in proteins. (A) Three types of N-glycans. (B) Eight core structures of O-glycans.
Figure 5.
Figure 5.
Spider graph showing the instrument requirements for glycomics, proteomics, glycoproteomics, and intact glycoprotein analysis.
Figure 6.
Figure 6.
Fragmentation behavior of native oligosaccharides under collision-induced dissociation conditions. Reprinted with permission from ref . Copyright 2011 Wiley Online Library.
Figure 7.
Figure 7.
IRMPD of glycopeptides yields low energy fragmentation corresponding to glycan and peptide fragments. Protonated species yield information regarding the peptide, while sodiated species yield fragmentation regarding the glycan. Conditions can be varied to yield both glycan and peptide fragmentation for protonated species under CID and IRMPD. Reprinted with permission from ref . Copyright 2008 American Chemical Society.
Figure 8.
Figure 8.
Comparison of (A) CID, (B) HCD, and (C) ETD fragmentation of an enriched plant glycopeptide. Reprinted with permission from ref . Under Creative Commons license (https://creativecommons.org/licenses/by/4.0/).
Figure 9.
Figure 9.
UVPD of a doubly deprotonated O-glycopeptide from κ casein yields fragmentation of both the glycan and peptide backbone. Reprinted with permission from ref . Copyright 2013 American Chemical Society.
Figure 10.
Figure 10.
A chemoenzymatic method for sequential releases and analyses of N-linked and O-linked glycans. Reprinted with permission from ref . Under Creative Commons license (CC BY 4.0) (http://creativecommons.org/licenses/by/4.0/).
Figure 11.
Figure 11.
Comparison of IgG N-glycans prepared by an automated workflow using a liquid handling robot and analyzed by (A) HILIC UHPLC after 2-AB labeling and (B) MALDI-TOF-MS after permethylation. Reprinted with permission from ref . Copyright 2016 American Chemical Society.
Figure 12.
Figure 12.
MALDI-TOF MS spectrum of human plasma N-glycans after linkage-specific sialic acid ethyl esterification. Reprinted with permission from ref . Copyright 2014 American Chemical Society.
Figure 13.
Figure 13.
PGC separation of N-linked glycans released from (A) human milk and (B) bovine milk. Reprinted with permission from ref . Copyright 2012 American Chemical Society.
Figure 14.
Figure 14.
MALDI-IMS for visualizations of the distribution of various N-glycans throughout a leiomyosarcoma tissue. Reprinted with permission from ref . Copyright 2016 American Chemical Society.
Figure 15.
Figure 15.
N-Glycan structural elucidation by exoglycosidase digestion and tandem MS. (A) EIC of m/z 814.29 shows two isomers for Hex4HexNAc4Fuc1. (B) HPLC fractionation to isolate one isomer. (C) EIC of exoglycosidase digestion product with m/z 631.7. (D) Differential tandem MS spectra for the identified isomers. Reprinted with permission from ref . Copyright 2012 American Chemical Society.
Figure 16.
Figure 16.
Comparison of different glycopeptide enrichment and detection methods showed ERLIC enrichment with HCD/ETD fragmention yielded the largest percentage and number of glycopeptides from palsma. Reprinted with permission from ref . Copyright 2017 American Chemical Society.
Figure 17.
Figure 17.
Bioorthogonal labeling of human prostate tissue slice cultures with azido sialic acids for imaging, enrichment, and LC-MS/MS analysis of sialoglycoproteins. Reprinted with permission from Spiciarich, ref . Copyright 2017 Wiley Online Library.
Figure 18.
Figure 18.
Analysis of the VVLHPNYSQVDIGLIK peptide of haptoglobin by nanoLC. Decreased retention of glycopeptides was observed with increasing number of neutral monosaccharide units. Reprinted with permission from ref . Copyright 2017 Wiley Online Library.
Figure 19.
Figure 19.
C18-PGC-LC-ESI-QTOF-MS/MS analysis of N- and O-glycopeptides from pronase digest of bovine fetuin. Reprinted with permission from ref . Copyright 2015 American Chemical Society.
Figure 20.
Figure 20.
Distinguishing sialic acid linkage isomers on glycopeptides using CID fragmentation and subsequent IM-MS analysis. Reprinted with permission from ref . Under Creative Commons Attribution 3.0 license (https://creativecommons.org/licenses/by/3.0/).
Figure 21.
Figure 21.
Comparison of nonspecific protease and trypsin for site-specific glycan mapping. Reprinted with permission from ref . Copyright 2015 American Chemical Society.
Figure 22.
Figure 22.
Overall strategy used for the site-specific glycopeptide analysis of cerebrospinal fluid samples. Reprinted with permission from ref . Copyright 2015 Wiley Online Library.
Figure 23.
Figure 23.
Workflow of glycoproteomics by combining proteomcis platform and novel algorism for intact glycopeptide identification. Reprinted with permission from ref . Copyright 2017 Springer Nature.
Figure 24.
Figure 24.
Overall flowchart of data collection for pGlyco 2.0 analysis. Reprinted with permission from ref . Under Creative Commons CC BY license (https://creativecommons.org/licenses/by/4.0/).
Figure 25.
Figure 25.
UVPD for middle-down analysis of a therapeutic monoclonal antibody. Reprinted with permission from ref . Copyright 2016 American Chemical Society.
Figure 26.
Figure 26.
Relative abundances of human serum N-glycans averaged for nine individual sera. Reprinted with permission from ref . Copyright 2015 American Chemical Society.
Figure 27.
Figure 27.
MALDI-TOF MS quantitation of IgG1 glycans using biantennary 13C-labeled N-glycans as internal standards (marked with an asterisk). Reprinted with permission from ref . Copyright 2015 American Chemical Society.
Figure 28.
Figure 28.
LC-MRM MS chromatograms for the relative quantitation of glycopeptides from abundant serum proteins. Both peptides (top) and glycopeptides can be monitored. Adapted from ref . Copyright 2018 American Chemical Society.
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