Chemical Strategies to Glycoprotein Analysis

Mertz, Joseph L.; Toonstra, Christian; Zhang, Hui

doi:10.1002/9781118970195.ch12

Cited by 4 publications

(3 citation statements)

References 130 publications

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“…Glycans inherently contain functional groups that can be directly targeted for enrichment ( 285 ). Historically, hydrazide chemistry has been widely used to profile sialylated glycoconjugates ( 286 ).…”

Section: Chemical Coupling Strategiesmentioning

confidence: 99%

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

Riley

Bertozzi

Pitteri

2021

Molecular & Cellular Proteomics

159

158

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Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry (MS)-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography (HILIC) and its derivatives, porous graphitic carbon (PGC), reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as MS instrumentation and software improve, so this review aims to help equip researchers with necessary information to choose appropriate enrichment strategies that best complement these efforts.

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Section: Chemical Coupling Strategiesmentioning

confidence: 99%

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

Riley

Bertozzi

Pitteri

2021

Molecular & Cellular Proteomics

159

158

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“…Innovations in affinity-based capture of glycans, glycopeptides, and glycoproteins by lectins, antibodies, or chemical affinity resins [ 31 , 32 ] have increased coverage of the glycoproteome [ 33 , 34 , 35 ]. Solid-phase extraction of N -linked glycoproteins (SPEG), which utilizes covalent bonds between hydrazide beads and oxidized glycan moieties of N -glycosylated peptides followed by the release by PNGase F was one of the first examples of such a workflow, and it has seen continued use since its introduction in 2003 [ 35 , 36 , 37 ]. Hydrophilic interaction liquid chromatography (HILIC), is a collective term for purification techniques that generally utilize solid-phase resins with neutral polar moieties to retain an aqueous layer, and thereby sequester glycopeptides via their hydrophilic glycan moieties from a less polar mobile phase [ 38 ].…”

Section: Introductionmentioning

confidence: 99%

“…Characterization of glycoproteins has now advanced to include intact glycopeptide analysis that can simultaneously analyze glycosites and their attached glycans [ 36 , 39 , 40 ]. The large search space created by the great diversity of the glycome makes it difficult to accurately identify intact glycopeptides and has led to various strategies to restrict or guide the peptide sequence matching process.…”

Section: Introductionmentioning

confidence: 99%

Comparison of Three Glycoproteomic Methods for the Analysis of the Secretome of CHO Cells Treated with 1,3,4-O-Bu3ManNAc

et al. 2020

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Comprehensive analysis of the glycoproteome is critical due to the importance of glycosylation to many aspects of protein function. The tremendous complexity of this post-translational modification, however, makes it difficult to adequately characterize the glycoproteome using any single method. To overcome this pitfall, in this report we compared three glycoproteomic analysis methods; first the recently developed N-linked glycans and glycosite-containing peptides (NGAG) chemoenzymatic method, second, solid-phase extraction of N-linked glycoproteins (SPEG), and third, hydrophilic interaction liquid chromatography (HILIC) by characterizing N-linked glycosites in the secretome of Chinese hamster ovary (CHO) cells. Interestingly, the glycosites identified by SPEG and HILIC overlapped considerably whereas NGAG identified many glycosites not observed in the other two methods. Further, utilizing enhanced intact glycopeptide identification afforded by the NGAG workflow, we found that the sugar analog 1,3,4-O-Bu3ManNAc, a “high flux” metabolic precursor for sialic acid biosynthesis, increased sialylation of secreted proteins including recombinant human erythropoietin (rhEPO).

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O-Glycoproteomics: Methods, Challenges, and New Opportunities

Riley,

Malaker

2024

Glycoprotein Analysis

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The field of O-glycoproteomics has seen many notable advances in the last decade, which have allowed the site-specific localization of O-glycans on a myriad of proteins. In this chapter, we describe the state-of-the-art techniques in glycoproteomics in the hope of providing a broad overview of the methods and practices used within the field. We first cover the digestion of O-glycoproteins with proteases, such as trypsin, Proteinase K, and the newly introduced class of O-glycoproteases. Then, given that glycopeptides are enriched to enhance their detection via mass spectrometry (MS), we also discuss enrichment methods involving lectins, antibodies, and/or solid-phase extraction. Importantly, we provide a broad overview of glycopeptide analysis within the MS, including dissociation via tandem MS, beam-type collision-induced dissociation (beamCID), electron transfer dissociation (ETD), and/or ETD with supplemental collisional activation (EThcD). Finally, we discuss the growing collection of glycoproteomic search algorithms, including some new programs specializing in O-glycoproteomics, that can assist in the analysis of resultant spectra. Taken together, this chapter serves as a broad overview of O-glycoproteomics, including current techniques, challenges, and emerging opportunities in the field.

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Chemical Strategies to Glycoprotein Analysis

Cited by 4 publications

References 130 publications

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

Comparison of Three Glycoproteomic Methods for the Analysis of the Secretome of CHO Cells Treated with 1,3,4-O-Bu3ManNAc

O-Glycoproteomics: Methods, Challenges, and New Opportunities

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