Mass Spectrometry for O-GlcNAcylation

Abstract

O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) at proteins with low-abundance expression level and species diversity, shows important roles in plenty of biological processes. O-GlcNAcylations with abnormal expression levels are associated with many diseases. Systematically profiling of O-GlcNAcylation at qualitative or quantitative level is vital for their function understanding. Recently, the combination of affinity enrichment, metabolic labeling or chemical tagging with mass spectrometry (MS) have made significant contributions to structure-function mechanism elucidating of O-GlcNAcylations in organisms. Herein, this review provides a comprehensive update of MS-based methodologies for quali-quantitative characterization of O-GlcNAcylation.

Tissue-specific O-GlcNAcylation profiling identifies substrates in translational machinery in Drosophila mushroom body contributing to olfactory learning

“…An obstacle to comprehensively identifying the O- GlcNAc conveyors of various cognitive functions is the lack of an effective tissue-specific O- GlcNAc profiling method. Given the structural diversity and relatively low abundance, enrichment of O- GlcNAc-modified proteins is required for mass spectrometry (MS)-based profiling of O- GlcNAcylation ( Yin et al, 2021 ). The enrichment strategies roughly fall into two categories.…”

“…The enrichment strategies roughly fall into two categories. One category involves direct capture of O- GlcNAcylated proteins by antibodies or lectins that recognize the GlcNAc moiety ( Yin et al, 2021 ; Hu et al, 2022 ; Dupas et al, 2022 ; Saha et al, 2021 ; Maynard and Chalkley, 2021 ; Ma et al, 2021a ; Ma et al, 2021b ). O- GlcNAc antibodies including RL2 and CTD110.6, as well as O- GlcNAc-binding lectins such as wheat germ agglutinin (WGA), are commonly used for enrichment.…”

“…In addition, the catalytic-dead mutant of Cp OGA that retains the ability to recognize O- GlcNAcylated substrates was successfully repurposed to concentrate many developmental regulators from Drosophila embryo lysates ( Selvan et al, 2017 ). Another category of enrichment strategies relies on chemoenzymatic or metabolic labeling ( Yin et al, 2021 ; Hu et al, 2022 ; Dupas et al, 2022 ; Saha et al, 2021 ; Maynard and Chalkley, 2021 ; Ma et al, 2021a ; Ma et al, 2021b ). Azido-modified intermediates, such as N -azidoacetylglucosamine (GlcNAz) and N -azidoacetylgalactosamine (GalNAz), are used to introduce specific tags (e.g.…”

Olfactory learning inDrosophilarequires O-GlcNAcylation of mushroom body ribosomal subunits

“…An obstacle for comprehensively identifying the O-GlcNAc conveyors of various cognitive functions is the lack of an effective tissue-specific O-GlcNAc profiling method. Given the structural diversity and relatively low abundance, enrichment of O-GlcNAc modified proteins is required for mass spectrometry (MS)-based profiling of O-GlcNAcylation 27 . The enrichment strategies roughly fall into two categories.…”

Mapping the O-GlcNAc Modified Proteome: Applications for Health and Disease

“…Various O-GlcNAc antibody reagents exist, but to our knowledge, a comparison between these antibodies in their capability to map the O-GlcNAc modified proteome has not been formally investigated. For a more complete review of enrichment techniques for O-GlcNAc modified peptides, readers should refer to other reviews ( Ma and Hart, 2014 ; Maynard and Chalkley, 2021 ; Yin et al, 2021 ; Zhu and Yi, 2021 ).…”