O-GlcNAc profiling: from proteins to proteomes

Abstract

O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) onto serine and threonine residues of proteins is an important post-translational modification (PTM), which is involved in many crucial biological processes including transcription, translation, proteasomal degradation, and signal transduction. Aberrant protein O-GlcNAcylation is directly linked to the pathological progression of chronic diseases including diabetes, cancer, and neurodegenerative disorders. Identification, site mapping, and quantification of O-GlcNAc proteins are a prerequisite to decipher their functions. In this review, we mainly focus on technological developments regarding O-GlcNAc protein profiling. Specifically, on one hand, we show how these techniques are being used for the comprehensive characterization of certain targeted proteins in which biologists are most interested. On the other hand, we present several newly developed approaches for O-GlcNAcomic profiling as well as how they provide us with a systems perspective to crosstalk amongst different PTMs and complicated biological events. Promising technical trends are also highlighted to evoke more efforts by diverse laboratories, which would further expand our understanding of the physiological and pathological roles of protein O-GlcNAcylation in chronic diseases.

Figure 1

Respective spectra from CID (A), ETD (B), HCD (C), and zoomed in HCD (D) of standard O-GlcNAc modified peptide CKII (PGGSTPV s SANMM, where ‘s’ represents the O-GlcNAc modified Ser). Note: “-HexNAc” or “-H2O” indicates the loss of HexNAc or H2O. Low m/z range HCD displays a distinctive pattern of HexNAc fragments (D). (Adapted from [53], with the permission from American Chemical Society)

Figure 2

Scheme for the enrichment of O-GlcNAcylated proteins/peptides. Most commonly used strategies with antibody enrichment (1), lectin enrichment (2), BEMAD (3), chemoenzymatic labeling (4) and metabolic labeling (5) are illustrated. In (1), proteins are captured onto antibody/antibodies-conjugated beads, and the enriched ones are digested and identified by tandem mass spectrometry. In (2), (3), and (4), proteins are digested into peptides, which are captured with lectin-conjugated resin (2), thio-capture column after BEMAD (3), and streptavidin-conjugated beads after chemoenzymatic labeling (4), with the enriched peptides identified by tandem mass spectrometry. In (5), cells are fed with GlcNAc analogs GlcNAz and GlcNAlk, and the GlcNAz- and GlcNAlk-containing proteins are subjected to click chemistry, streptavidin-conjugated beads enrichment and digestion, with the digests analyzed by tandem mass spectrometry. Note: The cocktail usage of several methods (e.g., chemo-enzymatic/metabolic labeling and BEMAD) has also been applied in some cases.