Chemical Glycoproteomics

Abstract

Chemical tools have accelerated progress in glycoscience, reducing experimental barriers to studying protein glycosylation, the most widespread and complex form of posttranslational modification. For example, chemical glycoproteomics technologies have enabled the identification of specific glycosylation sites and glycan structures that modulate protein function in a number of biological processes. This field is now entering a stage of logarithmic growth, during which chemical innovations combined with mass spectrometry advances could make it possible to fully characterize the human glycoproteome. In this review, we describe the important role that chemical glycoproteomics methods are playing in such efforts. We summarize developments in four key areas: enrichment of glycoproteins and glycopeptides from complex mixtures, emphasizing methods that exploit unique chemical properties of glycans or introduce unnatural functional groups through metabolic labeling and chemoenzymatic tagging; identification of sites of protein glycosylation; targeted glycoproteomics; and functional glycoproteomics, with a focus on probing interactions between glycoproteins and glycan-binding proteins. Our goal with this survey is to provide a foundation on which continued technological advancements can be made to promote further explorations of protein glycosylation.

Figure 1

Glycan structures display a range of complexity. (a) Example of an N-glycan, with the conserved pentasaccharide (Man₃GlcNAc₂) core structure highlighted in red. This core structure can be further elaborated with N-acetyllactosamine (GalGlcNAc, green), sialic acid (purple), or other monosaccharides. (b) Example of a mucin-type O-glycan, with the conserved α-linked GalNAc residue highlighted in red. This monosaccharide can also be elaborated to generate higher-order glycans; depicted is the sialyl Lewis X antigen (blue). (c) Example of the O-GlcNAc modification, characterized by a single β-linked GlcNAc monosaccharide.

Figure 2

Schematic of the bottom-up shotgun proteomics method. (a) A mixture of proteins is subjected to proteolytic digestion, producing (b) a mixture of peptides. (c) The peptides are separated using LC, and (d) full-scan mass spectra are collected at regular intervals. (e) Typically, ions are selected from each full scan for fragmentation (i.e., tandem MS) in a data-dependent fashion (i.e., based on ion abundance). (f) These tandem mass spectra are analyzed using a database search to generate peptide and protein identifications.

Figure 3

Bioorthogonal reactions with aldehyde-, ketone-, azide-, or alkyne-functionalized monosaccharides. These chemical handles can be introduced into glycans through chemical, metabolic, or enzymatic methods. After an enrichment probe (red star) has been appended to glycoproteins or glycopeptides, these compounds can be enriched from complex mixtures and identified by MS. (a) Aldehydes and ketones are condensed with amine nucleophiles, including hydrazide compounds (top) and aminooxy compounds (bottom), to form stable hydrazone or oxime linkages, respectively. (b) Azides undergo the Staudinger ligation (top) with triarylphosphines or a [3 + 2] cycloaddition with terminal alkynes through the Cu-catalyzed azide–alkyne cycloaddition (CuAAC, middle) or strained alkynes in the absence of Cu (copper-free click chemistry, bottom). (c) Alkynes undergo a [3 + 2] cycloaddition with azides through the CuAAC reaction.

Figure 4

Enrichment of glycoproteins using carbonyl chemistry. (a) Schematic of hydrazide capture technology. (1) Glycoproteins are subjected to mild oxidants, most commonly sodium periodate, which oxidatively cleaves the cis diols (highlighted in a gray box) in glycans into an aldehyde group (highlighted in purple). (2) The aldehyde is condensed with a hydrazide-functionalized solid support to covalently capture and enrich glycoproteins. (3) The protein is released by deglycosylation from the solid support for identification by LC-MS. (b,c) Hydrazide capture technology can also target specific monosaccharides, including (b) sialic acid using optimized oxidation conditions and (c) galactose using galactose oxidase.

Figure 5

Enrichment of glycoproteins using metabolic labeling followed by bioorthogonal ligation. (a) Schematic of metabolic labeling to introduce an unnatural monosaccharide carrying a chemical reporter (i.e., azide or alkyne; purple triangle) directly into glycans. (1) Unnatural monosaccharides (blue hexagons) are accepted by the cell’s natural machinery and incorporated into the cell surface, secreted, and/or attached to intracellular glycoproteins (orange ovals). The natural monosaccharides are depicted as gray hexagons. (2) A bioorthogonal chemical reaction is performed to label glycoproteins with a probe molecule (red star). (b) Chemical structures of natural monosaccharides (left column) and their azide and alkyne analogues (functional groups highlighted in purple) that have been used for metabolic labeling and enrichment of glycoproteins or glycopeptides. Although the unnatural monosaccharides are depicted in a peracetylated form, after they have entered cells by passive diffusion, cytosolic esterases will cleave the acetyl groups.

Figure 6

Enrichment of glycoproteins using chemoenzymatic tagging followed by bioorthogonal ligation. (a) Schematic showing how chemoenzymatic tagging can be used to introduce a chemical reporter directly onto glycoproteins in vitro. (1) UDP-galactose analogues are appended onto O-GlcNAc conjugates using a mutant galactosyl transferase (e.g., Y298L GalT). (2) A bioorthogonal chemical reaction is performed to label glycans with a probe molecule (red star). (b) Chemical structures of Y298L GalT substrates, including UDP-galactose (UDP-Gal) and two analogues, UDP-2-keto-Gal and UDP-GalNAz. (c) Structure of an azide-functionalized trisaccharide used to identify glycoprotein targets of a viral trans-sialidase.

Figure 7

Examples of enzymatic and chemical deglycosylation. (a) N-glycans can be enzymatically removed using N-glycosidases or endoglycosidases, most commonly PNGase F (top), which converts asparagine into aspartic acid after cleaving off the entire glycan. If performed in the presence of ¹⁸O-labeled water, the heavy oxygen (highlighted in red) will mark the site of glycosylation. Alternatively, Endo H (bottom) cleaves between the two innermost GlcNAc resides, leaving a single peptide-bound GlcNAc residue to mark the site of glycosylation. (b) O-glycans can be chemically deglycosylated. Illustrated is a schematic of the β-elimination followed by Michael addition with dithiothreitol (DTT) (BEMAD) method. (1) β-elimination, most commonly with NaOH, converts glycosylated serine or threonine residues into dehydroalanine or dehydrobutyric acid, respectively. (2) Michael addition with amine or thiol nucleophiles, most commonly DTT, covalently marks the site of glycosylation.

Figure 8

Chemically directed glycoproteomics using chemical or metabolic methods to embed a dibromide or dibromide-like motif into glycans. (a) Two bromine atoms perturb a peptide’s isotopic envelope in a predictable way. Illustrated are simulated isotopic envelopes of an AVERAGEPEPTIDE, a dibromide (Br₂), and a dibromide-labeled AVERAGEPEPTIDE, the last of which can be computationally recognized with high sensitivity and specificity in complex mass spectra. (b) Chemical structure and mass spectrum of an iodoacetamide-derivatized dibromide tag that was used to chemically modify peptides by alkylating cysteine residues. (c) Chemical structure of a cleavable dibromide enrichment tag for isotope-targeted glycoproteomics. The silane linkage can be cleaved using 2% formic acid, producing a low-molecular-weight tag that remains on the labeled glycopeptide. (d) A GlcNAc isomix was used to metabolically install a dibromide-like motif directly into glycoproteins for high-confidence glycosite mapping without chemical tagging. The GlcNAc isomix consisted of three components and was designed to mimic the 1:2:1 peak intensity distribution of the dibromide triplet pattern using heavy nitrogen and carbon instead of bromine atoms.

Figure 9

Chemical tools for covalently capturing glycoproteins and glycan-binding proteins. (a) Chemical structure of the trifunctional chemoproteomics (TRICEPS) probe that was used to capture cell-surface glycoprotein receptors for a specific ligand. (b) Chemical structures of sialic acid derivatives bearing an aryl azide photo-activatable cross-linking functional group. (c) Chemical structures of sialic acid derivatives bearing a diazirine photoactivatable cross-linking functional group. (d) Chemical structures of GlcNAc derivatives bearing a diazirine photoactivatable cross-linking functional group.