Click‐iG: Simultaneous Enrichment and Profiling of Intact N‐linked, O‐GalNAc, and O‐GlcNAcylated Glycopeptides

Abstract

Proteins are ubiquitously modified with glycans of varied chemical structures through distinct glycosidic linkages, making the landscape of protein glycosylation challenging to map. Profiling of intact glycopeptides with mass spectrometry (MS) has recently emerged as a powerful tool for revealing matched information of the glycosylation sites and attached glycans (i.e., intact glycosites), but is largely limited to individual glycosylation types. Herein, we describe Click‐iG, which integrates metabolic labeling of glycans with clickable unnatural sugars, an optimized MS method, and a tailored version of pGlyco3 software to enable simultaneous enrichment and profiling of three types of intact glycopeptides: N‐linked, mucin‐type O‐linked, and O‐GlcNAcylated glycopeptides. We demonstrate the utility of Click‐iG by the identification of thousands of intact glycosites in cell lines and living mice. From the mouse lung, heart, and spleen, a total of 2053 intact N‐glycosites, 262 intact O‐GalNAc glycosites, and 1947 O‐GlcNAcylation sites were identified. Click‐iG‐enabled comprehensive coverage of the protein glycosylation landscape lays the foundation for interrogating crosstalk between different glycosylation pathways.

O-GlcNAcylation Facilitates the Interaction between Keratin 18 and Isocitrate Dehydrogenases and Potentially Influencing Cholangiocarcinoma Progression

“…Given that disrupted O -GlcNAcylation influences the course of cancer progression in CCA, our subsequent investigation aimed to adopt Click-iG (Figure a), a chemical glycoproteomic platform, to systematically enrich, identify, and profile intact O -GlcNAcylated glycopeptides with matched information on glycosylation sites and glycan composition. , In contrast to established techniques, Click-iG provides extensive coverage of the protein glycosylation landscape, laying a foundation for investigating the interplay between various glycosylation pathways. In brief, we utilized 1,6-di- O -propionyl- N -azidoacetylgalactosamine (1,6-Pr 2 GalNAz), an optimized O -GlcNAc chemical reporter with minimal nonspecific modification and cytotoxicity (Figure S13), for metabolic incorporation into various glycoconjugates in HuCCT1 or HIBEpiC cells.…”

“…Therefore, we manually annotate the O-GlcNAcylation site with the aid of glycoprotein subcellular location and the presence of N-(4-aminomethyl)triazolylacetylglucosamine (GlcNAt). 22 Keratin 18 is Mainly O-GlcNAcylated at Ser 30. We noticed that Keratin 18 (K18) is on the list of up-regulated O-GlcNAcylated proteins in HuCCT1 cells compared to HIBEpiC control cells (Figure 2f, Tables S1 and S2).…”

“…LC–MS/MS analysis of the glycopeptides enriched above was performed as previously described . In brief, all samples were resuspended with 0.1% FA, analyzed by an Orbitrap Fusion Lumos Tribrid Mass Spectrometer with a Nanospray Flex ionization source (Thermo Fisher Scientific), and coupled online to a nanoflow LC system (EASY-nLC 1200, Thermo Fisher Scientific).…”

“…The raw data processing was performed using pGlyco3 () under the “HCD + EThcD” mode as previously described. , Briefly, the MS/MS spectrum were searched against the SwissPort Human sapiens proteome database downloaded from Uniprot () on 2016-11-4. The N -glycans and O -glycans were searched against the pGlyco- N -glycan mode and the pGlyco- O -glycan mode in the pGlyco3 software with modified glycan databases, respectively.…”

“…20,21 In addition, our collaborators recently reported the Click-iG strategy, which amalgamates the metabolic oligosaccharide engineering (MOE) of selected, clickable unnatural sugar probes for O-GlcNAcylated protein enrichment, and a customized pGlyco3 search engine for intact glycopeptide annotation (Figure 2a). 22,23 Click-iG outperforms pioneering workflows like IsoTag strategy 24 and software iterations such as MetaMorpheus O-Pair, 25 MSFragger-Glyco, 26 and StrucGP. 27 It allows for simultaneous and comprehensive profiling of multiple protein glycosylation types at the intact glycosite level in a single experiment, offering enhanced coverage of the protein glycosylation landscape.…”