Combining High-Energy C-Trap Dissociation and Electron Transfer Dissociation for Protein O-GlcNAc Modification Site Assignment

Abstract

Mass spectrometry-based studies of proteins that are post-translationally modified by O-linked β-N-acetylglucosamine (O-GlcNAc) are challenged in effectively identifying the sites of modification while simultaneously sequencing the peptides. Here we tested the hypothesis that a combination of high-energy C-trap dissociation (HCD) and electron transfer dissociation (ETD) could specifically target the O-GlcNAc modified peptides and elucidate the amino acid sequence while preserving the attached GlcNAc residue for accurate site assignment. By taking advantage of the recently characterized O-GlcNAc-specific IgG monoclonal antibodies and the combination of HCD and ETD fragmentation techniques, O-GlcNAc modified proteins were enriched from HEK293T cells and subsequently characterized using the LTQ Orbitrap Velos™ ETD (Thermo Fisher Scientific) mass spectrometer. In our dataset, 83 sites of O-GlcNAc modification are reported with high confidence confirming that the HCD/ETD combined approach is amenable to the detection and site assignment of O-GlcNAc modified peptides. Realizing HCD triggered ETD fragmentation on a linear ion trap/Orbitrap platform for more in-depth analysis and application of this technique to other post-translationally modified proteins are currently underway. Furthermore, this report illustrates that the O-GlcNAc transferase appears to demonstrate promiscuity with regards to the hydroxyl-containing amino acid modified in short stretches of primary sequence of the glycosylated polypeptides.

Tandem mass spectrometry identifies many mouse brain O -GlcNAcylated proteins including EGF domain-specific O -GlcNAc transferase targets

“…S4). The results of this evaluation indicate that combining tandem MS methods to obtain both site-specific (ETD) and diagnostic (HCD or CID) information can increase the confidence of O-GlcNAcylated peptide identification and site localization, consistent with previous studies (10,13). In addition, alternating CID-ETD analyses have been advocated as a superior method for identification of labile phosphopeptides (24).…”

“…Of the three fragmentation methods, only ETD provided information regarding the location of the modification sites. The basic tag (AMT-GalNAz) increased peptide fragment efficiency of both ETD and HCD compared with that for untagged, native O-GlcNAc peptides (13). Overall, ETD generated the greatest number of O-GlcNAcylated peptides, followed by HCD and CID (Fig.…”

“…Additionally, identifying specific Ser and Thr residues that are O-GlcNAcylated is difficult, because O-GlcNAc is readily lost as an oxonium ion during collision-induced dissociation (CID), a widely used fragmentation mode for peptide sequencing by MS (12). Alternative higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) MS methods have improved detection or have facilitated site-specific identification, but challenges remain (13).…”

“…Immunoprecipitation, using O-GlcNAc-specific monoclonal antibodies (13,16), identified 83 O-GlcNAcylated sites from a HEK293T cell extract (13). A recent metabolic labeling study that used alkyne-modified GlcNAc incorporated into OGT substrates and Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC) to a chemically cleavable biotin-azide probe, enabled identification of 374 putative O-GlcNAc modified proteins, but yielded no information on sites of O-GlcNAc modification (17).…”

“…A recent metabolic labeling study that used alkyne-modified GlcNAc incorporated into OGT substrates and Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC) to a chemically cleavable biotin-azide probe, enabled identification of 374 putative O-GlcNAc modified proteins, but yielded no information on sites of O-GlcNAc modification (17). In addition to limited coverage of O-GlcNAcylation sites, a drawback to using these approaches is that they typically require milligram quantities of protein (4,10,13,14,16) or are limited to cultured cells (17), which makes them generally ill-suited for clinically derived tissue samples often available in small amounts.…”