Mass Spectrometric Analysis, Automated Identification and Complete Annotation of O-Linked Glycopeptides

Abstract

Complex mixtures containing O-linked glycopeptides bearing SA 1-0 GalGalNAc structures, or single GalNAc units were subjected to CID and ETD analysis on a linear ion trap -Orbitrap mass spectrometer and the resulting data was analyzed using the Protein Prospector software. An overview of the structural information provided by the different fragmentation techniques, as well as their limitations is presented. We illustrate the importance of the complementary information in the MS survey scans as well as the different MS/MS techniques. We also present some unique features offered by Protein Prospector that are advantageous in glycopeptide analysis: i) considering a modification that will produce a neutral loss, without "labeling" the original modification site; ii) merging CID and ETD search results; iii) permitting the comparison of different modification site-assignments. Although these data were obtained from secreted glycopeptides, the observations and conclusions are also valid for the intracellular regulatory O-GlcNAc modification.

N- and O-Glycosylation in the Murine Synaptosome

“…Unfortunately, the sugars are usually completely eliminated via gas-phase rearrangement in such a fashion that the site(s) of modification cannot be determined when there are multiple possibilities (20,23). However, the mass spectrometric and proteomic search engines Protein Prospector (30) and ByOnic (31) can handle neutral losses and thus, identify O-glycopeptides. Both software tools are also able to assign the site(s) of modification(s) in those cases where fragment ions that retain the sugar are detected.…”

Enhanced Mass Spectrometric Mapping of the Human GalNAc-type O-Glycoproteome with SimpleCells

“…However, even electron capture dissociation/ETD-MS/MS has its limitations, with heavy glycosylation and/or a lack of abundant precursors with required multiple charge states (e.g. ϩ3, ϩ4, and ϩ5) being two typical reasons for failure to observe sufficient fragmentation for site-specific sequence analysis (5,12,13). The limitations can become acute when there are many adjacent Ser and Thr residues with potential for glycosylation and many Pro residues interrupting the continuity of c/z fragmentation, both situations being frequent in O-glycosylated peptides.…”

“…One approach utilized is the exo-glycosidase treatment prior to LC-MS/MS analysis (12,13). We have recently introduced a genetic engineering approach to transform the entire GalNActype O-glycoproteome of a cell into homogeneous simple truncated O-glycans with GalNAc (Tn antigen) and/or NeuAc␣2-6GalNAc (STn antigen) structures (5).…”

Protein O-glycosylation analysis

“…ESI-ion trap-CID tandem mass spectrometry (MS/MS) of glycopeptides results mainly in fragments arising from glycosidic bond cleavage, allowing the determination of the glycan structure with the peptide moieties staying largely intact (Perdivara et al , 2009 ;Darula et al , 2010 ;Zauner et al , 2010 ). When multiple sugar units are attached to a peptide, ion series (appearing often in different charge states) are measured due to sequential carbohydrate losses (Darula et al , 2010 ). Under ion trap-CID conditions, O-linked glycopeptides often undergo gas-phase deglycosylation that completely eliminates the carbohydrate, leaving the unmodifi ed and thus ' unlabeled ' Ser or Thr residue(s) behind Darula et al , 2011 ).…”

“…Electron transfer triggers a fragmentation almost exclusively along the peptide backbone, leaving the glycan mass intact and therefore allows the identifi cation of the peptide sequence. A good-quality ETD spectrum permits the identifi cation of the peptide sequence, the glycan mass, and the unambiguous assignment of the modifi cation site ( Figure 5 ) (Darula et al , 2010 ).…”