Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)

Abstract

Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets.

Glycoproteogenomics characterizes the CD44 splicing code associated with bladder cancer invasion

“…Using HCD fragmentation, we could identify several product ions consistent with the presence of glycosylated moieties, together with some y- and b -type peptide fragments supporting these reporter glycopeptides, even though with some ambiguity in terms of glycosites assignment (Figure 4 C). The presence of GalNAc, the first sugar residue in O -glycans, could be further confirmed by several HexNAc cross ring fragments ( m/z 126.055, 168.066, 186.076, and 204.087; Figures 4 C-D and Figures S6 -7 and 11) and, in some cases, high 144.066/138.055 oxonium ions ratios 53 , 54 . We also explored HexNAc-triggered CID fragmentations for further glycopeptide validation, including the characterization of extended glycan chains ( Figure S6 ).…”

New insights into the type A glycan modification of Clostridioides difficile flagellar protein flagellin C by phosphoproteomics analysis

“…Originally, the type A structure was fully elucidated through NMR analysis, which showed that the HexNAc is a GlcNAc, and that the N -methylthreonine-phospho moiety is attached to the C3 position ( 19 ). It has recently been suggested that the ratio between the abundance of the HexNAc oxonium ions at m/z 138.055 and 144.065 in MS/MS spectra of O -glycosylated peptides can be used to discriminate between a GlcNAc and GalNAc ( 22 , 23 ). More specifically, a ratio >2 would be indicative of a GlcNAc, which is supported by the MS/MS spectra of type A-modified peptides presented in this article.…”

Revised Model for the Type A Glycan Biosynthetic Pathway in Clostridioides difficile Strain 630Δerm Based on Quantitative Proteomics of cd0241–cd0244 Mutant Strains

“…The ratio of the oxonium ions at m/z 138.055 and 144.066 is consistent with a GlcNAc. 24,25 Of note, a signal at m/z 126.055 was observed that corresponds to a GlcNAc oxonium ion, which is distinct from the 126C TMT reporter ion (m/z 126.128).…”