Exploration of quantitative site‐specific serum O‐glycoproteomics with isobaric labeling for the discovery of putative O‐glycoprotein biomarkers

Abstract Purpose Exploration study of site‐specific isobaric‐TMT‐labeling quantitative serum O‐glycoproteomics for the discovery of putative O‐glycoprotein cancer biomarkers. Experimental design Sera of 10 breast cancer patients was used as the exploration cohort. More abundant N‐glycosylation was first removed with PNGase F. After tryptic digestion of de‐N‐glycosylated serum proteome, the TMT‐labeled O‐glycopeptides mixture was prepared and analyzed with RPLC‐MS/MS. Site‐specific qualitative and quantitative database search of O‐glycopeptides was carried out with pGlyco 3.0. The same raw datasets were also searched with intact N‐glycopeptide search engine GPSeeker to exclude possible interference of N‐glycosylation. The final IDs were checked manually with GlcNAc‐containing glycosite‐determining fragment ions for confirmation. Results With the control of spectrum‐level FDR ≤ 1% and manual validation, 299 O‐glycopeptides corresponding to 83 O‐glycosites and 66 O‐glycoproteins were identified, and 13 O‐glycopeptides were found differentially expressed. Most interestingly, differential O‐glycosylation was observed for IgG1 and IgG3, which is an interesting putative biomarker panel. Conclusion and clinical relevance Isobaric‐labeling site‐specific quantitative O‐glycoproteomics is currently a state‐of‐the‐art instrumental platform for discovery of putative seral cancer biomarkers. Differential seral O‐glycosylation was observed in the IgG1 and IgG3.