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.

Advances in plasma proteomics: Moving from technology to precision medicine

“…The latter group was prepared by mixing equal volume of plasma from ten healthy volunteers, including IgG1, IgG3, CO4, HP, ANT3, IC1 and FINC. [6] In addition to profiling changes in disease-related protein levels, there is growing interest in studying adaptive immunity to evaluate the effect of auto-reactive antibodies. [7] These circulating autoantibodies can provide opportunities for disease early risk assessment, diagnosis, and prediction of therapeutic responses.…”

“…The latter group was prepared by mixing equal volume of plasma from ten healthy volunteers, including IgG1, IgG3, CO4, HP, ANT3, IC1 and FINC. [ 6 ]…”

Analysis of N‐ and O‐linked site‐specific glycosylation by ion mobility mass spectrometry: State of the art and future directions

“…Subtle structural differences in glycan moieties, constitute a major problem in MS analysis of glycopeptides [92, 93]. Isomeric interferences occur when multiple isomeric glycoforms have the m/z ratio, resulting from variations in glycan stereochemistry, attachment sites, glycan linkages, or branching patterns.…”