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Investigation of Sialylation Aberration in N-linked Glycopeptides by Lectin and Tandem Labeling (LTL) Quantitative Proteomics

Investigation of Sialylation Aberration in N-linked Glycopeptides by Lectin and Tandem Labeling (LTL) Quantitative Proteomics

Vivekananda Shetty, Zacharie Nickens, Punit Shah, Gomathinayagam Sinnathamby, O John Semmes, Ramila Philip

Anal Chem. 2010 Nov 15;82(22):9201-10.

PMID: 20923142

Abstract:

The accuracy in quantitative analysis of N-linked glycopeptides and glycosylation site mapping in cancer is critical to the fundamental question of whether the aberration is due to changes in the total concentration of glycoproteins or variations in the type of glycosylation of proteins. Toward this goal, we developed a lectin-directed tandem labeling (LTL) quantitative proteomics strategy in which we enriched sialylated glycopeptides by SNA, labeled them at the N-terminus by acetic anhydride ((1)H(6)/(2)D(6)) reagents, enzymatically deglycosylated the differentially labeled peptides in the presence of heavy water (H(2)(18)O), and performed LC/MS/MS analysis to identify glycopeptides. We successfully used fetuin as a model protein to test the feasibility of this LTL strategy not only to find true positive glycosylation sites but also to obtain accurate quantitative results on the glycosylation changes. Further, we implemented this method to investigate the sialylation changes in prostate cancer serum samples as compared to healthy controls. Herein, we report a total of 45 sialylated glycopeptides and an increase of sialylation in most of the glycoproteins identified in prostate cancer serum samples. Further quantitation of nonglycosylated peptides revealed that sialylation is increased in most of the glycoproteins, whereas the protein concentrations remain unchanged. Thus, LTL quantitative technique is potentially an useful method for obtaining simultaneous unambiguous identification and reliable quantification of N-linked glycopeptides.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP285977788	Acetic anhydride-1,1′-13C2,d6		285977-78-8	Price

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