Capture and dimethyl labeling of glycopeptides on hydrazide beads for quantitative glycoproteomics analysis

Incorporation of isotopic tag onto peptides via chemical labeling is a popular approach for quantitative proteomics. Chemical labeling via solution based methods usually lead to a tedious process and sample loss because several sample preparation steps including buffer exchange and desalting are performed. In this study, a solid phase based labeling approach by integration of glycopeptide enrichment and stable isotope labeling on hydrazide beads was developed for relative quantification of protein glycosylation, by which enrichment, washing, labeling, and release of the glycopeptides were all performed on the hydrazide beads sequentially. This approach was proved to be accurate in quantitative glycoproteome analysis and have good linearity range with 2 orders of magnitude for quantification of glycopeptides. Compared with dimethyl labeling conventionally performed in solution, the developed approach has better enrichment recovery (10-330% improvement) and high detection sensitivity in which 42% of annotated glycosites (vs 26%) still can be quantified using only 10 μg of four standard glycoprotein mixtures and 400 μg of bovine serum album interference as starting sample. The applicability of the approach for quantitative glycopeptide profiling was also explored by differential analysis of glycoproteome between human normal serum and liver cancer serum.