Title : Site-specific glycan-peptide analysis for determination of N-glycoproteome heterogeneity
Abstract :
- A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture
- Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography-tandem mass spectrometry ( PGC-LC-MS/MS) and the data used to create an N-glycan modification database
- In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography ( Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC; pH 7.9)
- The resulting fractions were each separated into two equal aliquots
- The first set of aliquots were treated with peptide-N-glycosidase F ( PNGase F) to remove N-glycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2 .7) and identified by Mascot database search
- This enabled the creation of a glycopeptide-centric concatenated database for each fraction
- The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2 .7), employing fragmentation by CID and HCD
- The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the N-glycan modification database
- CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences
- This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins