Detergents assisted glycoprotein capture
A detergents assisted glycoprotein capture method was developed to increase the coverage of N-glycoproteome of various samples.Application of this approach in the larger scale N-glycoproteomics analysis of the HEK 293 cell membrane led to the identification of 2253 unique N-glycosites from 953 proteins. Application of this approach to human serum resulted in the identification of 850 N-glycosylation sites without any immuno-depletion and fractionation.
Sample Processing Protocol
Glycoproteins were captured on the hydrazide beads followed by trypsin digestion and PNGase F deglycosylation. Isolated glycopeptides were analyzed by LC-MS/MS wiht Orbitrap Velos mass spectrometry
Data Processing Protocol
Data files for human samples were processed with MaxQuant (126.96.36.199). The resulting precursor masses were matched to the Uniprot human database (downloaded on August 27th, 2015 with 73018 entries), complemented with the standard MaxQuant contaminant database. For MaxQuant, Andromeda  was used to search the acquired CID spectra against the in-silico fragmentation spectra of the matching database entries. Mass tolerances were 6 ppm and 0.5 Da for the precursor and fragment, respectively. Enzyme specificity was set as KR/P, and a maximum of two missed cleavages was allowed. Cysteine residue was set as a static modification of 57.0215 Da, and the methionine oxidation and asparagine deamination was set as a variable modification of 15.9949 Da and 0.9840 Da, respectively. The false discovery rate cut-off for both peptides and proteins was set at 1%. Peptides with deamidated asparagine within N-X-S/T motif(X could be any amino acid except proline) were considered as glycopeptides. The results from MaxQuant were directly exported to an Excel file.
Rui Chen, University of Ottawa
Daniel Figeys, Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology and Department of Chemistry and Biomolecular Sciences, University of Ottawa ( lab head )
Chen R, Zou H, Figeys D. Detergent-Assisted Glycoprotein Capture: A Versatile Tool for In-Depth N-Glycoproteome Analysis. J Proteome Res. 2016 Jun 3;15(6):2080-6 PubMed: 27147131