IQ-GPA (GlycoProteome Analyzer):
Automatic Identification and Quantification of Site-specific N- and O-Glycoproteins
We have developed MS based high-throughput method for the analysis of N- and O-glycoproteome, named IQ-GPA, which can automatically identify the N- and O-glycopeptides including glycan compositions as well as amino acid sequences, and quantify multiple samples in high speed. The efficiency of IQ-GPA was demonstrated by the analysis of standard alpha1-acid glycoprotein and human plasma.
The resultant data were then computationally analyzed using specific algorithms within the IQ-GPA suite: glycopeptides were identified against the GPA database by id-GPA, quantified by q-GPA, and finally compared between multiple samples by c-GPA.
In id-GPA, scoring entailed three steps:
1) Selection of N- and O-glycopeptide from 17 glycan-specific oxonium ions using HCD-MS/MS spectra; (M-score);
2) Selection of candidates by matching the isotope pattern to intact N-glycopeptides against the GPA-DB (S-score); and
3) Identification of N- and O-glycopeptide from CID and HCD-MS/MS fragment ions (Y-score) with estimated false discovery rate (FDR) < 1%.
For the automated label-free quantitation,
we used the combined intensities of top three isotope peaks at three highest MS spectral points (3TIQ).