Purpose: We have developed Integrated GlycoProteome Analyzer (I-GPA)[1] for high throughput analysis of N-glycoproteome, which combines methods for tandem mass spectrometry with a database search and algorithmic suite. We created novel scoring algorithms with calculation of false discovery rate (FDR) and label-free quantification method using the combined intensities of top three isotope peaks at three highest MS spectral points (3TIQ).

Methods: The resultant data were then computationally analyzed using specific algorithms within the I-GPA suite: glycopeptides were identified against the GPA database (id-GPA), quantitated (q-GPA) using the 3TIQ, and finally compared between multiple samples (c-GPA). In I-GPA, scoring entailed three steps: 1) Selection of N-glycopeptide from 15 glycan-specific oxonium ions using HCD-MS/MS spectra; (M-score); 2) Selection of candidates by matching the isotope pattern to intact N-glycopeptides in the GPA-DB (S-score); and 3) Identification of N-glycopeptide from CID and HCD-MS/MS fragment ions (Y-score) with (FDR) < 1%.

Results: Our method identified 123 N-glycoproteins present in plasma at concentration ranges over five orders of magnitude from highly abundant proteins such as immunoglobulin G (IgG, ~1 mg/ml) to low-abundance proteins such as AFP (~10 ng/ml).