Characterization of inter-alpha-trypsin inhibitor heavy chain h4 glycoprotein microheterogeneity using glycopeptide cid tandem mass spectra and glycan database searching

Collision-induced dissociation tandem mass spectrometry (CID MS/MS) is an effective tool for site-specific analysis of glycopeptides and a powerful means of studying glycoprotein microheterogeneity. However, manual interpretation of CID MS/MS datasets can be time-consuming. We propose a strategy for automated data analysis that matches N-glycopeptide spectra to peptide-glycan pairs by searching glycan structure databases. We test this strategy on serial proteolytic digests of Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), a liver-secreted glycoprotein with biomarker potential. We perform glycosidase-assisted LC-MS/MS analyses of HILIC-enriched glycopeptides from recombinant and serum-derived ITIH4 followed by automated glycopeptide analysis. The automated analysis matched 193 recombinant ITIH4 N-glycopeptide spectra representing 57 distinct peptide-glycan pairs with a false discovery rate (FDR) of 5.4%. Next, we applied our search strategy to the analysis of O-glycopeptides, and matched 26 O-glycopeptide spectra representing six distinct peptide-glycan pairs with an FDR of 1.1%. Recombinant and serum-derived ITIH4 primarily contain N-glycopeptides with complex glycan structures; recombinant ITIH4 glycoforms are less sialylated and more fucosylated compared to serum. O-glycopeptides carry core-1 O-glycans; however, sites of attachment differ between the recombinant and serum-derived material. The newly identified O-glycoforms are isoform-specific. Analysis of detached, permethylated N- and O-glycans confirms the glycopeptide observations. We also confirm that the four ITIH4 N-X-S/T sequons (N81, N207, N517 and N577) are glycosylated by treating ITIH4 tryptic/GluC and tryptic/chymotryptic glycopeptides with PNGaseF in the presence of 18O water. A fifth glycosylation site was discovered at N274 with the rare NVV motif. While occupancy of N-X-S/T sequons of ITIH4 is high (>80%) in both recombinant and serum-derived protein, glycan microheterogeneity of ITIH4 from the two sources differs. Our results demonstrate that exoglycosidase-assisted LC-MS/MS of glycopeptides combined with automated analysis using glycan structure database searching is a powerful workflow for studying glycosylation microheterogeneity. Using this workflow we observe site-specific differences in ITIH4 glycosylation, including differences in sialylation, branching, and core- and outer arm fucosylation between two biologically distinct sources of ITIH4. This study is significant because this automated workflow can be used to evaluate differences in protein glycosylation - in the future, this workflow can be extended to investigate disease-associated changes in glycosylation.