Comprehensive Characterization of Glycosylation and Hydroxylation of Basement Membrane Collagen IV by High‐Resolution Mass Spectrometry

Collagen IV is the main structural protein that provides a scaffold for assembly of basement membrane proteins. Posttranslational modifications such as hydroxylation of proline and lysine and glycosylation of lysine are essential for the functioning of collagen IV triple‐helical molecules. These modifications are highly abundant posing a difficult challenge for in‐depth characterization of collagen IV using conventional proteomics approaches. Herein, we implemented an integrated pipeline combining high‐resolution mass spectrometry with different fragmentation techniques and an optimized bioinformatics workflow to study posttranslational modifications in mouse collagen IV. We achieved 82% sequence coverage for the alpha1 chain, mapping 39 glycosylated hydroxylysine, 153 4‐hydroxyproline and 7 3‐hydroxyproline residues. Further, we employed our pipeline to map the modifications on human collagen IV and achieved 85% sequence coverage for the alpha1 chain, mapping 35 glycosylated hydroxylysine, 161 4‐hydroxyproline and 14 3‐hydroxyproline residues. Although lysine glycosylation heterogeneity was observed in both mouse and human, 21 conserved sites were identified. Likewise, 5 3‐hydroxyproline residues were conserved between mouse and human, suggesting that these modification sites are important for collagen IV function. Collectively, these are the first comprehensive maps of hydroxylation and glycosylation sites in collagen IV, which lay the foundation for dissecting the key role of these modifications in health and disease. Support or Funding Information This work was supported in part by NIH R01 grants DK099467 and DK065138.PTM map of full length human col4a1 chain