Enrichment and Analysis of Non-enzymatically Glycated Peptides: Boronate Affinity Chromatography Coupled with Electron Transfer Dissociation Mass Spectrometry

• Published in: Journal of proteome research Vol. 6; no. 6; pp. 2323 - 2330
• Main Authors: Zhang, Qibin, Tang, Ning, Brock, Jonathan W. C., Mottaz, Heather M., Ames, Jennifer M., Baynes, John W., Smith, Richard D., Metz, Thomas O.
• Format: Journal Article
• Language: English
• Published: 09-05-2007
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr070112q

Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated
in vitro
. Enriched peptides were subsequently analyzed by alternating electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.