Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry.
Nonenzymatic 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.
Zhang
Qibin
Q
Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Tang
Ning
N
Brock
Jonathan W C
JW
Mottaz
Heather M
HM
Ames
Jennifer M
JM
Baynes
John W
JW
Smith
Richard D
RD
Metz
Thomas O
TO
D000595:Amino Acid Sequence; D001897:Boronic Acids / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D004579:Electron Transport; D005947:Glucose / Q000032:analysis*; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods*
D001897:Boronic Acids; D006020:Glycopeptides; D005947:Glucose