Journal
JOURNAL OF PROTEOME RESEARCH
Volume 6, Issue 6, Pages 2323-2330Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr070112q
Keywords
nonenzymatic glycation; boronate affinity enrichment; electron-transfer dissociation; collision-induced dissociation; post-translational modification; liquid chromatography; mass spectrometry
Categories
Funding
- NATIONAL CENTER FOR RESEARCH RESOURCES [P41RR018522] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [R33DK071283, R37DK019971, R21DK071283, R01DK019971] Funding Source: NIH RePORTER
- NCRR NIH HHS [RR018522, P41 RR018522, P41 RR018522-05] Funding Source: Medline
- NIDDK NIH HHS [R33 DK071283-03, 5R21DK071283, R33 DK071283, R37 DK019971, R21 DK071283, DK-19971] Funding Source: Medline
- Wellcome Trust [06564] Funding Source: Medline
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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 posttranslational 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.
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