Analysis of amadori peptides enriched by boronic acid affinity chromatography.
Glycation of peptides and proteins by D-glucose is a universal, nonenzymatic reaction with important implications for the pathogenesis and diagnosis of many diseases, including diabetes mellitus. Whereas some modification sites have been identified in serum albumin and hemoglobin, a general approach to map glycation sites for nonabundant proteins present in complex mixtures, such as serum, is still missing. Here, we describe a universal enrichment procedure for glycated peptides using boronic acid affinity chromatography in the first dimension followed by reversed-phase chromatography, coupled either online to electrospray ionization mass spectrometry (ESI-MS) or offline to matrix-assisted laser desorption/ionization (MALDI) MS. This two-dimensional approach was optimized for high recoveries and low cross reactivities. For bovine serum albumin, a total of 31 Amadori peptides were identified in a tryptic digest corresponding to 26 different glycation sites.
Frolov
Andrej
A
Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Faculty of Chemistry and Mineralogy, Leipzig University, Leipzig, Germany.
Hoffmann
Ralf
R
D001897:Boronic Acids; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry*
D001897:Boronic Acids; D010455:Peptides; D012710:Serum Albumin, Bovine