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a Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Faculty of Chemistry and Mineralogy, Leipzig University, Leipzig, Germany
Key Words: m-aminophenylboronic acid • ESI • glycation • MALDI • mass spectrometry
Address for correspondence: Professor Dr. Ralf Hoffmann, Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Faculty of Chemistry and Mineralogy, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany. Voice: +49-0-341 9731331; fax: +49-0-341 9731330. Hoffmann{at}chemie.uni-leipzig.de
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.
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