Abstract
Nonenzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. Herein we report improved methods for the enrichment and analysis of glycated peptides using boronate affinity chromatography and electron-transfer dissociation mass spectrometry, respectively. The enrichment of glycated peptides was improved by replacing an off-line desalting step with an online wash of column-bound glycated peptides using 50 mM ammonium acetate, followed by elution with 100 mM acetic acid. The analysis of glycated peptides by MS/MS was improved by considering only higher charged (> or = 3) precursor ions during data-dependent acquisition, which increased the number of glycated peptide identifications. Similarly, the use of supplemental collisional activation after electron transfer (ETcaD) resulted in more glycated peptide identifications when the MS survey scan was acquired with enhanced resolution. Acquiring ETD-MS/MS data at a normal MS survey scan rate, in conjunction with the rejection of both 1+ and 2+ precursor ions, increased the number of identified glycated peptides relative to ETcaD or the enhanced MS survey scan rate. Finally, an evaluation of trypsin, Arg-C, and Lys-C showed that tryptic digestion of glycated proteins was comparable to digestion with Lys-C and that both were better than Arg-C in terms of the number of glycated peptides and corresponding glycated proteins identified by LC-MS/MS.