@article {357, title = {Improved methods for the enrichment and analysis of glycated peptides.}, journal = {Anal Chem}, year = {2008}, 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.

}, keywords = {Blood Proteins, Boronic Acids, Chromatography, Affinity, Glycosylation, Humans, Peptide Fragments, Proteomics, Spectrometry, Mass, Electrospray Ionization}, doi = {10.1021/ac801704j}, author = {Zhang, Qibin and Schepmoes, Athena A and Brock, Jonathan W C and Wu, Si and Moore, Ronald J and Purvine, Samuel O and Baynes, John W and Smith, Richard D and Metz, Thomas O} } @article {365, title = {Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry.}, journal = {J Proteome Res}, year = {2007}, abstract = {

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.

}, keywords = {Amino Acid Sequence, Boronic Acids, Chromatography, Affinity, Electron Transport, Glucose, Glycopeptides, Glycosylation, Mass Spectrometry, Molecular Sequence Data, Proteomics}, doi = {10.1021/pr070112q}, author = {Zhang, Qibin and Tang, Ning and Brock, Jonathan W C and Mottaz, Heather M and Ames, Jennifer M and Baynes, John W and Smith, Richard D and Metz, Thomas O} }