Development of an automated high-throughput sample preparation protocol for LC-MS/MS analysis of glycated peptides.

Advanced glycation end products (AGEs) are known to play a leading part in the pathogenesis of human diseases, such as diabetes, Alzheimer's disease, lateral sclerosis, and atherosclerosis. It is for this reason that research on AGEs is crucial and large-scale studies are needed for the treatment of diseases. The aim of this study was to develop a reproducible method to analyze Amadori compounds using an automated enrichment protocol for high-throughput analysis of clinical samples. The developed method enabled the enrichment of Amadori compounds simultaneously from 96 samples, and it was applied to the discovery of biomarkers in AGEs related diseases. In this study, ten human serum samples were processed using automated filter-aided sample preparation (aFASP) in a 96-well filter plate, and the eluted peptide mixtures were enriched by Affinity Cellufine PB in a fritted 96-well filter plate using a liquid handling robotic system. The eluted glycated peptides were analyzed by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. A total of 982 unique glycated peptides, corresponding to 524 unique glycated proteins, were identified from the analysis of ten human samples. The advantages and potentials of the automated sample preparation system were demonstrated through label free quantification of the glycated peptides.