Analysis of Glycated Albumin by On-Line Two-Dimensional Liquid Chromatography Mass Spectrometry

Nonenzymatic reaction between glucose and proteins leads to glycated proteins. There is evidence to suggest that protein glycation may be involved in the long-term complications of diabetes mellitus. Knowledge about which proteins are glycated and the sites of glycation may provide new insight in understanding the mechanisms of diabetic complications. Therefore, the development of methods for comprehensive identification of glycated proteins is crucial to elucidate their biological effect. In this report, boronate affinity chromatography (BAC) was coupled on-line to LC-MS for comprehensive analysis of the in vitro glycated bovine serum albumin (BSA). Additionally, in comparison to analysis by conventional collision induced dissociation (CID) MS2 and neutral loss-triggered MS3 (NLMS3), multistage activation (MSA) significantly improved the number of identified glycated peptides. In summary, the established on-line BAC LC-MS approach identified 37 glycation sites in glycated BSA.