Proteomic profiling of nonenzymatically glycated proteins in human plasma and erythrocyte membranes

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. In this work, we report the first proteomics-based characterization of nonenzymatically glycated proteins in human plasma and erythrocyte membranes from individuals with normal glucose tolerance, impaired glucose tolerance, and type 2 diabetes mellitus. Phenylboronate affinity chromatography was used to enrich glycated proteins and glycated tryptic peptides from both human plasma and erythrocyte membranes. The enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation-tandem mass spectrometry, resulting in the confident identification of 76 and 31 proteins from human plasma and erythrocyte membranes, respectively. Although most of the glycated proteins could be identified in samples from individuals with normal glucose tolerance, slightly higher numbers of glycated proteins and more glycation sites were identified in samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus.

Figure 1

Flowchart depicting glycated peptide identification from blood plasma and erythrocyte membranes of NGT, IGT, and T2DM individuals. Pooled samples from each patient group were processed and analyzed individually as outlined in the chart.

Figure 2

(a) Boronate affinity chromatograms of whole plasma from different volunteer groups. The inset is a zoomed view of binding fractions, which are labeled with the respective sample name. Peaks at approximately 2 min are nonglycated proteins, and glycated proteins elute near 15 min. (b) Boronate affinity chromatograms showing the enrichment of glycated peptides from tryptic digests of erythrocyte membranes. The glycated peptides elute slightly after 15 min, and the nonglycated peptides are washed out near 2 min. The chromatograms in both (a) and (b) were normalized based on the most intense peak in each trace, that is, the peak corresponding to the flow-through fraction.