Comprehensive Identification of Glycated Peptides and Their Glycation Motifs in Plasma and Erythrocytes of Control and Diabetic Subjects

Abstract

Non-enzymatic glycation of proteins sets the stage for formation of advanced glycation end-products and development of chronic complications of diabetes. In this report, we extended our previous methods on proteomics analysis of glycated proteins to comprehensively identify glycated proteins in control and diabetic human plasma and erythrocytes. Using immunodepletion, enrichment, and fractionation strategies, we identified 7749 unique glycated peptides, corresponding to 3742 unique glycated proteins. Semi-quantitative comparisons showed that glycation levels of a number of proteins were significantly increased in diabetes and that erythrocyte proteins were more extensively glycated than plasma proteins. A glycation motif analysis revealed that some amino acids were favored more than others in the protein primary structures in the vicinity of the glycation sites in both sample types. The glycated peptides and corresponding proteins reported here provide a foundation for potential identification of novel markers for diabetes, hyperglycemia, and diabetic complications in future studies.

Plasma Proteins Modified by Advanced Glycation End Products (AGEs) Reveal Site-specific Susceptibilities to Glycemic Control in Patients with Type 2 Diabetes

“…Enzymatic acetylation (and formylation) are regulatory functions in nucleus, liver cell cytosols, and mitochondria (54), but such activities have not been reported to our knowledge in plasma. In contrast, all HSA and apolipoprotein A-II sites identified here as modified by amideAGEs are known to be glycated in plasma from patients with T2DM (55,56), indicating that these sites are susceptible to glycation finally leading to AGEs.…”

“…This might be attributed to nearby residues or more generally the local environment, i.e. reagent accessibility and nearby functional groups affecting the pK a of the Lys residue or acting as local acid/base catalysts in glycation reactions (56,58,59). Furthermore, the differences were relatively moderate (3-6-fold compared with the other cohorts) and were detected only in one peptide per modification type, which suggests that the changes would not be detectable with an analytical approach relying on protein hydrolysis.…”

Oxidative degradation of N ε-fructosylamine-substituted peptides in heated aqueous systems

“…Previous studies indicate different lysine reactivities in glycation, depending on neighboring residues affecting the pK a of lysine, or acting in local catalysis either directly or via their ability to bind catalytically active phosphate (Zhang et al 2011). For example, aspartatic and glutamatic acids are overrepresented near 'reactive' lysines (Watkins et al 1985;Zhang et al 2011).…”

“…For example, aspartatic and glutamatic acids are overrepresented near 'reactive' lysines (Watkins et al 1985;Zhang et al 2011). Here, aspartic or glutamic acid did not majorly affect Amadori degradation kinetics, but favored carboxymethylation and deglycation (Fig.…”

Development of Diagnostic Fragment Ion Library for Glycated Peptides of Human Serum Albumin: Targeted Quantification in Prediabetic, Diabetic, and Microalbuminuria Plasma by Parallel Reaction Monitoring, SWATH, and MSE

“…Therefore, to achieve efficient identification, enrichment of glycated peptides using boronate affinity chromatography (BAC) was adopted prior to MS analysis (25). Further, by using a combination of immunodepletion, enrichment and fractionation strategies, a total of 7,749 unique glycated peptides corresponding to 1,095 native human plasma proteins, 1,592 in vitro glycated human plasma proteins, and 1,664 erythrocyte proteins were identified (26). In these lines, we have previously reported a database search approach for the identification of glycated peptide in a crude or nonenriched sample by untargeted MS/MS or data-independent workflow (27).…”