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.

Synthesis of boronate-silica hybrid affinity monolith via a one-pot process for specific capture of glycoproteins at neutral conditions

“…The mechanism can be explained that the boronic acid can covalently bind with cis-diols to form five-or six-membered cyclic esters in a basic aqueous media; the reversible disassociation can be performed under acidic conditions. The excellent specificity makes boronate groups potential affinity ligands for specific recognition and isolation of cis-diol-containing compounds, including catechols, nucleotides, carbohydrates, nucleosides, glycopeptides, and glycoproteins [11][12][13][14]. In addition, the moderate acidic elution condition make boronate affinity well coupled with mass spectrometry for analysis of cis-diol biomolecules in -omics [14][15][16].…”

Glycation of antibodies: Modification, methods and potential effects on biological functions

“…However, the sensitivity of low abundance protein or peptide for ETD is low because of the low fragmentation efficiency. 53,54,[56][57][58] These issues often require extra experiments to confirm or detect glycated peptides. 31 Another way of improving the sensitivity and reducing the neutral loss of the glycated peptide is by using sodium borohydride or sodium cyanoborohydride reduction followed by trypsin cleavage and peptide map analysis with MS/MS detection.…”

“…The NBT reduction assay for measuring protein glycation was first introduced by Johnson, et al 61 in 1983. 58 NBT is reduced by the ketoamine form of glycated protein, which results in a change in absorbance at 525 nm. This method has been used to measure poly-lysine 62 and glycated albumin.…”

Advances in purification and separation of posttranslationally modified proteins

“…Another method for glycoproteome enrichment is based on the reversible reaction of boronic acid with 1,2-cis-diols [192]. These moieties are present in glycan components like mannose, galactose, glucose, and sialic acid, but are also preserved in glycated proteins, and boronate affinity chromatography is currently the only antibody-independent method for enriching glycated proteins [166,193]. Several derivatives of boronic acid have been introduced for this purpose, including bisboronic compounds, which can also be conjugated with a fluorescence tag and used for detecting protein glycosylation/glycation [194], and both sulfonyl-and sulfonamide-phenylboronic acid derivatives, which can be used at physiological pH.…”