Analysis of protein glycation using phenylboronate acrylamide gel electrophoresis

Abstract

The incorporation of the specialized carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for SDS-PAGE analysis has been successful for the separation of carbohydrates and has here been adapted for the analysis of post-translationally modified proteins. While conventional SDS-PAGE analysis cannot distinguish between glycated and unglycated proteins, methacrylamido phenylboronate acrylamide gel electrophoresis (mP-AGE) in low loading shows dramatic retention of delta-gluconolactone modified proteins, while the mobility of the unmodified proteins remains unchanged. With gels containing 1% methacrylamido phenylboronate, mP-AGE analysis of gluconoylated recombinant protein Sbi results in the retention of the modified protein at a position expected for a protein that has quadrupled its expected molecular size. Subsequently, mP-AGE was tested on HSA, a protein that is known to undergo glycation under physiological conditions. mP-AGE could distinguish between various carbohydrate-protein adducts, using in vitro glycated HSA, and discriminate early from late glycation states of the protein. Enzymatically glycosylated proteins show no altered retention in the phenylboronate-incorporated gels, rendering this method highly selective for glycated proteins. We reveal that a trident interaction between phenylboronate and the Amadori cis 1,2 diol and amine group provides the molecular basis of this specificity. These results epitomize mP-AGE as an important new proteomics tool for the detection, separation, visualization and identification of protein glycation. This method will aid the design of inhibitors of unwanted carbohydrate modifications in recombinant protein production, ageing, diabetes, cardiovascular diseases and Alzheimer's disease.

Analysis of protein glycation using fluorescent phenylboronate gel electrophoresis

“…The Flu-PAGE results observed with saccharide-incubated serum samples are consistent with those found when analysing HSA glycation using mP-AGE19, showing strongest interactions between boronic acid and cis -1,2-diol-containing fructosamine adducts (resulting from glycation with glucose, mannose and galactose), stabilised by an electrostatic interaction between the protonated amino group and the negatively charged boronate moiety. When using Flu-PAGE in conjunction with mP-AGE analysis of the serum sample, the highest fluorescence intensity can be observed in the glycated HSA fraction that is retained in its electrophoretic mobility via interaction with the gel-incorporated MPBA (Supplementary Fig.…”

“…We first tested this technique on simple carbohydrates and showed that by using the same basic principle as BAC, exploiting the reversible covalent interaction between boronic acid and cis -diols1718, the MPBA-incorporated acrylamide gels enabled the improved separation of saccharides, and the differentiation between monosaccharides and dissacharides. Later we adapted this boronate-assisted saccharide electrophoresis (BASE) method to allow the separation of glycated from non-glycated proteins by incorporating MPBA into polyacrylamide gels for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis1920. This method, coined mP-AGE, showed that under electrophoresis conditions the polyacrylamide incorporated MPBA is specific for fructosamine modified proteins, via interactions with the cis -1,2-diol-containing fructosamine adducts and by further stabilisation through an electrostatic interaction between the protonated amino group and the negatively charged boronate moiety19.…”

“…Later we adapted this boronate-assisted saccharide electrophoresis (BASE) method to allow the separation of glycated from non-glycated proteins by incorporating MPBA into polyacrylamide gels for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis1920. This method, coined mP-AGE, showed that under electrophoresis conditions the polyacrylamide incorporated MPBA is specific for fructosamine modified proteins, via interactions with the cis -1,2-diol-containing fructosamine adducts and by further stabilisation through an electrostatic interaction between the protonated amino group and the negatively charged boronate moiety19. This method enables the differentiation between early and late glycated adducts and has now been successfully used to analyse glycated human serum albumin (HSA) in serum from diabetes sufferers21.…”

Review: Glycation of human serum albumin

“…One current clinical assay based on an enzymatic method utilizes an albumin-specific proteinase (i.e., ketoamine oxidase) to digest glycated HSA, followed by treatment with a fructosaminase to oxidize the glycated amino acids and produce hydrogen peroxide, which is then measured [59–61]. Raman spectroscopy [63], refractive index measurements [64], capillary electrophoresis [65] and other electrophoretic methods [66,67] have also been explored as alternative techniques for measuring glycated albumin.…”

Ex vivo instability of glycated albumin: A role for autoxidative glycation

“…Since albumin is the most abundant protein in P/S, fructosamine is predominantly a measure of GA—though other circulating proteins such as glycated lipoproteins and glycated globulins do contribute to the total concentration of fructosamine [25]. The concentration of GA can be directly measured by several methods, including gel electrophoresis, enzymatic methods, colorimetry, and immunoassays [18, 19, 26, 27]. HbA1c remains the primary test for protein glycation that is used to monitor diabetes; however, studies have shown that GA may be more reliable than HbA1c in certain instances—such as specific clinical conditions in which HbA1c does not work properly (e.g., iron deficiency, pregnancy, and end-stage renal disease) [28].…”