Analysis of protein glycation using phenylboronate acrylamide gel electrophoresis.
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.
Morais
Marta P Pereira
MP
Department of Biology and Biochemistry, University of Bath, Bath, UK.
Mackay
Julia D
JD
Bhamra
Savroop K
SK
Buchanan
J Grant
JG
James
Tony D
TD
Fossey
John S
JS
van den Elsen
Jean M H
JM
D001897:Boronic Acids / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000191:economics / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D015394:Molecular Structure; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000191:economics / Q000379:methods*; D012709:Serum Albumin / Q000032:analysis* / Q000378:metabolism
D001897:Boronic Acids; D012709:Serum Albumin; C010686:benzeneboronic acid