Takátsy A, Böddi K, Nagy LV, Nagy G, Szabó S, Markó L, Wittmann I, Ohmacht R, Ringer T, Bonn GK, Gjerde D, Szabó Z
Enrichment of Amadori-products derived from the non-enzymatic glycation of proteins using microscale boronate affinity chromatography. [JOURNAL ARTICLE]
Anal Biochem 2009 Jun 11.
Amadori-peptides were enriched using boronate affinity tips and measured by MALDI-TOF/MS. As demonstrated by electrochemical measurements, the tips show the highest binding efficiency for glucose at pH 8.2 employing ammonium chloride/ammonia buffer with ionic strenght of 150 mM , exceeding taurine buffer at the same concentration. The bound constituents were released by sorbitol and formic acid. It has also been demonstrated that elution with sorbitol at 1.2 M is superior to acidic media. Comparison of results was based on the numbers of detected peptides and their glycated sites. ng sorbitol for elution requires desalting prior to analysis. Therefore three different sorbents were tested: fullerene-derivatised silica, Zip Tip (C18) and C18-silica. Fullerene-derivatised silica and Zip Tip showed the same performance regarding the numbers of glycated peptides and sites were better than C18-silica. The elaborated off-line method was compared to LC-MS/MS measurements, by which considerable less modified peptides were detected. Affinity tips used under optimized conditions were tested for the analysis of HSA from sera of healty and diabetic individuals. A peptide with the mass of 1783.9 Da could only be detected in samples of diabetic patients and could therefore be a very interesting biomarker candidate.
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