Enrichment of Amadori products derived from the nonenzymatic glycation of proteins using microscale boronate affinity chromatography

Amadori peptides were enriched using boronate affinity tips and measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF/MS). As demonstrated by electrochemical measurements, the tips show the highest binding efficiency for glucose at pH 8.2 employing ammon...

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Bibliographic Details
Published in:Analytical biochemistry 2009-10, Vol.393 (1), p.8-22
Main Authors: Takátsy, Anikó, Böddi, Katalin, Nagy, Lívia, Nagy, Géza, Szabó, Szilvia, Markó, Lajos, Wittmann, István, Ohmacht, Róbert, Ringer, Thomas, Bonn, Günther K., Gjerde, Douglas, Szabó, Zoltán
Format: Article
Language:English
Subjects:
Amadori products
Boronate affinity chromatography
Boronic Acids - chemistry
Chromatography, Affinity - instrumentation
Chromatography, Affinity - methods
Glycation
Glycopeptides - chemistry
Glycopeptides - isolation & purification
Glycoproteins - chemistry
Humans
MALDI–TOF/MS
Molecular Structure
Peptides - chemistry
Peptides - isolation & purification
Ribonuclease, Pancreatic - analysis
Serum Albumin - analysis
Type 2 diabetes mellitus
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Summary:Amadori peptides were enriched using boronate affinity tips and measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (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 strength of 150 mM, exceeding taurine buffer at the same concentration. The bound constituents were released by sorbitol and formic acid. It was also 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. Using sorbitol for elution requires desalting prior to analysis. Therefore, three different sorbents were tested: fullerene-derivatized silica, ZipTip (C18), and C18 silica. Fullerene-derivatized silica and ZipTip showed the same performance regarding the numbers of glycated peptides, and sites were better than C18 silica. The elaborated off-line method was compared with liquid chromatography–tandem mass spectrometry (LC–MS/MS) measurements, by which considerable less modified peptides were detected. Affinity tips used under optimized conditions were tested for the analysis of human serum albumin (HSA) from sera of healthy and diabetic individuals. A peptide with a mass of 1783.9 Da could be detected only in samples of diabetic patients and, therefore, could be a very interesting biomarker candidate.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2009.06.007