Differentiation and Quantitation of Coeluting Isomeric Amadori and Heyns Peptides Using Sugar-Specific Fragment Ion Ratios

Abstract

D-glucose and D-fructose present in blood, tissues, and organs of all mammals can react with amino groups, leading to glucated (Amadori) and fructated (Heyns) products, i.e., proteins glycated at lysine residues. While typically present at low concentration in humans, metabolic diseases including diabetes elevate sugar levels, favoring glycation and consecutive reactions leading to advanced glycation end products (AGEs) linked to diabetic complications and cardiovascular diseases. Analytical methods able to differentiate and to individually quantify Amadori-and Heyns-modified proteins in complex sample mixtures, e.g., serum, are still very limited. Here, we show that the reported and supposedly specific neutral losses displayed in tandem mass spectra of Heyns peptides cannot be used for a reliable differentiation as they were also observed for Amadori peptides. However, the combination of several neutral loss signals in fragment ion ratios at both precursor and fragment ion signals allowed the differentiation and relative quantitation of coeluting isomeric Amadori and Heyns peptides at different concentrations and peptide ratios. This was also true for digested human plasma. Thus, the presented strategy allows the quantitation of Amadori and Heyns peptides in complex samples, especially by spiking isotope-labeled peptides. This will allow searching for glucated and fructated biomarkers in clinical samples.

Chromatographic separation of glycated peptide isomers derived from glucose and fructose

“…However, late-eluting fructated peptides #6c and #7c were partially separated with retention time differences of 0.5 min and 0.9 min (R s = 2.4), respectively. The characteristic fragmentation pattern confirmed fructated peptides in both peaks and excluded a glucated peptide (data not shown) [14]. These peaks may represent tautomeric forms, i.e., α-or β-pyranosyl forms, or more likely epimers, i.e., glucosyl-/mannosyllysine considering a previous report [23].…”

“…All columns were equipped with Security Guard columns from Phenomenex Ltd. (Aschaffenburg, Germany). Amadori, Heyns, and the corresponding unmodified peptides were synthesized on solid phase, as previously described [4,14,23]. Briefly, peptides were synthesized employing Fmoc/ t Bu chemistry and DIC/HOBt activation on Fmoc-l-Lys(Boc)-or Fmoc-l-Arg(Pbf)-Wang resins.…”

“…While stronger ion-pair reagents are advantageous for the separation of unmodified and glycated peptides, they do not improve the separation of glucose-and fructose-derived peptide isomers, even for pentapeptides and shallow gradients [12]. Coeluting glucated and fructated amino acids or peptides can be analyzed by multiple reaction monitoring (MRM) in ESI-MS using isomer-typical transitions [11,13,14]. However, these transitions are unspecific and misleading for many sequences [11,14], which necessitates at least a partial chromatographic separation.…”

“…Coeluting glucated and fructated amino acids or peptides can be analyzed by multiple reaction monitoring (MRM) in ESI-MS using isomer-typical transitions [11,13,14]. However, these transitions are unspecific and misleading for many sequences [11,14], which necessitates at least a partial chromatographic separation.…”