Chromatographic separation of glycated peptide isomers derived from glucose and fructose

Abstract

Amino groups in proteins can react with aldehyde groups in aldoses or keto groups in ketoses, e.g., d-glucose and d-fructose, yielding Schiff bases that rearrange to more stable Amadori and Heyns products, respectively. Analytical strategies to identify and quantify each glycation product in the presence of the corresponding isomer are challenged by similar physicochemical properties, impeding chromatographic separations, and by identical masses including very similar fragmentation patterns in tandem mass spectrometry. Thus, we studied the separation of seven peptide families, each consisting of unmodified, glucated, and fructated 15mer to 22mer peptides using reversed-phase (RP) and hydrophilic interaction chromatography (HILIC). In RP-HPLC using acidic acetonitrile gradients, unglycated peptides eluted ~ 0.1 to 0.8 min after the corresponding glycated peptides with four of seven peptides being baseline separated. Isomeric glucated and fructated peptides typically coeluted, although two late-eluting peptides were partially separated. Neutral eluents (pH 7.2) improved the chromatographic resolution (Rs), especially in the presence of phosphate, providing good and often even baseline separations for six of the seven isomeric glycated peptide pairs with fructated peptides eluting earlier (Rs = 0.7 to 1.5). Some glucated and unmodified peptides coeluted, but they can be distinguished by mass spectrometry. HILIC separated glycated and unmodified peptides well, whereas glucated and fructated peptides typically coeluted. In conclusion, HILIC efficiently separated unmodified and the corresponding glycated peptides, while isomeric Amadori and Heyns peptides were best separated by RP-HPLC using phosphate buffered eluents.

The NMR signature of maltose-based glycation in full-length proteins

“…Glucose-based glycation is very well studied, also because it is a critical quality attribute in therapeutic proteins (Alt et al 2016 ; Sjögren et al 2016 ). It is mainly detected and monitored by mass-spectrometry coupled with high-performance liquid chromatography (Schmutzler and Hoffmann 2022 ; Rabbani et al 2016 ; Soboleva et al 2017 ; Zhang et al 2011 ) but also recently by NMR spectroscopy (Kaufmann et al 2016 ; Moises et al 2022 ).…”