Separation of Amadori peptides from their unmodified analogs by ion-pairing RP-HPLC with heptafluorobutyric acid as ion-pair reagent |
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Authors: | Andrej Frolov Ralf Hoffmann |
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Institution: | (1) Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Faculty of Chemistry and Mineralogy, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany |
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Abstract: | Glycation is a common class of nonenzymatic posttranslational modifications relevant for several diseases and cell aging in
general, such as D-glucose-derived modifications at the ɛ-amino groups of lysine residues in blood proteins, especially albumin, immunoglobulin,
and hemoglobin, for diabetic patients. These Amadori compounds are identified on the peptide level after enzymatic digestion
and chromatographic separation by mass spectrometry. Their syntheses usually rely on a global glycation approach. Both areas
require the reliable separation of glycated peptides from their unmodified congeners present in different ratios, which is
typically not achieved by standard eluent systems in ion-pairing RP-HPLC (IP-RPLC). Here, we compare aqueous acetonitrile
and methanol gradients containing either trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) as ion-pairing agents
to separate such peptide pairs. TFA-containing eluents resulted in rather low resolutions, and the glycated and unglycated
peptides often coeluted. HFBA increased the retention times of the unmodified peptide more than for the glycated peptide thereby
improving the separation of all eight studied peptide pairs, even achieving baseline separations for some sequences. Thus
the use of HFBA as ion-pair reagent provides a universally applicable eluent system in IP-RPLC to separate glycated peptides
from their unmodified counterparts, even at the preparative scale required for synthetic peptides. |
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Keywords: | Electrospray ionization (ESI) Glycation Mass spectrometry (MS) Matrix-assisted laser desorption/ionization (MALDI) Solid-phase peptide synthesis (SPPS) |
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