Selective Enrichment of Multiply Phosphorylated Peptides Using Polyarginine-coated Nanodiamonds Prior to Liquid Chromatography Mass Spectrometric Analysis.
Date Issued
2010
Date
2010
Author(s)
Hsieh, Feng-Jen
Abstract
Identification of multi-phosphorylated peptides by liquid chromatography-tandem mass spectrometry（LC-MS/MS）remains a challenging task due to the poor ionization of the species. For this reason, various kits to selectively enrich phosphopeptides prior to MS analysis have been developed and commercialized, but most of them are selective only for singly phosphorylated peptides. Interestingly, poly-arginine-coated nanodiamond（PA-ND）shows a high selectivity for multi-phosphorylated peptides and this has been demonstrated by Chang et al. For further applications in real sample analysis with LC-MS/MS, an effective elution buffer to separate the target molecules from the PA-ND substrate has yet to be found.
In this work, we have successfully synthesized a much highly selective probe by coating PA on ND（average diameter: 86.9 nm → 140.6 nm）in 0.1 M MES buffer. The zeta potential in deionized water increased from -27.6 mV to +43.29 mV, better than the previous one, ~0 mV. Furthermore, we found a better enrichment condition, with 40 % acetonitrile containing 1 % trifluoroacetic acid, that allows us to selectively probe multi-phosphorylated peptides in very complex samples（with impurities ≥5000 higher than of the target analyte）. Finally, we also found that 12.5 % ammonium water containing 0.5 M potassium phosphate can easily elute the enriched substance from the probe. LC-MS/MS can therefore successfully identify a triply phosphorylated peptide extracted from a highly mixture by the newly synthesized PA-ND particles and with the newly developed protocols.
Subjects
nanodiamond
nanoprobe
phosphorylated peptides
LC-MSMS
proteomics
Type
thesis
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