Anion-Exchange Chromatography of Phosphopeptides:Weak Anion Exchange versus Strong Anion Exchange and Anion-ExchangeChromatography versus Electrostatic Repulsion–Hydrophilic InteractionChromatography

摘要

Most phosphoproteomics experiments rely on prefractionation of tryptic digests before online liquid chromatography-mass spectrometry. This study compares the potential and limitations of electrostatic repulsion–hydrophilic interaction chromatography (ERLIC) and anion-exchange chromatography (AEX). At a pH higher than 5, phosphopeptides have two negative charges per residue and are well-retained in AEX. However, peptides with one or two phosphate groups are not separated from peptides with multiple Asp or Glu residues, interfering with the identification of phosphopeptides. At a pH of 2, phosphate residues have just a single negative charge but Asp and Glu are uncharged. This facilitates the separation of phosphopeptides from unmodified acidic peptides. Singly phosphorylated peptides are retained weakly under these conditions, due to electrostatic repulsion, unless hydrophilic interaction is superimposed in the ERLIC mode. Weak anion-exchange (WAX) and strong anion-exchange (SAX) columns were compared, with both peptide standards and a HeLa cell tryptic digest. The SAX columnexhibited greater retention at pH 6 than did the WAX column. However,only about 60% as many phosphopeptides were identified with SAX atpH 6 than via ERLIC at pH 2. In one ERLIC run, 12 467 phosphopeptideswere identified, including 4233 with more than one phosphate. We concludethat chromatography of phosphopeptides is best performed at low pHin the ERLIC mode. Under those conditions, the performances of theSAX and WAX materials were comparable. The data have been depositedwith the ProteomeXchange with identifier PXD001333.