Anion-exchange chromatography of phosphopeptides: weak anion exchange versus strong anion exchange and anion-exchange chromatography versus electrostatic repulsion-hydrophilic interaction chromatography.

ABSTRACT: 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 re

SUBMITTER: Alpert AJ 

PROVIDER: S-EPMC4423237 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature