MassIVE MSV000080774

Imported Reanalysis Dataset Public PXD001333

Anion-Exchange Chromatography of Tryptic and Phosphopeptides: WAX vs. SAX and AEX vs. ERLIC

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Description

At a pH > 5, phosphopeptides have two negative charges per residue and are well-retained in anion-exchange chromatography. However, the peptides with one or two phosphate groups are not separated from the peptides with multiple Asp or Glu residues, which interfere with the identification of phosphopeptides in tryptic digests. At a pH around 2, phosphate residues have just a single negative charge but Asp and Glu have none. This facilitates the separation of phosphopeptides from unmodified acidic peptides. The singly phosphorylated peptides are retained too weakly under these conditions, due to electrostatic repulsion, unless hydrophilic interaction is superimposed in the ERLIC mode (electrostatic repulsion-hydrophilic interaction chromatography). Weak anion-exchange (WAX) and strong anion-exchange (SAX) columns were compared, both with peptide standards and with a complex tryptic digest. The SAX column exhibited higher capacity at pH 6 than did the WAX column. However, only about 60% as many phosphopeptides were identified at pH 6 than via ERLIC at pH 2. In one run, 12,467 phosphopeptides were identified, including 4233 with more than one phosphate. We conclude that chromatography of phosphopeptides is best performed at low pH in the ERLIC mode. Under those conditions the performances of the SAX and WAX materials were comparable. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: chromatography ERLIC SAX WAX AEX

Contact

Principal Investigators:
(in alphabetical order) 		Andrew Alpert, PolyLC Inc., 9151 Rumsey Road, ste. 175, Columbia, MD 21045 USA, N/A
Submitting User: ccms

Publications

Alpert AJ, Hudecz O, Mechtler K.
Anion-exchange chromatography of phosphopeptides: weak anion exchange versus strong anion exchange and anion-exchange chromatography versus electrostatic repulsion-hydrophilic interaction chromatography.
Anal. Chem. 2015;87(9):4704-11. Epub 2015 Apr 17.

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