title: 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 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 column exhibited greater retention at pH 6 than did the WAX column. However, only about 60% as many phosphopeptides were identified with SAX at pH 6 than via ERLIC at pH 2. In one ERLIC 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. The data have been deposited with the ProteomeXchange with identifier PXD001333.
authors.affiliation: †PolyLC Inc., 9151 Rumsey Road, Ste. 175, Columbia, Maryland 21045, United States.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Alpert
authors.affiliation: ‡Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.|§Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Dr. Bohr-Gasse 3, 1030 Vienna, Austria.
authors.forename: Otto
authors.initials: O
authors.lastname: Hudecz
authors.affiliation: ‡Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.|§Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Dr. Bohr-Gasse 3, 1030 Vienna, Austria.
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
mesh_terms: D002852:Chromatography, Ion Exchange; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification*; D055672:Static Electricity
chemical_list: D010748:Phosphopeptides
references.citation: Anal Chem. 2007 May 15;79(10):3623-34
references.pmid: 17411013
references.citation: Mol Cell Proteomics. 2010 Jun;9(6):1314-23
references.pmid: 20164059
references.citation: Mol Cell Proteomics. 2003 Nov;2(11):1234-43
references.pmid: 14506206
references.citation: J Proteome Res. 2008 Nov;7(11):4869-77
references.pmid: 18828627
references.citation: Proteomics. 2008 Apr;8(7):1346-61
references.pmid: 18318008
references.citation: J Proteome Res. 2010 Sep 3;9(9):4585-94
references.pmid: 20681634
references.citation: Sci Signal. 2010 May 25;3(123):ra41
references.pmid: 20501937
references.citation: J Chromatogr A. 2011 Dec 9;1218(49):8890-6
references.pmid: 21530976
references.citation: Anal Biochem. 1985 Feb 15;145(1):47-56
references.pmid: 2988370
references.citation: Anal Chem. 2011 Sep 15;83(18):7137-43
references.pmid: 21815630
references.citation: J Proteome Res. 2011 Sep 2;10(9):4041-53
references.pmid: 21736374
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Jan 1;879(1):25-34
references.pmid: 21130716
references.citation: Nucleic Acids Res. 2013 Jan;41(Database issue):D1063-9
references.pmid: 23203882
references.citation: Anal Biochem. 1983 Aug;133(1):251-9
references.pmid: 6638483
references.citation: Anal Chem. 2008 Jan 1;80(1):62-76
references.pmid: 18027909
references.citation: J Proteome Res. 2012 Aug 3;11(8):4269-76
references.pmid: 22768876
references.citation: J Proteome Res. 2012 Dec 7;11(12):5663-77
references.pmid: 23106611
references.citation: Cell Mol Life Sci. 2011 Jun;68(11):1983-2002
references.pmid: 20953656
references.citation: Nat Methods. 2007 Nov;4(11):923-5
references.pmid: 17952086
references.citation: Biochim Biophys Acta. 2015 May;1854(5):460-8
references.pmid: 25619855
references.citation: Mol Cell Proteomics. 2011 Nov;10(11):O111.011064
references.pmid: 21788404
references.citation: J Chromatogr A. 2000 Nov 3;897(1-2):99-111
references.pmid: 11128229
references.citation: J Proteome Res. 2011 Dec 2;10(12):5354-62
references.pmid: 22073976
references.citation: Mol Cell Proteomics. 2010 Apr;9(4):635-47
references.pmid: 20047950
references.citation: PLoS One. 2011 Feb 23;6(2):e16884
references.pmid: 21373199
references.citation: J Chromatogr A. 2001 Jan 26;908(1-2):149-61
references.pmid: 11218117
references.citation: Anal Chem. 2010 Jun 15;82(12):5253-9
references.pmid: 20481592
references.citation: J Proteome Res. 2014 Aug 1;13(8):3679-84
references.pmid: 24909410
references.citation: J Proteome Res. 2009 Jan;8(1):133-41
references.pmid: 19053533
references.citation: Anal Chem. 2015 Feb 3;87(3):1596-604
references.pmid: 25405705
references.citation: J Sep Sci. 2010 Jul;33(13):1879-87
references.pmid: 20533337
references.citation: J Proteome Res. 2013 Dec 6;12(12):5989-95
references.pmid: 24144214