title: Enhanced detection of multiply phosphorylated peptides and identification of their sites of modification.
abstract: Phosphorylation is an important post-translational modification that rapidly mediates many cellular events. A key to understanding the dynamics of the phosphoproteome is localization of the modification site(s), primarily determined using LC-MS/MS. A major technical challenge to analysis is the formation of phosphopeptide-metal ion complexes during LC which hampers phosphopeptide detection. We have devised a strategy that enhances analysis of phosphopeptides, especially multiply phosphorylated peptides. It involves treatment of the LC system with EDTA and 2D-RP/RP-nanoUPLC-MS/MS (high pH/low pH) analysis. A standard triphosphorylated peptide that could not be detected with 1D-RP-nanoUPLC-MS/MS, even if the column was treated with EDTA-Na2 or if 25 mM EDTA-Na2 was added to the sample, was detectable at less than 100 fmol using EDTA-2D-RP/RP-nanoUPLC-MS/MS. Digests of α-casein and ß-casein were analyzed by EDTA-1D-RP-nanoUPLC, 2D-RP/RP-nanoUPLC, and EDTA-2D-RP/RP-nanoUPLC to compare their performance in phosphopeptide analysis. With the first two approaches, no tri- and tetraphosphopeptides were identified in either α- or ß-casein sample. With the EDTA-2D-RP/RP approach, 13 mono-, 6 di-, and 3 triphosphopeptides were identified in the α-casein sample, while 19 mono-, 8 di-, 4 tri-, and 3 tetraphosphopeptides were identified in the ß-casein sample. Using EDTA-2D-RP/RP-nanoUPLC-MS/MS to examine 500 μg of a human foreskin fibroblast cell lysate a total of 1,944 unique phosphopeptides from 1,087 unique phosphoproteins were identified, and 2,164 unique phosphorylation sites were confidently localized (Ascore ≥20). Of these sites 79% were mono-, 20% di-, and ∼1% were tri- and tetraphosphopeptides, and 78 novel phosphorylation sites in human proteins were identified.
authors.affiliation: Program in Genomics and Proteomics, University of Lille , France.
authors.forename: Antoine
authors.initials: A
authors.lastname: Fleitz
authors.forename: Edward
authors.initials: E
authors.lastname: Nieves
authors.forename: Carlos
authors.initials: C
authors.lastname: Madrid-Aliste
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Fentress
authors.forename: L David
authors.initials: LD
authors.lastname: Sibley
authors.forename: Louis M
authors.initials: LM
authors.lastname: Weiss
authors.forename: Ruth Hogue
authors.initials: RH
authors.lastname: Angeletti
authors.forename: Fa-Yun
authors.initials: FY
authors.lastname: Che
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010748:Phosphopeptides
references.citation: Biochemistry. 1979 Sep 4;18(18):3865-79
references.pmid: 314815
references.citation: Mol Cell Proteomics. 2003 Nov;2(11):1143-55
references.pmid: 12966146
references.citation: J Proteome Res. 2009 Aug;8(8):3852-61
references.pmid: 19534553
references.citation: Cell Signal. 2011 Aug;23(8):1387-95
references.pmid: 21447384
references.citation: Anal Chem. 2008 Dec 15;80(24):9735-47
references.pmid: 19012417
references.citation: Cell. 2000 Jan 7;100(1):113-27
references.pmid: 10647936
references.citation: Proteomics. 2011 Jul;11(13):2639-53
references.pmid: 21630456
references.citation: Nucleic Acids Res. 2009 Apr;37(5):1701-12
references.pmid: 19155274
references.citation: J Proteome Res. 2009 Jan;8(1):133-41
references.pmid: 19053533
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10762-7
references.pmid: 18669648
references.citation: Nat Cell Biol. 2002 May;4(5):E127-30
references.pmid: 11988757
references.citation: Nat Protoc. 2008;3(10):1630-8
references.pmid: 18833199
references.citation: J Proteome Res. 2007 Nov;6(11):4150-62
references.pmid: 17924679
references.citation: Sci Signal. 2010 Dec 21;3(153):rs4
references.pmid: 21177495
references.citation: Proteomics. 2001 Feb;1(2):200-6
references.pmid: 11680867
references.citation: Anal Chem. 2010 Jan 1;82(1):53-6
references.pmid: 19950968
references.citation: Curr Biol. 2006 Nov 7;16(21):2150-5
references.pmid: 17084700
references.citation: J Proteomics. 2011 Nov 18;74(12):2786-97
references.pmid: 21839867
references.citation: Mol Cell Proteomics. 2008 May;7(5):971-80
references.pmid: 18212344
references.citation: Methods. 2011 Aug;54(4):379-86
references.pmid: 21440633
references.citation: Anal Chem. 2003 Jun 1;75(11):2724-9
references.pmid: 12948142
references.citation: Anal Chem. 2008 Apr 1;80(7):2419-25
references.pmid: 18321079
references.citation: Nat Biotechnol. 2009 Jan;27(1):77-83
references.pmid: 19098899
references.citation: Mol Cell Proteomics. 2011 Dec;10(12):M111.009910
references.pmid: 21917720
references.citation: Mol Cell Proteomics. 2008 Apr;7(4):661-71
references.pmid: 18039691
references.citation: Cell. 2012 Jul 20;150(2):413-25
references.pmid: 22817900
references.citation: Mol Cell Proteomics. 2010 Oct;9(10):2162-72
references.pmid: 20562096
references.citation: Rapid Commun Mass Spectrom. 2005;19(19):2747-56
references.pmid: 16136520
references.citation: Mol Cell Proteomics. 2012 Feb;11(2):M111.010132
references.pmid: 22101235
references.citation: Mol Cell Proteomics. 2011 Nov;10(11):O111.011064
references.pmid: 21788404
references.citation: J Proteome Res. 2009 Jan;8(1):418-24
references.pmid: 19053530
references.citation: Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3811-6
references.pmid: 17360435
references.citation: J Biol Chem. 2009 Sep 18;284(38):25723-34
references.pmid: 19635792
references.citation: Anal Biochem. 1996 Aug 1;239(2):180-92
references.pmid: 8811904
references.citation: J Proteome Res. 2008 Oct;7(10):4215-24
references.pmid: 18785766
references.citation: J Proteome Res. 2011 Mar 4;10(3):1088-97
references.pmid: 21210654
references.citation: Sci Signal. 2011 Aug 9;4(185):ra52
references.pmid: 21868358
references.citation: J Mass Spectrom. 2009 Jun;44(6):861-78
references.pmid: 19504542
references.citation: Nat Biotechnol. 2006 Oct;24(10):1285-92
references.pmid: 16964243
references.citation: Anal Chem. 2010 Aug 1;82(15):6409-14
references.pmid: 20593796
references.citation: Protein Sci. 1993 Jan;2(1):86-92
references.pmid: 8443592
references.citation: Nat Biotechnol. 2004 Sep;22(9):1139-45
references.pmid: 15314609
references.citation: Mol Cell Proteomics. 2009 Dec;8(12):2809-26
references.pmid: 19674963
references.citation: Sci Signal. 2011 Mar 15;4(164):rs3
references.pmid: 21406692
references.citation: Anal Chem. 2010 Apr 1;82(7):2873-8
references.pmid: 20210298
references.citation: J Proteome Res. 2010 Dec 3;9(12):6786-94
references.pmid: 20873877