title: Probing the complementarity of FAIMS and strong cation exchange chromatography in shotgun proteomics.
abstract: High field asymmetric waveform ion mobility spectrometry (FAIMS), also known as differential ion mobility spectrometry, coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) offers benefits for the analysis of complex proteomics samples. Advantages include increased dynamic range, increased signal-to-noise, and reduced interference from ions of similar m/z. FAIMS also separates isomers and positional variants. An alternative, and more established, method of reducing sample complexity is prefractionation by use of strong cation exchange chromatography. Here, we have compared SCX-LC-MS/MS with LC-FAIMS-MS/MS for the identification of peptides and proteins from whole cell lysates from the breast carcinoma SUM52 cell line. Two FAIMS approaches are considered: (1) multiple compensation voltages within a single LC-MS/MS analysis (internal stepping) and (2) repeat LC-MS/MS analyses at different and fixed compensation voltages (external stepping). We also consider the consequence of the fragmentation method (electron transfer dissociation or collision-induced dissociation) on the workflow performance. The external stepping approach resulted in a greater number of protein and peptide identifications than the internal stepping approach for both ETD and CID MS/MS, suggesting that this should be the method of choice for FAIMS proteomics experiments. The overlap in protein identifications from the SCX method and the external FAIMS method was ~25% for both ETD and CID, and for peptides was less than 20%. The lack of overlap between FAIMS and SCX highlights the complementarity of the two techniques. Charge state analysis of the peptide assignments showed that the FAIMS approach identified a much greater proportion of triply-charged ions.
authors: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham, B15 2TT, UK.
authors: Andrew J
authors: AJ
authors: Creese
authors: Neil J
authors: NJ
authors: Shimwell
authors: Katherine P B
authors: KP
authors: Larkins
authors: John K
authors: JK
authors: Heath
authors: Helen J
authors: HJ
authors: Cooper
mesh_terms: D001940:Breast / Q000737:chemistry / Q000473:pathology; D001943:Breast Neoplasms / Q000737:chemistry* / Q000473:pathology; D045744:Cell Line, Tumor; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005260:Female; D006801:Humans; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010455:Peptides; D011506:Proteins
references: J Proteome Res. 2012 Feb 3;11(2):927-40
references: 22059388
references: Anal Chem. 2005 Apr 1;77(7):2176-86
references: 15801752
references: J Proteome Res. 2009 Dec;8(12):5475-84
references: 19821632
references: Mol Cell Proteomics. 2009 May;8(5):904-12
references: 19131326
references: Nature. 2008 Oct 30;455(7217):1251-4
references: 18820680
references: Mol Cell Proteomics. 2010 Dec;9(12):2840-52
references: 20829449
references: J Proteome Res. 2011 Aug 5;10(8):3690-700
references: 21702434
references: Rapid Commun Mass Spectrom. 2009 Jul;23(13):1963-9
references: 19504484
references: Expert Rev Proteomics. 2006 Dec;3(6):611-9
references: 17181475
references: J Am Soc Mass Spectrom. 2002 Nov;13(11):1282-91
references: 12443018
references: Anal Chem. 2007 Jan 15;79(2):477-85
references: 17222010
references: Anal Chem. 2011 Sep 15;83(18):6918-23
references: 21863819
references: Mol Cell Proteomics. 2012 Apr;11(4):M111.014985
references: 22186714
references: J Proteome Res. 2011 Sep 2;10(9):3929-43
references: 21678914
references: Anal Chem. 2008 Jul 1;80(13):4825-35
references: 18540640
references: Anal Chem. 2000 Oct 1;72(19):4577-84
references: 11028613
references: Mol Cell Proteomics. 2007 Nov;6(11):1942-51
references: 17673454
references: J Chromatogr A. 2004 Nov 26;1058(1-2):3-19
references: 15595648
references: Anal Chem. 2008 Sep 15;80(18):7137-43
references: 18652493
references: J Proteome Res. 2011 Apr 1;10(4):1785-93
references: 21309581
references: Anal Chem. 2010 Oct 1;82(19):8327-34
references: 20843012
references: J Proteome Res. 2009 Jul;8(7):3355-66
references: 19469569
references: J Proteome Res. 2011 May 6;10(5):2377-88
references: 21413819
references: Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12130-5
references: 15302935
references: Anal Chem. 2012 Mar 6;84(5):2597-601
references: 22280549