title: Dual-pressure linear ion trap mass spectrometer improving the analysis of complex protein mixtures.
abstract: The considerable progress in high-throughput proteomics analysis via liquid chromatography-electrospray ionization-tandem mass spectrometry over the past decade has been fueled to a large degree by continuous improvements in instrumentation. High-throughput identification experiments are based on peptide sequencing and are largely accomplished through the use of tandem mass spectrometry, with ion trap and trap-based instruments having become broadly adopted analytical platforms. To satisfy increasingly demanding requirements for depth of characterization and throughput, we present a newly developed dual-pressure linear ion trap mass spectrometer (LTQ Velos) that features increased sensitivity, afforded by a new source design, and demonstrates practical cycle times 2 times shorter than that of an LTQ XL, while improving or maintaining spectral quality for MS/MS fragmentation spectra. These improvements resulted in a substantial increase in the detection and identification of both proteins and unique peptides from the complex proteome of Caenorhabditis elegans, as compared to existing platforms. The greatly increased ion flux into the mass spectrometer in combination with improved isolation of low-abundance precursor ions resulted in increased detection of low-abundance peptides. These improvements cumulatively resulted in a substantially greater penetration into the baker's yeast (Saccharomyces cerevisiae) proteome compared to LTQ XL. Alternatively, faster cycle times on the new instrument allowed for higher throughput for a given depth of proteome analysis, with more peptides and proteins identified in 60 min using an LTQ Velos than in 180 min using an LTQ XL. When mass analysis was carried out with resolution in excess of 25,000 full width at half-maximum (fwhm), it became possible to isotopically resolve a small intact protein and its fragments, opening possibilities for top down experiments.
authors.affiliation: Thermo Fisher Scientific, San Jose, California 95134, USA.
authors.forename: Tonya Pekar
authors.initials: TP
authors.lastname: Second
authors.forename: Justin D
authors.initials: JD
authors.lastname: Blethrow
authors.forename: Jae C
authors.initials: JC
authors.lastname: Schwartz
authors.forename: Gennifer E
authors.initials: GE
authors.lastname: Merrihew
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacCoss
authors.forename: Danielle L
authors.initials: DL
authors.lastname: Swaney
authors.forename: Jason D
authors.initials: JD
authors.lastname: Russell
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
authors.forename: Vlad
authors.initials: V
authors.lastname: Zabrouskov
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006736:Horses; D009211:Myoglobin / Q000032:analysis; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation* / Q000379:methods
chemical_list: D009211:Myoglobin; D010455:Peptides; D011506:Proteins
references.citation: Genome Res. 2008 Oct;18(10):1660-9
references.pmid: 18653799
references.citation: Mass Spectrom Rev. 2010 Mar-Apr;29(2):294-312
references.pmid: 19391099
references.citation: Mol Cell Proteomics. 2006 Jul;5(7):1326-37
references.pmid: 16635985
references.citation: Nature. 2008 Oct 30;455(7217):1251-4
references.pmid: 18820680
references.citation: Nature. 2003 Oct 16;425(6959):737-41
references.pmid: 14562106
references.citation: Mol Cell Proteomics. 2009 Aug;8(8):1908-20
references.pmid: 19429919
references.citation: J Proteome Res. 2009 Apr;8(4):1870-5
references.pmid: 19256536
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10762-7
references.pmid: 18669648
references.citation: Curr Opin Biotechnol. 2007 Aug;18(4):378-84
references.pmid: 17698335
references.citation: J Proteome Res. 2007 Sep;6(9):3549-57
references.pmid: 17676885
references.citation: Proc Natl Acad Sci U S A. 2007 Jan 30;104(5):1488-93
references.pmid: 17242355
references.citation: J Proteome Res. 2008 Jan;7(1):29-34
references.pmid: 18067246
references.citation: Mol Cell Proteomics. 2009 Jul;8(7):1751-64
references.pmid: 19369195
references.citation: Nat Methods. 2007 Nov;4(11):923-5
references.pmid: 17952086
references.citation: Genome Res. 2009 Apr;19(4):657-66
references.pmid: 19181841
references.citation: J Proteome Res. 2008 Jan;7(1):40-4
references.pmid: 18052118
references.citation: Rapid Commun Mass Spectrom. 2004;18(18):2162-8
references.pmid: 15317041
references.citation: Proc Natl Acad Sci U S A. 2008 Oct 7;105(40):15281-6
references.pmid: 18829439
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9440-5
references.pmid: 12883005
references.citation: Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11969-74
references.pmid: 12163647