A Dual Pressure Linear Ion Trap Orbitrap Instrument with Very High Sequencing Speed

Since its introduction a few years ago, the linear ion trap Orbitrap (LTQ Orbitrap) instrument has become a powerful tool in proteomics research. For high resolution mass spectrometry measurements ions are accumulated in the linear ion trap and passed on to the Orbitrap analyzer. Simultaneously with acquisition of this signal, the major peaks are isolated in turn, fragmented and recorded at high sensitivity in the linear ion trap, combining the strengths of both mass analyzer technologies. Here we describe a next generation LTQ Orbitrap system termed Velos, with significantly increased sensitivity and scan speed. This is achieved by a vacuum interface using a stacked ring radio frequency ion guide with 10-fold higher transfer efficiency in MS/MS mode and 3–5-fold in full scan spectra, by a dual pressure ion trap configuration, and by reduction of overhead times between scans. The first ion trap efficiently captures and fragments ions at relatively high pressure whereas the second ion trap realizes extremely fast scan speeds at reduced pressure. Ion injection times for MS/MS are predicted from full scans instead of performing automatic gain control scans. Together these improvements routinely enable acquisition of up to ten fragmentation spectra per second. Furthermore, an improved higher-energy collisional dissociation cell with increased ion extraction capabilities was implemented. Higher-collision energy dissociation with high mass accuracy Orbitrap readout is as sensitive as ion trap MS/MS scans in the previous generation of the instrument. Since its introduction a few years ago, the linear ion trap Orbitrap (LTQ Orbitrap) instrument has become a powerful tool in proteomics research. For high resolution mass spectrometry measurements ions are accumulated in the linear ion trap and passed on to the Orbitrap analyzer. Simultaneously with acquisition of this signal, the major peaks are isolated in turn, fragmented and recorded at high sensitivity in the linear ion trap, combining the strengths of both mass analyzer technologies. Here we describe a next generation LTQ Orbitrap system termed Velos, with significantly increased sensitivity and scan speed. This is achieved by a vacuum interface using a stacked ring radio frequency ion guide with 10-fold higher transfer efficiency in MS/MS mode and 3–5-fold in full scan spectra, by a dual pressure ion trap configuration, and by reduction of overhead times between scans. The first ion trap efficiently captures and fragments ions at relatively high pressure whereas the second ion trap realizes extremely fast scan speeds at reduced pressure. Ion injection times for MS/MS are predicted from full scans instead of performing automatic gain control scans. Together these improvements routinely enable acquisition of up to ten fragmentation spectra per second. Furthermore, an improved higher-energy collisional dissociation cell with increased ion extraction capabilities was implemented. Higher-collision energy dissociation with high mass accuracy Orbitrap readout is as sensitive as ion trap MS/MS scans in the previous generation of the instrument. Proteomics experiments typically involve the analysis of peptide mixtures obtained by the enzymatic digestion of proteomes that can be as complex as complete cell lysates (1Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5484) Google Scholar, 2Yates 3rd, J.R. Gilchrist A. Howell K.E. Bergeron J.J. Proteomics of organelles and large cellular structures.Nat. Rev. Mol. Cell Biol. 2005; 6: 702-714Crossref PubMed Scopus (340) Google Scholar). Dynamic range of peptide abundances and the sheer number of peptides encountered in these mixtures require extremely sensitive and fast peptide detection and fragmentation (3Mann M. Kelleher N.L. 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Mass Spectrom. 2002; 13: 659-669Crossref PubMed Scopus (608) Google Scholar) with a Fourier transform (FT) 1The abbreviations used are:FTFourier transformS-lensstacked ring ion guideCIDcollision-induced dissociationHCDhigher-energy collisional dissociationBSAbovine serum albuminMS/MStandem mass spectrometryLTQlinear quadrupole ion trapLC-MS/MSliquid chromatography tandem mass spectrometryAGCautomatic gain controlRTroom temperatureRFradio frequencySILACstable isotope labeling by amino acids in cell cultureHPLChigh pressure liquid chromatography.1The abbreviations used are:FTFourier transformS-lensstacked ring ion guideCIDcollision-induced dissociationHCDhigher-energy collisional dissociationBSAbovine serum albuminMS/MStandem mass spectrometryLTQlinear quadrupole ion trapLC-MS/MSliquid chromatography tandem mass spectrometryAGCautomatic gain controlRTroom temperatureRFradio frequencySILACstable isotope labeling by amino acids in cell cultureHPLChigh pressure liquid chromatography. mass spectrometer has rapidly become a popular technological platform in proteomics because it combines the sensitivity, speed, and robustness of ion traps with the high resolution capabilities of FT instruments. The first implementation of this principle used an ion cyclotron resonance instrument with a 7T magnet as the high resolution device (7Syka J.E. Marto J.A. Bai D.L. Horning S. Senko M.W. Schwartz J.C. Ueberheide B. Garcia B. Busby S. Muratore T. Shabanowitz J. Hunt D.F. Novel linear quadrupole ion trap/FT mass spectrometer: performance characterization and use in the comparative analysis of histone H3 post-translational modifications.J. Proteome Res. 2004; 3: 621-626Crossref PubMed Scopus (332) Google Scholar). Later, the OrbitrapTM analyzer developed by Makarov was coupled to the LTQ, combining the linear ion trap with a very small and powerful analyzer (8Hardman M. Makarov A.A. Interfacing the orbitrap mass analyzer to an electrospray ion source.Anal. Chem. 2003; 75: 1699-1705Crossref PubMed Scopus (245) Google Scholar, 9Hu Q. Noll R.J. Li H. Makarov A. Hardman M. Graham Cooks R. The Orbitrap: a new mass spectrometer.J. Mass Spectrom. 2005; 40: 430-443Crossref PubMed Scopus (963) Google Scholar, 10Makarov A. Denisov E. Kholomeev A. Balschun W. Lange O. Strupat K. Horning S. Performance evaluation of a hybrid linear ion trap/orbitrap mass spectrometer.Anal. Chem. 2006; 78: 2113-2120Crossref PubMed Scopus (579) Google Scholar, 11Makarov A. Denisov E. Lange O. Horning S. Dynamic range of mass accuracy in LTQ Orbitrap hybrid mass spectrometer.J. Am. Soc. Mass Spectrom. 2006; 17: 977-982Crossref PubMed Scopus (327) Google Scholar). Fourier transform stacked ring ion guide collision-induced dissociation higher-energy collisional dissociation bovine serum albumin tandem mass spectrometry linear quadrupole ion trap liquid chromatography tandem mass spectrometry automatic gain control room temperature radio frequency stable isotope labeling by amino acids in cell culture high pressure liquid chromatography. Fourier transform stacked ring ion guide collision-induced dissociation higher-energy collisional dissociation bovine serum albumin tandem mass spectrometry linear quadrupole ion trap liquid chromatography tandem mass spectrometry automatic gain control room temperature radio frequency stable isotope labeling by amino acids in cell culture high pressure liquid chromatography. Here we describe a next generation linear ion trap-Orbitrap instrument with significant improvements in ion source transmission and with a new ion trap configuration. We show that this instrument, termed the LTQ Orbitrap Velos, is capable of much higher scan speeds compared with the current LTQ Orbitrap. Furthermore, we implemented more efficient ion extraction for the higher-energy collisional dissociation (HCD) cell (12Olsen J.V. Macek B. Lange O. Makarov A. Horning S. Mann M. Higher-energy C-trap dissociation for peptide modification analysis.Nat. Methods. 2007; 4: 709-712Crossref PubMed Scopus (702) Google Scholar). Due to this improvement and the 10-fold higher transmission of ions from atmosphere, high resolution and high mass accuracy MS/MS can now routinely be obtained at very high sensitivity and at scan speeds of up to 5 Hz acquisition rates. A related instrument, the LTQ-Velos, which does not contain the Orbitrap analyzer for high resolution measurements, has been described very recently (13Second T.P. Blethrow J.D. Schwartz J.C. Merrihew G.E. MacCoss M.J. Swaney D.L. Russell J.D. Coon J.J. Zabrouskov V. Dual-pressure linear ion trap mass spectrometer improving the analysis of complex protein mixtures.Anal. Chem. 2009; 81: 7757-7765Crossref PubMed Scopus (121) Google Scholar). The LTQ Orbitrap Velos instrument described in this paper is a further development of the LTQ Orbitrap product (10Makarov A. Denisov E. Kholomeev A. Balschun W. Lange O. Strupat K. Horning S. Performance evaluation of a hybrid linear ion trap/orbitrap mass spectrometer.Anal. Chem. 2006; 78: 2113-2120Crossref PubMed Scopus (579) Google Scholar). The three major novel hardware elements are (i) the introduction of a more efficient ion transfer system (Stacked Ring Ion Guide or “S-lens”), (ii) a dual pressure ion trap, and (iii) a more efficient HCD cell (Fig. 1) .