Abstract
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.
Highlights
Introduction of anS-lens—The tube lens/skimmer assembly has been replaced by a set of stainless steel apertures to which an RF voltage is applied to form a so-called S-lens
We describe a 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
As no direct current gradient appeared to be necessary in such a construction, all odd-numbered apertures have been connected to one phase of a RF voltage and the even-numbered apertures to the other
Summary
Construction of an Improved Linear Ion Trap Orbitrap Instrument— The LTQ Orbitrap Velos instrument described in this paper is a further development of the LTQ Orbitrap product [10]. Penetration of the resulting electric field distribution up to the axis of the device has been ensured by locating the printed circuit board in the center of each gap between four RF-only rods of the cell This design enables rapid extraction of all ions from the HCD cell and allows increasing speed and sensitivity of the instrument in HCD mode. The software controlling the instrument includes a new mode of predictive automatic gain control (pAGC) In this mode the injection times of precursor ions for MS/MS are no longer determined by a dedicated pre-scan in the LTQ, but from a full-MS spectrum acquired from either the linear trap or the Orbitrap mass analyzer. Raw LC-MS Data Analysis—Fragmented peptides were identified and quantified by MASCOT and MaxQuant; Raw Orbitrap full-scan MS and ion trap CID-MS/MS and Orbitrap HCD spectra from the SILAC-labeled HeLa cytoplasmic extracts were processed by MaxQuant as described [20, 21].
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