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. 2009 Dec;8(12):2759-69.
doi: 10.1074/mcp.M900375-MCP200. Epub 2009 Oct 14.

A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed

Jesper V Olsen  1 , Jae C Schwartz, Jens Griep-Raming, Michael L Nielsen, Eugen Damoc, Eduard Denisov, Oliver Lange, Philip Remes, Dennis Taylor, Maurizio Splendore, Eloy R Wouters, Michael Senko, Alexander Makarov, Matthias Mann, Stevan Horning
Affiliations

A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed

Jesper V Olsen et al. Mol Cell Proteomics. 2009 Dec.

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.

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Figures

Fig. 1.
Fig. 1.
Schematic of the LTQ Orbitrap Velos MS instrument with three new hardware implementations. A, the stacked ring ion guide (S-Lens) increases the ion flux from the electrospray ion source into the instrument by a factor 5–10. B, the dual linear ion trap design enables efficient trapping and activation in the high-pressure cell (left) and fast scanning and detection in the low pressure cell (right). C, the combo C-trap and HCD collision cell with an applied axial field with improved fragment ion extraction and trapping capabilities.
Fig. 2.
Fig. 2.
Top-down analysis of intact bovine carbonic anhydrase II by LTQ Orbitrap Velos. A, Orbitrap MS full scan of (M + 24 H)24+ acquired at full isotopic resolution. Inset, deconvolution of base peak. Note that the mass error is less than one p.p.m. with external calibration. Red dotted line indicates the in silico simulation of carbonic anhydrase (M + 24 H)24+ at resolving power of 72,000. B, HCD of [M + 34 H]34+ of carbonic anhydrase. The inset shows the cleavage coverage of the protein sequence by HCD.
Fig. 3.
Fig. 3.
A, S-lens ion injection times for CID-MS/MS compared with the previous ion source configuration. The same SILAC-labeled HeLa extract separated with a 2 h LC gradient were analyzed on an LTQ Orbitrap XL instrument (blue lines) and an LTQ Orbitrap Velos instrument (red lines) with ion trap-CID MS/MS. Injection times of the top5 most intense peaks from each scan cycle are shown as running medians of 1000 scans. B, LC-MS/MS of serial dilutions of a simple BSA digest on both the XL and Velos type LTQ Orbitraps under identical conditions: 0.05, 0.5, 5, and 50 fmol of the digest loaded on column and analyzed by top5 CID in the ion trap. Unique peptides identified by the XL at the different concentrations are indicated in the red bars, whereas peptide identifications from the Velos are displayed in the blue bars.
Fig. 4.
Fig. 4.
CID-MS/MS in the dual-pressure linear ion trap top20 cycle times for shotgun proteomics. A, full scan data from a complex SILAC-labeled HeLa cell cytoplasmic fraction using a top10 CID method on the LTQ Orbitrap XL instrument. B, full scan data from the same HeLa sample analyzed with a top20 method on the LTQ Orbitrap Velos instrument.
Fig. 5.
Fig. 5.
Profile data of MS/MS detected in the linear ion trap and in the Orbitrap instrument. A, CID-MS/MS spectrum acquired in the linear ion trap. 5000 ions of a doubly charged BSA peptide at m/z 740.40 from a 25 fmol/μl BSA standard mixture infusion are isolated in 4 ms and fragmented by 10 ms resonant excitation in the high-pressure trap using normalized collision energy of 35%. Fragment ions are moved to the low-pressure trap and scanned out to the two electron multiplier detectors with a scan speed of 33,300 amu/sec. The spectrum is recorded from m/z 200 to 2000, which results in a scan time of 50 ms. Note that the natural isotope cluster of doubly charged fragment ions are resolved (inset). B, low-energy CID spectrum of the same peptide acquired in the Orbitrap analyzer. 50,000 ions are isolated in the low pressure linear ion trap by 50 ms injection time and activated by 35% normalized collision energy. The fragment ions are detected in the Orbitrap analyzer with a resolution of 7500, which is achieved by 100 ms transient detection time. All fragment ion isotope clusters are baseline resolved (inset).
Fig. 6.
Fig. 6.
HCD fragmentation and extraction efficiency. A, HCD scan at zero collision energy. 50,000 ions of a doubly charged BSA peptide at m/z 740.40 from a 25 fmol BSA digest/μl direct infusion experiment are isolated in 48 ms and transferred to the C-trap before detection in the Orbitrap analyzer at a resolution of 7500 at m/z 400. B, HCD of the same peptide. 50,000 ions of the doubly charged BSA peptide at m/z 740.40 are isolated in 43 ms and fragmented in the HCD collision cell by 37 eV acceleration before detection in the Orbitrap analyzer at a resolution of 7500 at m/z 400. Natural isotope clusters of fragment ions (inset) are baseline resolved.
Fig. 7.
Fig. 7.
HCD scan cycle times. HeLa cytoplasmic extract was analyzed with 2 h gradient using a top10 HCD method. Orbitrap analyzer full scans had a resolution of 30,000 (0.5 s), and HCD spectra 7500.
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