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Ultra high resolution linear ion trap Orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes.

Michalski A, Damoc E, Lange O, Denisov E, Nolting D, Müller M, Viner R, Schwartz J, Remes P, Belford M, Dunyach JJ, Cox J, Horning S, Mann M, Makarov A - Mol. Cell Proteomics (2011)

Bottom Line: Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas.For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s-increasing protein identifications in complex mixtures by about 30%.The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.

View Article: PubMed Central - PubMed

Affiliation: Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.

ABSTRACT
Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm-incorporating phase information-further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s-increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.

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Related in: MedlinePlus

Parallel CID top20 method and ultra high resolution survey scans.A, High resolution MS scan at three different transient lengths followed by 20 CID MS/MS scans in the linear ion trap. Note that cycle time is unaffected by the resolution of the full scan. Preview refers to the portion of the survey scan that is used to select precursor ions for fragmentation. B, LC MS heat map of peptides eluting over a 3 min elution time interval in a 40 Th range. More detail is visible in the ultra high resolution setting (left panel) compared with the normal resolution setting (right panel). C, Separation of isobaric species in standard LC MS/MS analysis. The 34S isotope containing peak is clearly resolved from the 13C2 isotope.
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Figure 4: Parallel CID top20 method and ultra high resolution survey scans.A, High resolution MS scan at three different transient lengths followed by 20 CID MS/MS scans in the linear ion trap. Note that cycle time is unaffected by the resolution of the full scan. Preview refers to the portion of the survey scan that is used to select precursor ions for fragmentation. B, LC MS heat map of peptides eluting over a 3 min elution time interval in a 40 Th range. More detail is visible in the ultra high resolution setting (left panel) compared with the normal resolution setting (right panel). C, Separation of isobaric species in standard LC MS/MS analysis. The 34S isotope containing peak is clearly resolved from the 13C2 isotope.

Mentions: To investigate the instrument capability for the analysis of very complex peptide mixtures such as HeLa cell lysate, we started with a digested standard of 400 ng that was analyzed using different methods. Comparison of the normal CID and rapid CID scan modes revealed that rCID produced significantly more fragmentation events and therefore this mode was chosen for all subsequent experiments. The scheme in Fig. 4A shows the timing sequence of MS and MS/MS scans at different MS transient lengths. For a 192 ms survey scan, resolution is 60,000 and there is no parallel operation between MS and MS/MS scans. Due to rCID an entire top20 method takes 2.7 s, easily compatible with peptide LC elution profiles. At 384 ms resolution is 120,000 and a few MS/MS scans are performed in parallel with the survey scan. At the full 768 ms transient (240,000 resolution), about six CID spectra are performed in parallel, while total cycle time is still unchanged (Fig. 4A). Therefore, the longest transients appear to be advantageous because the increased resolution comes “for free” as it does not cost extra measurement time.


Ultra high resolution linear ion trap Orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes.

Michalski A, Damoc E, Lange O, Denisov E, Nolting D, Müller M, Viner R, Schwartz J, Remes P, Belford M, Dunyach JJ, Cox J, Horning S, Mann M, Makarov A - Mol. Cell Proteomics (2011)

Parallel CID top20 method and ultra high resolution survey scans.A, High resolution MS scan at three different transient lengths followed by 20 CID MS/MS scans in the linear ion trap. Note that cycle time is unaffected by the resolution of the full scan. Preview refers to the portion of the survey scan that is used to select precursor ions for fragmentation. B, LC MS heat map of peptides eluting over a 3 min elution time interval in a 40 Th range. More detail is visible in the ultra high resolution setting (left panel) compared with the normal resolution setting (right panel). C, Separation of isobaric species in standard LC MS/MS analysis. The 34S isotope containing peak is clearly resolved from the 13C2 isotope.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3316736&req=5

Figure 4: Parallel CID top20 method and ultra high resolution survey scans.A, High resolution MS scan at three different transient lengths followed by 20 CID MS/MS scans in the linear ion trap. Note that cycle time is unaffected by the resolution of the full scan. Preview refers to the portion of the survey scan that is used to select precursor ions for fragmentation. B, LC MS heat map of peptides eluting over a 3 min elution time interval in a 40 Th range. More detail is visible in the ultra high resolution setting (left panel) compared with the normal resolution setting (right panel). C, Separation of isobaric species in standard LC MS/MS analysis. The 34S isotope containing peak is clearly resolved from the 13C2 isotope.
Mentions: To investigate the instrument capability for the analysis of very complex peptide mixtures such as HeLa cell lysate, we started with a digested standard of 400 ng that was analyzed using different methods. Comparison of the normal CID and rapid CID scan modes revealed that rCID produced significantly more fragmentation events and therefore this mode was chosen for all subsequent experiments. The scheme in Fig. 4A shows the timing sequence of MS and MS/MS scans at different MS transient lengths. For a 192 ms survey scan, resolution is 60,000 and there is no parallel operation between MS and MS/MS scans. Due to rCID an entire top20 method takes 2.7 s, easily compatible with peptide LC elution profiles. At 384 ms resolution is 120,000 and a few MS/MS scans are performed in parallel with the survey scan. At the full 768 ms transient (240,000 resolution), about six CID spectra are performed in parallel, while total cycle time is still unchanged (Fig. 4A). Therefore, the longest transients appear to be advantageous because the increased resolution comes “for free” as it does not cost extra measurement time.

Bottom Line: Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas.For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s-increasing protein identifications in complex mixtures by about 30%.The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.

View Article: PubMed Central - PubMed

Affiliation: Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.

ABSTRACT
Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm-incorporating phase information-further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s-increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.

Show MeSH
Related in: MedlinePlus