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The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics.

Beck S, Michalski A, Raether O, Lubeck M, Kaspar S, Goedecke N, Baessmann C, Hornburg D, Meier F, Paron I, Kulak NA, Cox J, Mann M - Mol. Cell Proteomics (2015)

Bottom Line: The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222.We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell.Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum-the highest proteome coverage reported with a QTOF instrument so far.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;

No MeSH data available.


Related in: MedlinePlus

Ion transfer efficiency of mass range most relevant in shotgun-proteomics experiments (m/z 100–1500). Inset shows net analyte ion currents at the indicated measurement points and transfer efficiencies at the orthogonal accelerator at various stages along the flight path of the impact II instrument.
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Figure 3: Ion transfer efficiency of mass range most relevant in shotgun-proteomics experiments (m/z 100–1500). Inset shows net analyte ion currents at the indicated measurement points and transfer efficiencies at the orthogonal accelerator at various stages along the flight path of the impact II instrument.

Mentions: The number of ions that successfully pass through the instrument and are finally recorded determine a mass spectrometer's sensitivity. We were interested in the transfer efficiencies along the ion path, from entering the vacuum system to the detector. To experimentally determine this, we infused a 1 pmol/μl BSA solution or blank solution and measured the difference in the ion current between these conditions. When operating the outlet of the capillary and the funnel region as a Faraday cage, we measured a net ion current of 63 pA, which we defined as the starting value (100%) (Fig. 3). The large ion acceptance aperture of the first funnel efficiently captures in the ion flux leaving the capillary. It transfers the ions to the second funnel, which also passes the ions in an almost lossless manner through the next stage as evidenced by a net current reading of 57 pA (>90%) after the hexapole. Likewise, more than 90% of the ions are transferred through the quadrupole (in nonmass selecting operation) and the collision cell. More than 60% of these ions are transmitted into the flight tube by the orthogonal accelerator (see above). The reflectron contains two grids, which are each passed twice, leading to a geometrically defined overall transmission of 74%. Although all these ions hit the MCP, not all of them enter the channels and not all of them result in secondary electrons (detector quantum yield). However, when secondary ions are generated they are greatly amplified (>106 fold), and can be efficiently discriminated from electronic noise (zero noise detection). Together, this leads to an estimated detection probability of 30% for our MCP detector. Although not all of these measurements and estimates are very precise, they suggest an overall detection probability of ions transmitted into the vacuum system of about 10%. This excellent number is because of the fact that the continuous beam generated by the ES source can be utilized and that all the ion guiding elements have been successfully optimized for high transmission.


The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics.

Beck S, Michalski A, Raether O, Lubeck M, Kaspar S, Goedecke N, Baessmann C, Hornburg D, Meier F, Paron I, Kulak NA, Cox J, Mann M - Mol. Cell Proteomics (2015)

Ion transfer efficiency of mass range most relevant in shotgun-proteomics experiments (m/z 100–1500). Inset shows net analyte ion currents at the indicated measurement points and transfer efficiencies at the orthogonal accelerator at various stages along the flight path of the impact II instrument.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587313&req=5

Figure 3: Ion transfer efficiency of mass range most relevant in shotgun-proteomics experiments (m/z 100–1500). Inset shows net analyte ion currents at the indicated measurement points and transfer efficiencies at the orthogonal accelerator at various stages along the flight path of the impact II instrument.
Mentions: The number of ions that successfully pass through the instrument and are finally recorded determine a mass spectrometer's sensitivity. We were interested in the transfer efficiencies along the ion path, from entering the vacuum system to the detector. To experimentally determine this, we infused a 1 pmol/μl BSA solution or blank solution and measured the difference in the ion current between these conditions. When operating the outlet of the capillary and the funnel region as a Faraday cage, we measured a net ion current of 63 pA, which we defined as the starting value (100%) (Fig. 3). The large ion acceptance aperture of the first funnel efficiently captures in the ion flux leaving the capillary. It transfers the ions to the second funnel, which also passes the ions in an almost lossless manner through the next stage as evidenced by a net current reading of 57 pA (>90%) after the hexapole. Likewise, more than 90% of the ions are transferred through the quadrupole (in nonmass selecting operation) and the collision cell. More than 60% of these ions are transmitted into the flight tube by the orthogonal accelerator (see above). The reflectron contains two grids, which are each passed twice, leading to a geometrically defined overall transmission of 74%. Although all these ions hit the MCP, not all of them enter the channels and not all of them result in secondary electrons (detector quantum yield). However, when secondary ions are generated they are greatly amplified (>106 fold), and can be efficiently discriminated from electronic noise (zero noise detection). Together, this leads to an estimated detection probability of 30% for our MCP detector. Although not all of these measurements and estimates are very precise, they suggest an overall detection probability of ions transmitted into the vacuum system of about 10%. This excellent number is because of the fact that the continuous beam generated by the ES source can be utilized and that all the ion guiding elements have been successfully optimized for high transmission.

Bottom Line: The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222.We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell.Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum-the highest proteome coverage reported with a QTOF instrument so far.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;

No MeSH data available.


Related in: MedlinePlus