title: The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics.
abstract: Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. 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. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. 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.
authors.affiliation: From the ‡Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
authors.forename: Scarlet
authors.initials: S
authors.lastname: Beck
authors.affiliation: §Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany;
authors.forename: Annette
authors.initials: A
authors.lastname: Michalski
authors.affiliation: §Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany;
authors.forename: Oliver
authors.initials: O
authors.lastname: Raether
authors.affiliation: §Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany;
authors.forename: Markus
authors.initials: M
authors.lastname: Lubeck
authors.affiliation: §Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany;
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kaspar
authors.affiliation: §Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany;
authors.forename: Niels
authors.initials: N
authors.lastname: Goedecke
authors.affiliation: §Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany;
authors.forename: Carsten
authors.initials: C
authors.lastname: Baessmann
authors.affiliation: From the ‡Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
authors.forename: Daniel
authors.initials: D
authors.lastname: Hornburg
authors.affiliation: From the ‡Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
authors.forename: Florian
authors.initials: F
authors.lastname: Meier
authors.affiliation: From the ‡Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
authors.forename: Igor
authors.initials: I
authors.lastname: Paron
authors.affiliation: From the ‡Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
authors.forename: Nils A
authors.initials: NA
authors.lastname: Kulak
authors.affiliation: ¶Computational Systems Biochemistry, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Juergen
authors.initials: J
authors.lastname: Cox
authors.affiliation: From the ‡Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany; mmann@biochem.mpg.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
mesh_terms: D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid; D004171:Diploidy; D006238:Haploidy; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D013058:Mass Spectrometry; D051379:Mice; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013997:Time Factors
chemical_list: D007477:Ions; D010455:Peptides; D020543:Proteome