The quadrupole Orbitrap mass spectrometer (Q Exactive) made a powerful proteomics instrument available in a benchtop format. It significantly boosted the number of proteins analyzable per hour and has now evolved into a proteomics analysis workhorse for many laboratories. Here we describe the Q Exactive Plus and Q Exactive HF mass spectrometers, which feature several innovations in comparison to the original Q Exactive instrument. A low-resolution pre-filter has been implemented within the injection flatapole, preventing unwanted ions from entering deep into the system, and thereby increasing its robustness. A new segmented quadrupole, with higher fidelity of isolation efficiency over a wide range of isolation windows, provides an almost 2-fold improvement of transmission at narrow isolation widths. Additionally, the Q Exactive HF has a compact Orbitrap analyzer, leading to higher field strength and almost doubling the resolution at the same transient times. With its very fast isolation and fragmentation capabilities, the instrument achieves overall cycle times of 1 s for a top 15 to 20 higher energy collisional dissociation method. We demonstrate the identification of 5000 proteins in standard 90-min gradients of tryptic digests of mammalian cell lysate, an increase of over 40% for detected peptides and over 20% for detected proteins. Additionally, we tested the instrument on peptide phosphorylation enriched samples, for which an improvement of up to 60% class I sites was observed. The quadrupole Orbitrap mass spectrometer (Q Exactive) made a powerful proteomics instrument available in a benchtop format. It significantly boosted the number of proteins analyzable per hour and has now evolved into a proteomics analysis workhorse for many laboratories. Here we describe the Q Exactive Plus and Q Exactive HF mass spectrometers, which feature several innovations in comparison to the original Q Exactive instrument. A low-resolution pre-filter has been implemented within the injection flatapole, preventing unwanted ions from entering deep into the system, and thereby increasing its robustness. A new segmented quadrupole, with higher fidelity of isolation efficiency over a wide range of isolation windows, provides an almost 2-fold improvement of transmission at narrow isolation widths. Additionally, the Q Exactive HF has a compact Orbitrap analyzer, leading to higher field strength and almost doubling the resolution at the same transient times. With its very fast isolation and fragmentation capabilities, the instrument achieves overall cycle times of 1 s for a top 15 to 20 higher energy collisional dissociation method. We demonstrate the identification of 5000 proteins in standard 90-min gradients of tryptic digests of mammalian cell lysate, an increase of over 40% for detected peptides and over 20% for detected proteins. Additionally, we tested the instrument on peptide phosphorylation enriched samples, for which an improvement of up to 60% class I sites was observed. Mass spectrometry (MS)-based 1The abbreviations used are:MSmass spectrometryMS/MStandem mass spectrometryACNacetonitrile.1The abbreviations used are:MSmass spectrometryMS/MStandem mass spectrometryACNacetonitrile. proteomics aims at the comprehensive analysis of proteins present in a biological sample (1Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5598) Google Scholar), and the field has expanded in many surprising directions (2Altelaar A.F. Heck A.J. Trends in ultrasensitive proteomics.Curr. Opin. Chem. Biol. 2012; 16: 206-213Crossref PubMed Scopus (116) Google Scholar). Application of the developed techniques has revealed novel insights into fundamental biology, as well as produced analysis techniques with implications for clinical applications. A major hurdle, however, is the complexity of the systems under scrutiny, as it has been shown that human cell lines, for instance, express at least 10,000 genes that are detectable as proteins (3Nagaraj N. Wisniewski J.R. Geiger T. Cox J. Kircher M. Kelso J. Paabo S. Mann M. Deep proteome and transcriptome mapping of a human cancer cell line.Mol. Syst. Biol. 2011; 7: 548Crossref PubMed Scopus (757) Google Scholar, 4Geiger T. Wehner A. Schaab C. Cox J. Mann M. Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins.Mol. Cell. Proteomics. 2012; (M111.014050)Abstract Full Text Full Text PDF Scopus (579) Google Scholar, 5Beck M. Schmidt A. Malmstroem J. Claassen M. Ori A. Szymborska A. Herzog F. Rinner O. Ellenberg J. Aebersold R. The quantitative proteome of a human cell line.Mol. Syst. Biol. 2011; 7: 549Crossref PubMed Scopus (588) Google Scholar). If we further consider all the peptides produced in bottom-up proteomics experiments, this hurdle is compounded, as ideally many hundreds of thousands of analytes should be characterized in order for the proteins giving rise to them to be fully reconstructed (6Nesvizhskii A.I. Aebersold R. Interpretation of shotgun proteomic data: the protein inference problem.Mol. Cell. Proteomics. 2005; 4: 1419-1440Abstract Full Text Full Text PDF PubMed Scopus (794) Google Scholar). In principle, issues of sample complexity and dynamic range could be addressed by a very high degree of up-front fractionation. However, this strategy faces diminishing returns and leads to unacceptably long analysis times for most purposes. Given the fact that even with optimal chromatographic resolution many peptides with abundance differences of many orders of magnitude elute within the same time frame, there remains a need to improve the mass spectrometric detection in terms of speed, resolution, and sensitivity. mass spectrometry tandem mass spectrometry acetonitrile. mass spectrometry tandem mass spectrometry acetonitrile. Nanoscale liquid chromatography coupled online to mass spectrometry is the current technique of choice for the analysis of complex peptide mixtures. In a top-N shotgun strategy, a full scan, providing a complete overview of isotope patterns resulting from ionized peptides, is followed by N fragmentation scans performed on the most abundant not-yet-sequenced isotope patterns currently visible in the full scan. During fragmentation, the goal is to cleanly isolate the intended precursor peptide ion, which today is generally done either by a linear ion trap or by a quadrupole mass filter. Fragment ions are then mass measured by an Orbitrap mass analyzer, a time-of-flight analyzer, or, less often, ion cyclotron resonance–Fourier transform or linear or three-dimensional ion traps. Apart from the MS instrumentation, recent developments in the proteomics workflow include a move toward automated online quality control systems (7Pichler P. Mazanek M. Dusberger F. Weilnbock L. Huber C.G. Stingl C. Luider T.M. Straube W.L. Kocher T. Mechtler K. SIMPATIQCO: a server-based software suite which facilitates monitoring the time course of LC-MS performance metrics on Orbitrap instruments.J. Proteome Res. 2012; 11: 5540-5547Crossref PubMed Scopus (43) Google Scholar, 8Scheltema R.A. Mann M. SprayQc: a real-time LC-MS/MS quality monitoring system to maximize uptime using off the shelf components.J. Proteome Res. 2012; 11: PubMed Scopus Google and N. Cox J. N. K. O. O. Mann M. analysis with complete of the proteome by on a Cell. Proteomics. 2011; Scholar), which very peptide chromatography T. R. Mechtler K. to an reveals a linear and number of Chem. 2011; PubMed Scopus (116) Google Scholar, Geiger T. P. F. Cox J. Mann M. Deep and proteome by LC-MS/MS Cell. Proteomics. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). The Orbitrap mass was almost and on this very in proteomics R.A. Orbitrap mass Chem. PubMed Scopus Google Scholar). of an mass spectrometer coupled to a which and the ion up to to injection into the Orbitrap to the Orbitrap and of this of the precursor the in the linear ion trap J. O. P. M. M. A. Mann M. S. A linear ion trap instrument with very high Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar), but a an instrument on a quadrupole Q Exactive mass developed A. O. A. A. N. Cox J. Mann M. S. Mass spectrometry-based proteomics using Q a benchtop quadrupole Orbitrap mass Cell. Proteomics. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). with the linear ion quadrupole mass the of of a mass by the a of ions to the we this mass and the of ions of ion ions of or precursor to analysis in the Orbitrap mass A. O. A. A. N. Cox J. Mann M. S. Mass spectrometry-based proteomics using Q a benchtop quadrupole Orbitrap mass Cell. Proteomics. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). However, that instrument the efficiency quadrupole S. C. M. T. proteomic on mass Cell. Proteomics. 2012; 11: Full Text Full Text PDF PubMed Scopus Google Scholar), and it the compact Orbitrap that been in the Orbitrap instrument A. O. M. R. J. P. M. Cox J. S. Mann M. A. high resolution linear ion trap Orbitrap mass spectrometer facilitates top and peptide fragmentation Cell. Proteomics. 2011; Google Scholar). Here we describe the into the Q Exactive Plus and the Q Exactive HF include by a low-resolution of the quadrupole, a segmented quadrupole, in the of the Q Exactive HF an mass Orbitrap analyzer, doubling resolution or We describe in the of complex analysis of peptides and The instrument is on the Q Exactive mass spectrometer A. O. A. A. N. Cox J. Mann M. S. Mass spectrometry-based proteomics using Q a benchtop quadrupole Orbitrap mass Cell. Proteomics. 2011; Full Text Full Text PDF PubMed Scopus Google with the same of an ion a ion and an injection in the a a at the and to them from entering further into the a segmented quadrupole mass a a higher energy collisional dissociation and an Orbitrap mass In to the the injection was with ion capabilities, providing a low-resolution for the of ions from the ion higher resolution in the quadrupole mass filter. The segmented quadrupole mass has ion transmission and mass the instrument be with a compact Orbitrap (Q Exactive leading to a higher field and higher of ion in leads to almost the resolution in the same time to the cell A. O. M. R. J. P. M. Cox J. S. Mann M. A. high resolution linear ion trap Orbitrap mass spectrometer facilitates top and peptide fragmentation Cell. Proteomics. 2011; Google Scholar). this be used to the at the same resolution, a strategy we The mass range in the Orbitrap is mass by the quadrupole is up to and isolation be and The instrument software the ion injection time to for of transmission the isolation with the Orbitrap remains the same as for the Orbitrap it from for at to 10,000 at to for at to at The in the Orbitrap be analysis of most analytes peptides and proteins. in 20 and at for with and was and cell in liquid and at of in 1 of then 1 of was and for on a at cycle and control and 1 of 20 of at of was to and at for 15 was then performed by of and for at in the The sample was using of to the to which the sample was for 1 at by at an of and then at the of at a of then with and with to the The peptide was using a from a of using the sample J.R. A. N. Mann M. sample for proteome PubMed Scopus Google Scholar). In cell in at for and at cycle and control The protein was from at using an of A of of protein was then on top of per and with of was performed with for at in the in the same further with of in and with of was performed by at an of and at The peptides from the with a and then further by at a of at for peptides in liquid and per in of and peptides by at for 1 into new and with of on a for from all the then and times with of and times with of then in of into J. Mann M. and for and sample in Chem. 2003; PubMed Scopus Google Scholar), and with from with of 60% in in 40% The was to to and up to with enriched peptides and with to the The peptide was using a The was to with and at chromatography was performed with the system coupled online to either an original Q Exactive or a Q Exactive HF with a with in A During analysis the was in a to a of A peptide of was the with A at a of resulting in a of and with a linear of to and at a of by over at a of to the and the time for an LC-MS/MS was to quality automated detection of and was performed with R.A. Mann M. SprayQc: a real-time LC-MS/MS quality monitoring system to maximize uptime using off the shelf components.J. Proteome Res. 2012; 11: PubMed Scopus Google Scholar). MS using a for the Q Exactive and a for the Q Exactive the most abundant not-yet-sequenced precursor ions from the scans was performed higher energy collisional dissociation fragmentation with a of ions with control of was performed with a of for the Q Exactive and for the Q Exactive of the quadrupole scans at a resolution of at on the Q Exactive and at on the Q Exactive HF and for was to at with a ion injection time of on the Q Exactive and at with ion injection time of on the Q Exactive HF The energy was for the Q Exactive and for the Q Exactive HF this was to in the instrument The the of the ion to be at the was as the was at which optimal transmission of the by the peptides from We precursor ions with or and higher from fragmentation In the comparison the Q Exactive and the Q Exactive we that there was a of the number of ions for fragmentation on the Q Exactive HF to the Q Exactive this an on the performance of the we performance on this we that the original Q Exactive of ions optimal for the Q Exactive Plus and Q Exactive HF with the proteomics analysis J. Mann M. high peptide identification mass and protein PubMed Scopus Google Scholar), with the J. N. A. R.A. Mann M. a peptide into the Proteome Res. 2011; PubMed Scopus Google Scholar). The was at for peptide and to a of and a mass of was used to fragmentation scans for identification on a with an mass of the precursor ion of up to mass The mass was 20 by in the human with common J. N. A. R.A. Mann M. a peptide into the Proteome Res. 2011; PubMed Scopus Google Scholar). was as to and at and a of We of as a and protein and as for the cell was as a for the phosphorylation enriched analysis of the was performed with for the of from the mass spectrometry on and with the and R. 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S. N. L. R. Aebersold R. of the by a new for and proteome Cell. Proteomics. 2012; Full Text Full Text PDF PubMed Scopus Google and of in ion monitoring which isolation in order to quantitative at to the quadrupole in the Q we isolation efficiency at the of the isolation Additionally, the transmission of the quadrupole for narrow isolation has been We measured this efficiency for the of ions and an almost 2-fold increase that the a transmission for isolation windows, for that is to the of the of the improvement to the increase in ion current to the doubling of the that the Orbitrap is of the time to the transient time in fully of the the isolation that we used transmission this A. Cox J. Mann M. detectable peptide elute in shotgun proteomics but the is to Proteome Res. 2011; PubMed Scopus Google Scholar). the segmented quadrupole the to narrow isolation windows, we tested a range of on a complex cell We the with and the in which to a peptide from scans of the with the peptide J. N. A. R.A. Mann M. a peptide into the Proteome Res. 2011; PubMed Scopus Google Scholar). The Q Exactive HF performed in terms of peptide at an isolation of with and the peptide for the Q Exactive this was The isolation in a to the peptide by the peptide of the of peptides that be in this the optimal of this was from at the optimal isolation of We the of isolation by the precursor ion A. Cox J. Mann M. detectable peptide elute in shotgun proteomics but the is to Proteome Res. 2011; PubMed Scopus Google for the same range of isolation the isolation of the of a precursor ion to degree be for tandem mass M. M. N. M. fragmentation and isolation for improvement of protein identification and of mass on mass Chem. 2011; PubMed Scopus Google Scholar, A. J. K. S. J. Schmidt T. R. C. mass a novel strategy for analysis of complex protein by Chem. 2003; PubMed Scopus Google Scholar). the optimal isolation of the precursor ion was to in the of this is well within the range of for a and fragmentation we this isolation as the The Orbitrap of an in of and a the of with the at the of the and the at a for A. O. M. R. J. P. M. Cox J. S. Mann M. A. high resolution linear ion trap Orbitrap mass spectrometer facilitates top and peptide fragmentation Cell. Proteomics. 2011; Google Scholar). In a standard Orbitrap analyzer, and 15 M. Schmidt A. Malmstroem J. Claassen M. Ori A. Szymborska A. Herzog F. Rinner O. Ellenberg J. Aebersold R. The quantitative proteome of a human cell line.Mol. Syst. Biol. 2011; 7: 549Crossref PubMed Scopus (588) Google Scholar), the is with and the is by a of A of the from to an increase in the field and the detected in to the the to The cell an increase of the injection ion energy of for the same on the the increase in in the by a of the by the even in the standard A. O. of a Orbitrap mass Mass PubMed Scopus Google Scholar). A provides of ions into the injection on of the injection ion from the the are the need for and a of with the of from the facilitates the of transform O. A. A. transform for Orbitrap mass J. Mass Scopus Google for all of in the same as in the Q Exactive instrument. 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