Mass Spectrometry-based Proteomics Using Q Exactive, a High-performance Benchtop Quadrupole Orbitrap Mass Spectrometer
Mass spectrometry-based proteomics has greatly benefitted from enormous advances in high resolution instrumentation in recent years. In particular, the combination of a linear ion trap with the Orbitrap analyzer has proven to be a popular instrument configuration. Complementing this hybrid trap-trap instrument, as well as the standalone Orbitrap analyzer termed Exactive, we here present coupling of a quadrupole mass filter to an Orbitrap analyzer. This “Q Exactive” instrument features high ion currents because of an S-lens, and fast high-energy collision-induced dissociation peptide fragmentation because of parallel filling and detection modes. The image current from the detector is processed by an “enhanced Fourier Transformation” algorithm, doubling mass spectrometric resolution. Together with almost instantaneous isolation and fragmentation, the instrument achieves overall cycle times of 1 s for a top10 higher energy collisional dissociation method. More than 2500 proteins can be identified in standard 90-min gradients of tryptic digests of mammalian cell lysate— a significant improvement over previous Orbitrap mass spectrometers. Furthermore, the quadrupole Orbitrap analyzer combination enables multiplexed operation at the MS and tandem MS levels. This is demonstrated in a multiplexed single ion monitoring mode, in which the quadrupole rapidly switches among different narrow mass ranges that are analyzed in a single composite MS spectrum. Similarly, the quadrupole allows fragmentation of different precursor masses in rapid succession, followed by joint analysis of the higher energy collisional dissociation fragment ions in the Orbitrap analyzer. High performance in a robust benchtop format together with the ability to perform complex multiplexed scan modes make the Q Exactive an exciting new instrument for the proteomics and general analytical communities. Mass spectrometry-based proteomics has greatly benefitted from enormous advances in high resolution instrumentation in recent years. In particular, the combination of a linear ion trap with the Orbitrap analyzer has proven to be a popular instrument configuration. Complementing this hybrid trap-trap instrument, as well as the standalone Orbitrap analyzer termed Exactive, we here present coupling of a quadrupole mass filter to an Orbitrap analyzer. This “Q Exactive” instrument features high ion currents because of an S-lens, and fast high-energy collision-induced dissociation peptide fragmentation because of parallel filling and detection modes. The image current from the detector is processed by an “enhanced Fourier Transformation” algorithm, doubling mass spectrometric resolution. Together with almost instantaneous isolation and fragmentation, the instrument achieves overall cycle times of 1 s for a top10 higher energy collisional dissociation method. More than 2500 proteins can be identified in standard 90-min gradients of tryptic digests of mammalian cell lysate— a significant improvement over previous Orbitrap mass spectrometers. Furthermore, the quadrupole Orbitrap analyzer combination enables multiplexed operation at the MS and tandem MS levels. This is demonstrated in a multiplexed single ion monitoring mode, in which the quadrupole rapidly switches among different narrow mass ranges that are analyzed in a single composite MS spectrum. Similarly, the quadrupole allows fragmentation of different precursor masses in rapid succession, followed by joint analysis of the higher energy collisional dissociation fragment ions in the Orbitrap analyzer. High performance in a robust benchtop format together with the ability to perform complex multiplexed scan modes make the Q Exactive an exciting new instrument for the proteomics and general analytical communities. Mass spectrometry-based proteomics often involves the analysis of complex mixtures of proteins derived from cell or tissue lysates or from body fluids, posing tremendous analytical challenges (1Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5598) Google Scholar, 2Yates 3rd, J.R. Gilchrist A. Howell K.E. Bergeron J.J. Proteomics of organelles and large cellular structures.Nat. Rev. 2005; 6: 702-714Crossref Scopus (345) Google Scholar, 3Walther T.C. Mann M. Mass spectrometry-based proteomics in cell biology.J. Cell Biol. 2010; 190: 491-500Crossref PubMed Scopus (307) Google Scholar). After proteolytic digestion, the resulting peptide mixtures are separated by liquid chromatography and online electrosprayed for mass spectrometric (MS) and tandem mass spectrometric (MS/MS) analysis. Because tens of thousands of peptides elute over a relatively short time and with ion signals different by many orders of magnitude (4Michalski A. Cox J. Mann M. More than 100,000 Detectable Peptide Species Elute in Single Shotgun Proteomics Runs but the Majority is Inaccessible to Data-Dependent LC-MS/MS.J. Proteome Res. 2011; 10: 1785-1793Crossref PubMed Scopus (480) Google Scholar, 5Köcher T. Swart R. Mechtler K. Ultra-High-Pressure RPLC Hyphenated to an LTQ-Orbitrap Velos Reveals a Linear Relation between Peak Capacity and Number of Identified Peptides.Anal. Chem. 2011; 83: 2699-2704Crossref PubMed Scopus (116) Google Scholar), mass spectrometers have been pushed to even higher sensitivity, sequencing speed, and resolution (6Domon B. Aebersold R. Mass spectrometry and protein analysis.Science. 2006; 312: 212-217Crossref PubMed Scopus (1619) Google Scholar, 7Mann M. Kelleher N.L. Precision proteomics: the case for high resolution and high mass accuracy.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 18132-18138Crossref PubMed Scopus (353) Google Scholar). In current shotgun proteomics there are mainly four mass spectrometric separation principles: quadrupole mass filters, time of flight (TOF) 1The abbreviations used are:TOFtime-of-flightAIFall ion fragmentationCIDcollision induced dissociationETDelectron transfer dissociationFDRfalse discovery rateFTFourier transformHCDhigher energy collisional dissociationHPLChigh performance liquid chromatographyLTQlinear trap quadrupoleMS/MStandem mass spectrometrypAGCpredictive automatic gain controlRFradio frequencySIMselected ion monitoring. mass analyzers, linear ion traps, and Orbitrap™ analyzers. These are typically combined in hybrid configurations. Quadrupole TOF instruments use a quadrupole mass filter to either transmit the entire mass range produced by the ion source (for analysis of all ions in MS mode) or to transmit only a defined mass window around a precursor ion of choice (MS/MS mode). In the latter case ions are activated in a collision cell and resulting fragments are analyzed in the TOF part of the instrument with very high repetition rate. This TOF part of quadrupole TOF instruments replaces the final quadrupole section of triple quadrupole instruments, which are today mainly used for targeted proteomics (8Wolf-Yadlin A. Hautaniemi S. Lauffenburger D.A. White F.M. Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5860-5865Crossref PubMed Scopus (431) Google Scholar, 9Addona T.A. Abbatiello S.E. Schilling B. Skates S.J. Mani D.R. Bunk D.M. Spiegelman C.H. Zimmerman L.J. Ham A.J. Keshishian H. Hall S.C. Allen S. Blackman R.K. Borchers C.H. Buck C. Cardasis H.L. Cusack M.P. Dodder N.G. Gibson B.W. Held J.M. Hiltke T. Jackson A. Johansen E.B. Kinsinger C.R. Li J. Mesri M. Neubert T.A. Niles R.K. Pulsipher T.C. Ransohoff D. Rodriguez H. Rudnick P.A. Smith D. Tabb D.L. Tegeler T.J. Variyath A.M. Vega-Montoto L.J. Wahlander A. Waldemarson S. Wang M. Whiteaker J.R. Zhao L. Anderson N.L. Fisher S.J. Liebler D.C. Paulovich A.G. Regnier F.E. Tempst P. Carr S.A. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.Nat. Biotechnol. 2009; 27: 633-641Crossref PubMed Scopus (865) Google Scholar, 10Picotti P. Bodenmiller B. Mueller L.N. Domon B. Aebersold R. Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics.Cell. 2009; 138: 795-806Abstract Full Full PubMed Scopus Google Scholar). all ion fragmentation collision induced dissociation transfer dissociation discovery Fourier higher energy collisional dissociation high performance liquid chromatography linear trap quadrupole tandem mass spectrometry automatic gain ion monitoring. 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Proteomics an Orbitrap benchtop mass Cell 2010; Full Full PubMed Scopus Google The combination of a quadrupole mass filter with an Orbitrap analyzer has been that a quadrupole trap combination and to the hybrid mass spectrometers In particular, a quadrupole Exactive instrument or “Q Exactive” be to ions because of the fast times of and be to fragment peptides in a fast time Furthermore, because of the and used in current quadrupole mass filters, this analyzer combination have a and be the ability to and MS and ranges at high resolution in the Orbitrap analyzer the of multiplexed scan modes in proteomics The Q Exactive instrument an ion source a ion in the source a quadrupole mass a an and an Orbitrap mass analyzer as in are at this in a ion a transfer to an in J. P. D. M. M. A. Mann M. 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The cell is separated from the only by a single of ions in the cell is by the of the and the to the collision as this to the and the all fragments the even the of the is This allows to multiple precursor ions and to fragment at collision energy the of The ion can be the the Orbitrap analyzer and analyzed in a single Orbitrap detection This the of modes of In the of ion for a single Orbitrap detection is by the of the times than the time for the Orbitrap new by the Orbitrap analyzer to a quadrupole is the automatic gain of ion This by an for a MS with of the ion currents for the signals the of in ion current The mass range by the instrument is with the range of mass ranges from for at to at to for at to 100,000 at in the Orbitrap is typically which the analyzer for high resolution analysis of large peptides and The ability to the cell or the with ions a previous Orbitrap detection cycle is is that allows to the of ion currents and of in the Orbitrap mass analyzer are processed an of Fourier for of and of the can be in A. of Orbitrap mass Mass 2010; Google Scholar). and make use of complex which can be by magnitude and or by and the of the ion to be of the ions in a very complex A.G. for collision analysis and of ion mass Chem. PubMed Scopus Google Scholar), have to be in the magnitude mode, which to the in Orbitrap mass spectrometers the A. of Mass and an of ion that is almost This allows in a that the of can be which in In a combination of the magnitude and the of the to mass and of is detection as as ion this of and Orbitrap analyzer to the between ion and of detection from almost to a of a of achieves between and of for the for rapidly for from the gain is to because of with The online is but fast to be in the MS time cycle time is by and ion times and by of the The is to of the instrument and that final mass is to that of magnitude in are to in and the in a and with for at followed by with for in the The with at for with with for at The by of in a The peptide J. Mann M. and for and in Chem. 2003; PubMed Scopus Google Scholar). peptides well with in in a and the final with in to instrument Fisher to a Q Exactive or an Orbitrap Velos mass from Fisher with a ion source with in The peptide a and separated with a linear of and at a of by over Because of and the time for an MS a top10 the precursor ions from the scan for Q Exactive to typically used an Orbitrap of the is in the Orbitrap Velos is with which allows of the of ions in a In of the of ions is the Q Exactive for the in for the s with the Orbitrap of with a window and with a mass of at a resolution of at the Q Exactive and at the Orbitrap Velos and and for of resolution to different for to at the Q Exactive and at the Orbitrap collision energy for the Q Exactive and for the Orbitrap are because of different in the instrument The which the of the to be at defined as the Q the Orbitrap Velos the for a precursor ion in the MS instruments with peptide but of and precursor ions only the Orbitrap This because of the higher sequencing of the Q Exactive and a different precursor for the in there between the with to the of identified peptides and Q Exactive resolution and in a new of ion monitoring a and over the entire the Q The range in which peptides of of of around precursor ions in time in a previous top10 of peptides the resulting from analysis. The for multiplexed the in the Q Exactive to at and a of ions for scan The ion time to for the scan and to for of the multiplexed The in the features and in the to of in the format as the standard top10 that in the of the to for the fragment ions of The mass spectrometric from top10 analyzed with the J. Mann M. enables high peptide mass and protein Biotechnol. 2008; PubMed Scopus Google Scholar). The discovery to for proteins and which to have a of used to peptides for a with an mass of the precursor ion of to The fragment mass of the the combined with the J. A. Mann M. Peptide the Proteome Res. 2011; Scopus Google Scholar). as to and at and a of of as and protein and as to the precursor masses for mass analysis of the by in the and R. R. for analysis and Scholar). The used for analysis are at to a high performance quadrupole Orbitrap mass in a of the are in but here we a by the Exactive The Exactive have mass and mainly for M. H. R. L. a benchtop Orbitrap mass Mass 2009; PubMed Scopus Google Scholar). can be with a higher energy collisional dissociation cell at the of the the detection of the Exactive allows fragmentation B. A. S. Mann M. dissociation for peptide 2007; PubMed Scopus Google mass This is for this instrument and can be used in proteomics T. Cox J. Mann M. Proteomics an Orbitrap benchtop mass Cell 2010; Full Full PubMed Scopus Google Scholar). mass in the Q Exactive, the from source to to for which we used the in the Orbitrap Velos J. P. D. M. M. A. Mann M. S. linear ion trap Orbitrap instrument with very high sequencing Cell 2009; Full Full PubMed Scopus Google Scholar). rapidly the instrument from ion the Orbitrap analyzer is the as in previous Orbitrap analyzers. The Orbitrap is as is the Exactive and higher than the Orbitrap The Q Exactive a ion from the source the mass analyzer in with the Exactive and in to the Orbitrap The of the Q Exactive with the Exactive is the of a mass quadrupole analyzer between the ion source and the This quadrupole is the as that used in triple quadrupole instruments, features a of of mass a of of the Q Exactive is to that of the The quadrupole mass filter has very and the instrument is in a In the analysis of complex peptides of mass often and resolution is a of a mass in M. Kelleher N.L. Precision proteomics: the case for high resolution and high mass accuracy.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 18132-18138Crossref PubMed Scopus (353) Google Scholar). 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Cox J. Mann M. More than 100,000 Detectable Peptide Species Elute in Single Shotgun Proteomics Runs but the Majority is Inaccessible to Data-Dependent LC-MS/MS.J. Proteome Res. 2011; 10: 1785-1793Crossref PubMed Scopus (480) Google Scholar). with top10 the Velos instrument, which have the of parallel the Q Exactive to be in J. P. D. M. M. A. Mann M. S. linear ion trap Orbitrap instrument with very high sequencing Cell 2009; Full Full PubMed Scopus Google Scholar). The for the very fast cycle times are allows short times ion filling is in parallel with detection times of have been precursor is in a and peptide fragmentation is Because of the parallel ion in the Q Exactive, times than the the overall cycle In with complex mixtures times for in the range of 1 to and for between and to parallel and detection in almost all The times here are to of the Orbitrap Velos J. P. D. M. M. A. Mann M. S. linear ion trap Orbitrap instrument with very high sequencing Cell 2009; Full Full PubMed Scopus Google Scholar), of instrument in scan and the performance of the Q Exactive for shotgun we a of a mammalian cell The peptide separated by in a by standard used in The entire analysis in and for an Orbitrap a of the MS signals by peptides from the over the The in the is a of a the of in this peptide derived from cell can be the MS and peptide well The MS in a single MS scan the and a precursor that for that the Q Exactive all fragmentation is by and are with high resolution. This enables of as in and high fragment mass The analyzed in with the J. Mann M. enables high peptide mass and protein Biotechnol. 2008; PubMed Scopus Google Scholar, J. A. Mann M. Peptide the Proteome Res. 2011; Scopus Google Scholar). the of the of the Q Exactive The of MS in of and the of in of that top10 sequencing is only there are peptide in the MS scan that for The of to a very high that the (4Michalski A. Cox J. Mann M. More than 100,000 Detectable Peptide Species Elute in Single Shotgun Proteomics Runs but the Majority is Inaccessible to Data-Dependent LC-MS/MS.J. Proteome Res. 2011; 10: 1785-1793Crossref PubMed Scopus (480) Google Scholar, 5Köcher T. Swart R. Mechtler K. Ultra-High-Pressure RPLC Hyphenated to an LTQ-Orbitrap Velos Reveals a Linear Relation between Peak Capacity and Number of Identified Peptides.Anal. Chem. 2011; 83: 2699-2704Crossref PubMed Scopus (116) Google Scholar), because of the short MS and cycle time of 1 peptides identified in for a of peptides in the analysis. 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This allows isolation and fragmentation in different of the instruments and in isolation and fragmentation many of operation modes are the Q Exactive, that this combination of a mass filter and an Orbitrap analyzer enables scan The scan modes is the that mass which is fast This almost complex and of MS and mass ranges followed by high resolution analysis in the Orbitrap analyzer. multiplexed scan at the MS and at the In ion monitoring a narrow mass range is to for ions of are in many but are often Orbitrap This is because the isolation of the mass range in the linear ion trap is relatively time there is a the of ions that can be and the analysis of even a single scan as the mass to the mass Li B. P. R. A. S. Mann M. mass an Orbitrap mass mass a Cell 2005; Full Full PubMed Scopus Google Scholar), multiple mass range filling of the Orbitrap has been The Q Exactive have the for allows mass ranges of In this mode, the is used as a which is with the of ions from to different These ions are together the Orbitrap analyzer and in the as mass Because times are typically than MS multiplexed use Orbitrap instrument time this in a cell in a that a gain TOF instruments because are by scan peptides to be of from a previous top10 to mass ranges the range MS In and a of the the peptides are with very to The ion time of scan than 1 ions for of the for for the this high sensitivity, peptides from the but from the targeted These multiplexed analyzed together in resolution to the overall cycle time to the quadrupole at mass window multiplexed an in targeted peptide analysis and peptide In complex sequencing can be a In fragmentation of with of the fragments can the of in with a linear ion in the multiple of peptide isolation and fragmentation an the The Q Exactive, can different precursor ions and fragment in the cell at an collision energy for of precursor ions is only the all fragment ions are in the cell and joint analysis in the Orbitrap analyzer we a top10 with of a of a multiplexed analyzed at the resolution of of the from of multiplexed as to is by the that the of ions can be for targeted is that multiplexed are the Q Exactive be to resulting gain in peptide sequencing is the only of multiplexed an the fragmentation of different of proteins have the and performance of a new of mass the combination of a quadrupole mass filter with the Orbitrap analyzer. The quadrupole is of the robust and mass combination with the relatively Orbitrap mass of a high performance instrument with a and These make the instrument an to the proteomics as proteomics is and by with or