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. 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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 Text Full Text PDF PubMed Scopus (647) Google Scholar). time-of-flight all ion fragmentation collision induced dissociation electron transfer dissociation false discovery rate Fourier transform higher energy collisional dissociation high performance liquid chromatography linear trap quadrupole tandem mass spectrometry predictive automatic gain control radio frequency selected ion monitoring. The quadrupole TOF instruments achieve peptide separation "in space", meaning the ions are separated nearly instantaneously by passing through either the quadrupole section, in which only a chosen small mass range has stable trajectories, or by traversing the TOF section. In contrast, trapping instruments such as linear ion traps separate ions "in time" by applying external RF-DC fields to a stationary ion population that allow only a certain ion population to stably remain in the trap (see ref (11Louris J.N. Cooks R.G. Syka J.E.P. Kelley P.E. Stafford G.C. Todd J.F.J. Instrumentation, applications, and energy deposition in quadrupole ion-trap tandem mass-spectrometry.Anal. Chem. 1987; 59: 1677-1685Crossref Scopus (454) Google Scholar)) for the concept of separation and fragmentation in time versus in space). The Orbitrap mass analyzer was developed about ten years ago by Makarov. It consists of a small electrostatic device into which ion packets are injected at high energies to orbit around a central, spindle-shaped electrode (12Hardman M. Makarov A.A. Interfacing the orbitrap mass analyzer to an electrospray ion source.Anal. Chem. 2003; 75: 1699-1705Crossref PubMed Scopus (248) Google Scholar, 13Makarov A. Electrostatic axially harmonic orbital trapping: a high-performance technique of mass analysis.Anal. Chem. 2000; 72: 1156-1162Crossref PubMed Scopus (646) Google Scholar, 14Scigelova M. Makarov A. Orbitrap mass analyzer–overview and applications in proteomics.Proteomics. 2006; 6: 16-21Crossref PubMed Scopus (174) Google Scholar). The image current of the axial motion of the ions is picked up by the detector and this signal is Fourier transformed (FT) to yield high resolution mass spectra. Commercially, the Orbitrap analyzer was first introduced in 2005 in a hybrid instrument (15Syka J.E. Marto J.A. Bai D.L. Horning S. Senko M.W. Schwartz J.C. Ueberheide B. Garcia B. Busby S. Muratore T. Shabanowitz J. Hunt D.F. Novel linear quadrupole ion trap/FT mass spectrometer: performance characterization and use in the comparative analysis of histone H3 post-translational modifications.J. Proteome Res. 2004; 3: 621-626Crossref PubMed Scopus (333) Google Scholar). In proteomics and related fields, this combination of a low resolution linear ion trap with the high resolution Orbitrap analyzer—termed "LTQ Orbitrap"—has now become widespread (16Makarov A. Denisov E. Lange O. Horning S. Dynamic range of mass accuracy in LTQ Orbitrap hybrid mass spectrometer.J. Am. Soc. Mass Spectrom. 2006; 17: 977-982Crossref PubMed Scopus (338) Google Scholar, 17Makarov A. Denisov E. Kholomeev A. Balschun W. Lange O. Strupat K. Horning S. Performance evaluation of a hybrid linear ion trap/orbitrap mass spectrometer.Anal. Chem. 2006; 78: 2113-2120Crossref PubMed Scopus (592) Google Scholar). The LTQ Orbitrap instruments represent a multistage trap combination (Fig. 1). In MS mode the linear trap performs the function of collecting the ion population, passing them on to an intermediate C-trap for injection and analysis in the Orbitrap analyzer at high resolution. In MS/MS mode the linear ion trap only retains a chosen mass window, which is activated by a supplemental RF field leading to fragmentation of the trapped precursor ions, and records the signal of a mass dependent scan at low resolution. Note that the high resolution MS scan can be performed at the same time as the low resolution MS/MS scans in the linear ion trap. Recently, an improved linear ion trap Orbitrap analyzer combination termed "LTQ Orbitrap Velos" has been introduced (18Olsen J.V. Schwartz J.C. Griep-Raming J. Nielsen M.L. Damoc E. Denisov E. Lange O. Remes P. Taylor D. Splendore M. Wouters E.R. Senko M. Makarov A. Mann M. Horning S. A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed.Mol. Cell Proteomics. 2009; 8: 2759-2769Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). It features an S-lens with up to 10-fold improved ion transmission from the atmosphere, a dual linear ion trap, and a more efficient Higher energy Collisional Dissociation (HCD) cell interfaced directly to the C-trap (18Olsen J.V. Schwartz J.C. Griep-Raming J. Nielsen M.L. Damoc E. Denisov E. Lange O. Remes P. Taylor D. Splendore M. Wouters E.R. Senko M. Makarov A. Mann M. Horning S. A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed.Mol. Cell Proteomics. 2009; 8: 2759-2769Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). HCD fragmentation is similar to the fragmentation in triple quadrupole or quadrupole TOF instruments and its products are analyzed with high mass accuracy in the Orbitrap analyzer (19Olsen J.V. Macek B. Lange O. Makarov A. Horning S. Mann M. Higher-energy C-trap dissociation for peptide modification analysis.Nat. Methods. 2007; 4: 709-712Crossref PubMed Scopus (720) Google Scholar). Thus, the LTQ Orbitrap or LTQ Orbitrap Velos instruments offer versatile fragmentation modes depending on the analytical problem (20Macek B. Waanders L.F. Olsen J.V. Mann M. Top-down protein sequencing and MS3 on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer.Mol. Cell Proteomics. 2006; 5: 949-958Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 21McAlister G.C. Berggren W.T. Griep-Raming J. Horning S. Makarov A. Phanstiel D. Stafford G. Swaney D.L. Syka J.E. Zabrouskov V. Coon J.J. A proteomics grade electron transfer dissociation-enabled hybrid linear ion trap-orbitrap mass spectrometer.J. Proteome Res. 2008; 7: 3127-3136Crossref PubMed Scopus (124) Google Scholar, 22McAlister G.C. Phanstiel D.H. Westphall M.S. Coon J.J. Higher-energy collision-activated dissociation without a dedicated collision cell.Mol. Cellular Proteomics. 2011; 10 (O111.009456)Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Taking advantage of the small size of the Orbitrap analyzer a standalone benchtop instrument termed "Exactive" has been introduced mainly for small molecule applications. However, because of the absence of mass selection, its use in proteomics is limited to non-mass selective fragmentation of the entire mass range (called "All Ion Fragmentation" (AIF) on this instrument (23Geiger T. Cox J. Mann M. Proteomics on an Orbitrap benchtop mass spectrometer using all-ion fragmentation.Mol. Cell Proteomics. 2010; 9: 2252-2261Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar)). The combination of a quadrupole mass filter with an Orbitrap analyzer has not yet been reported. We reasoned that such a quadrupole trap combination might offer unique and complementary advantages to the hybrid mass spectrometers described above. In particular, a quadrupole Exactive instrument or "Q Exactive" would be able to select ions virtually instantaneously because of the fast switching times of quadrupoles and it would be able to fragment peptides in HCD mode on a similarly fast time scale. Furthermore, because of the small size and mature technology used in current quadrupole mass filters, this analyzer combination should have a small footprint and be particularly robust. Finally, the ability to separate "in space" and analyze MS and MS/MS ranges at high resolution in the Orbitrap analyzer offers the promise of enabling efficient multiplexed scan modes not currently applied in proteomics research using trapping instruments. The Q Exactive instrument includes an atmospheric pressure ion source (API), a stacked-ring ion guide (S-lens) in the source region, a quadrupole mass filter, a C-trap, an HCD cell, and an Orbitrap mass analyzer as shown in Fig. 2. Ions are formed at atmospheric pressure (in this work in a nanoelectrospray ion source), pass through a transfer tube to an S-lens described in (18Olsen J.V. Schwartz J.C. Griep-Raming J. Nielsen M.L. Damoc E. Denisov E. Lange O. Remes P. Taylor D. Splendore M. Wouters E.R. Senko M. Makarov A. Mann M. Horning S. A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed.Mol. Cell Proteomics. 2009; 8: 2759-2769Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar) and then via an injection multipole into a bent flatapole. The bent flatapole has 2-mm gaps between its rods, oriented in such a way that the line of sight from the S-lens is open for clusters and droplets to fly unimpeded out of the flatapole. After collisional cooling in the bent flatapole, ions are transmitted via a lens into a hyperbolic quadrupole (r0 = 4 mm), capable of isolating ions down to an isolation width of 0.4 Th at m/z 400. The quadrupole is followed by its exit lens combined with a split lens used to gate the incoming ion beam. A short octapole then brings ions into the C-trap interfaced to an HCD cell with axial field (18Olsen J.V. Schwartz J.C. Griep-Raming J. Nielsen M.L. Damoc E. Denisov E. Lange O. Remes P. Taylor D. Splendore M. Wouters E.R. Senko M. Makarov A. Mann M. Horning S. A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed.Mol. Cell Proteomics. 2009; 8: 2759-2769Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). The gas-filled HCD cell is separated from the C-trap only by a single diaphragm, allowing easy HCD tuning. Fragmentation of ions in the HCD cell is achieved by adjusting the offset of the RF rods and the axial field to provide the required collision energy. As long as this offset remains negative relative to the C-trap and the HCD exit lenses, all fragments remain trapped inside the HCD cell, even if the offset of the RF rods is varied. This allows to introduce multiple precursor ions and to fragment them at their optimum collision energy without compromising the storage of preceding injections. The summed ion population can then be transferred back into the C-trap, ejected into the Orbitrap analyzer and analyzed in a single Orbitrap detection cycle. This opens the possibility of fundamentally new, "multiplexing" modes of operation. In practice, the useful number of ion injections for a single Orbitrap detection is limited by the sum of the individual inject times being lower than the time for the Orbitrap scan. A new challenge posed by interfacing the Orbitrap analyzer to a quadrupole is the automatic gain control (AGC) of weak ion signals. This problem was addressed by using an AGC pre-scan for a full MS spectrum with subsequent prediction of the ion currents for the weak signals on the basis of their share in total ion current (predictive AGC). The mass range covered by the instrument is m/z 50–4000, with the range of mass selection reaching m/z 2500. Acquisition speed ranges from 12 Hz for resolving power 17,500 at m/z 200 (corresponding to 12,500 at m/z 400) to 1.5 Hz for resolving power 140,000 at m/z 200 (corresponding to 100,000 at m/z 400). Vacuum in the Orbitrap compartment is typically below 7 × 10−10 mBar, which makes the analyzer adequate for high resolution analysis of most analytes, including large peptides and small proteins. The ability to fill the HCD cell or the C-trap with ions while a previous Orbitrap detection cycle is still ongoing is another important innovation that allows to significantly reduce the influence of low ion currents on acquisition speed and quality of spectra. Transients detected in the Orbitrap mass analyzer are processed using an enhanced version of Fourier Transformation (eFT™) for conversion of transients into frequency and then m/z. Details of the technique can be found in (24Lange O. Makarov A. Denisov E. Balschun W. Accelerating spectral acquisition rate of Orbitrap mass spectrometry.Proc. 58th Conf. Amer. Soc. Mass Spectrom. 2010; Google Scholar). Both eFT and conventional FT make use of complex numbers, which can be represented by magnitude and phase, or by real and imaginary components. As the initial phase of the ion package appears to be dependent on initial parameters of the ions in a very complex way (25Vining B.A. Bossio R.E. Marshall A.G. Phase correction for collision model analysis and enhanced resolving power of fourier transform ion cyclotron resonance mass spectra.Anal. Chem. 1999; 71: 460-467Crossref PubMed Scopus (29) Google Scholar), FT spectra have to be presented in the so-called magnitude mode, which amounts to disregarding the phase information. However, in Orbitrap mass spectrometers the built-in excitation-by-injection mechanism (26Makarov A. Practical Aspects of Trapped Ion Mass Spectrometry.in: March R.E. Todd J.F.J. Theory and Instrumentation. Vol 4. CRC Press (Taylor & Francis), 2009Google Scholar) provides an initial phase of ion oscillations that is almost m/z independent. This synchronization allows converting spectra in such a way that the real component of data can be utilized, which results in narrower peaks. In practice, eFT uses a combination of the magnitude and the real component of the signal to improve mass accuracy and peak shape. Better accuracy of synchronization is achieved, if detection starts as early as possible after ion injection. For this reason, modifications of preamplifier and Orbitrap analyzer were introduced to reduce the delay between ion injection and start of transient detection from almost 10 ms to a fraction of a millisecond. Practical implementation of eFT achieves between 1.8- and 2-fold increase of resolving power for the same transient (except for rapidly decaying signals, for example from proteins, where the gain is reduced to about 1.4-fold because of "hard sphere" collisions with background gas). The dual-spectrum online processing is computationally demanding but still fast enough to be completed in the LC MS time scale. Thus cycle time is still determined by transient acquisition and ion injection times and not by processing of the data. The eFT method is sensitive to precise synchronization of the instrument electronics and remaining shot-to-shot jitter, so that final mass accuracy is comparable to that of traditional magnitude mode FT spectra. Side-lobes in eFT spectra are comparable to those in conventional FT spectra. HeLa cells were lysed and the pellet was dissolved in a urea (6 m) and thiourea (2 m) solution. Proteins were reduced with dithiotreitol (1 mm) for 30 min at room temperature followed by alkylation with iodoacetamide (55 mm) for 20 min in the dark. The mixture was incubated with LysC (1 μg/50 μg protein) (Wako, Richmond, VA) at room temperature for 3 h before 1:4 dilution with water. Incubation with trypsin (1 μg/50 μg protein) (Promega, Madison, WI) was carried out for 12 h at room temperature. The digestion was stopped by addition of formic acid (3%). Organic solvent was removed in a SpeedVac concentrator. The peptide mixture was desalted on reversed phase C18 StageTips (27Rappsilber J. Ishihama Y. Mann M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.Anal. Chem. 2003; 75: 663-670Crossref PubMed Scopus (1804) Google Scholar). Directly before analysis, peptides were eluted into 8 well autosampler vials with 60 μl buffer B (80% acetonitrile in 0.5% acetic acid). Organic solvent was removed in a SpeedVac concentrator and the final sample volume was adjusted with buffer A* (2% acetonitrile in 0.1% trifluoroacetic acid) to 12 μl. A nanoflow HPLC instrument (Easy nLC, Proxeon Biosystems, now Thermo Fisher Scientific) was coupled on-line to a Q Exactive or an LTQ Orbitrap Velos mass spectrometer (both from Thermo Fisher Scientific) with a nanoelectrospray ion source (Proxeon). Chromatography columns were packed in-house with ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH) in buffer A (0.5% acetic acid). The peptide mixture (5 μg) was loaded onto a C18-reversed phase column (15 cm long, 75 μm inner diameter) and separated with a linear gradient of 5–60% buffer B (80% acetonitrile and 0.5% acetic acid) at a flow rate of 250 nL/min controlled by IntelliFlow technology over 90 min. Because of loading and washing steps, the total time for an LC MS/MS run was about 40–50 min longer. MS data was acquired using a data-dependent top10 method dynamically choosing the most abundant precursor ions from the survey scan (300–1650 Th) for HCD fragmentation. Target values on Q Exactive were similar to those typically used on an LTQ Orbitrap Velos. Determination of the target value is based on predictive Automatic Gain Control (pAGC) in both instruments. However, the LTQ Orbitrap Velos is equipped with electron multipliers, which allows scaling of the number of ions in a direct manner. In contrast, scaling of the number of ions is more indirect on the Q Exactive accounting for the difference in target values for the same S/N. Dynamic exclusion duration was 60 s with early expiration disabled on the LTQ Orbitrap Velos. Isolation of precursors was performed with a 4-Th window and MS/MS scans were acquired with a starting mass of 100 Th. Survey scans were acquired at a resolution of 70,000 at m/z 200 on the Q Exactive and 30,000 at m/z 400 on the LTQ Orbitrap Velos (see Results and Discussion and Table I for conversion of resolution values to different m/z values). Resolution for HCD spectra was set to 17,500 at m/z 200 on the Q Exactive and 7500 at m/z 400 on the LTQ Orbitrap Velos. Normalized collision energy was 30 eV for the Q Exactive and 35 eV for the LTQ Orbitrap Velos—they are not identical because of different scaling functions in the instrument software. The underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1% on the Q Exactive. For the LTQ Orbitrap Velos the lower threshold for targeting a precursor ion in the MS scans was 5,000 counts. Both instruments were run with peptide recognition mode enabled, but exclusion of singly charged and unassigned precursor ions was only enabled on the LTQ Orbitrap Velos. This was because of the higher sequencing speed of the Q Exactive and a slightly different precursor selection algorithm for the data-dependent scans. However, in practice there was not much difference between the settings with regard to the number of identified unique peptides and proteins.Table IFour Q Exactive resolution settings and transient timesResolution @ m/z = 200 ThResolution @ m/z = 400 ThTransient length17,50012,50064 ms35,00025,000128 ms70,00050,000256 ms140,000100,000512 ms Open table in a new tab To demonstrate multiplexing of selected ion monitoring (SIM) scans, a method alternating full scans and SIM scans over the entire gradient was set up on the Q Exactive. The 92 min range in which peptides eluted was divided into 23 segments of 4 min duration. For each of these segments, three SIM windows of 2 Th width were defined, centered around 69 randomly chosen, low abundance precursor ions observed in these elution time windows in a previous top10 run. Pre-selection of these low abundance peptides was carried out manually based on the msms.txt file resulting from MaxQuant analysis. The method for multiplexed SIM scans was specified using the "Targeted SIM" template in the Q Exactive method editor. Resolution was set to 140,000 at m/z 200 and a target value of 1e6 ions for both scan types was chosen. The maximum ion injection time was set to 10 ms for the full scan and to 100 ms for each of the multiplexed SIMs. The inclusion list was saved in the global list features and in the data-dependent settings page "inclusion" was set to "on." Multiplexing of MS/MS spectra was done in exactly the same format as the standard top10 method, except that "msx" in the method setup of the data-dependent scans was set to 2 for multiplexing the fragment ions of two consecutively selected precursors. The mass spectrometric raw data from top10 methods were analyzed with the MaxQuant software (developmental version 1.1.1.32) (28Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9223) Google Scholar). The false discovery rate (FDR) was set to 0.01 for proteins and peptides, which had to have a minimum length of 6 amino acids. MaxQuant was used to score peptides for identification based on a search with an initial allowed mass deviation of the precursor ion of up to 7 ppm. The allowed fragment mass deviation was 20 ppm. Search of the MS/MS spectra against the International Protein Index human data base (version 3.68, 87,061 entries) combined with 262 common contaminants was performed using the Andromeda search engine (29Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: A Peptide Search Engine Integrated into the MaxQuant Environment.J. Proteome Res. 2011; 11: 1794-1805Crossref Scopus (3474) Google Scholar). Enzyme specificity was set as C-terminal to Arg and Lys, also allowing cleavage at proline bonds and a maximum of two missed cleavages. Carbamidomethylation of cysteine was set as fixed modification and N-terminal protein acetylation and methionine oxidation as variable modifications. MaxQuant applied time-dependent recalibration to the precursor masses for improved mass accuracy. Further analysis of the data provided by MaxQuant was performed in the R scripting and statistical environment (30Ihaka