Abstract

In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, whereas mass spectra are acquired in about 100 microseconds with time-of-flight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive because of its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al., PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 600,000 fragmentation spectra in standard 120 min LC runs are achievable, which can be used for near exhaustive precursor selection in complex mixtures or accumulating the signal of weak precursors. In 120 min single runs of HeLa digest, MaxQuant identified more than 6,000 proteins without matching to a library and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,500 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored. In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, whereas mass spectra are acquired in about 100 microseconds with time-of-flight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive because of its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al., PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 600,000 fragmentation spectra in standard 120 min LC runs are achievable, which can be used for near exhaustive precursor selection in complex mixtures or accumulating the signal of weak precursors. In 120 min single runs of HeLa digest, MaxQuant identified more than 6,000 proteins without matching to a library and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,500 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored. Jointly, proteins form a cellular machinery—the proteome—that orchestrates essentially all biological processes in health and disease. Studying it on a system-wide scale holds great promise to advance our understanding of cellular biology and disease mechanisms (1Altelaar A.F.M. Munoz J. Heck A.J.R. Next-generation proteomics: towards an integrative view of proteome dynamics.Nat. Rev. Genet. 2012; 14: 35-48Crossref PubMed Scopus (521) Google Scholar, 2Larance M. Lamond A.I. Multidimensional proteomics for cell biology.Nat. Rev. Mol. Cell Biol. 2015; 16: 269-280Crossref PubMed Scopus (291) Google Scholar, 3Aebersold R. Mann M. Mass-spectrometric exploration of proteome structure and function.Nature. 2016; 537: 347-355Crossref PubMed Scopus (1113) Google Scholar). However, as compared with genomics and transcriptomics technologies, proteomics still lags in terms of coverage, throughput, and sensitivity. Virtually complete measurements of mammalian proteomes have become possible (4Bekker-Jensen D.B. Kelstrup C.D. Batth T.S. Larsen S.C. Haldrup C. Bramsen J.B. Sørensen K.D. Høyer S. Ørntoft T.F. Andersen C.L. Nielsen M.L. Olsen J.V. An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes.Cell Syst. 2017; 4: 587-599.e4Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), but have mostly involved laborious sample preparation workflows, days of measurement time and substantial amounts of starting material. Furthermore, current high-performance instrumentation often requires expert knowledge and extensive maintenance, which impedes widespread adaptation of proteomics in nonspecialized laboratories. In bottom-up workflows, proteins are extracted from a biological sample of interest and enzymatically cleaved to make them more amenable to mass spectrometric (MS) analysis. The resulting complex peptide mixtures are typically separated via nano-flow liquid chromatography (LC) 1The abbreviations used are:LCliquid chromatographyCCScollisional cross sectionIMSion mobility spectrometryPASEFparallel accumulation–serial fragmentationTIMStrapped ion mobility spectrometry. 1The abbreviations used are:LCliquid chromatographyCCScollisional cross sectionIMSion mobility spectrometryPASEFparallel accumulation–serial fragmentationTIMStrapped ion mobility spectrometry., ionized by electrospray and mass analyzed. In "data-dependent" or "topN" acquisition schemes, the mass spectrometer detects suitable peptide precursor ions in full scans (MS) and selects them for fragmentation in N consecutive MS/MS scans. High resolution and high mass accuracy analyzers detect hundreds of thousands of distinct molecular features in single LC-MS experiments, of which only a minority is identified and quantified (5Michalski 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). These co-eluting peptides with abundances ranging over many orders of magnitude present a formidable analytical challenge, which has constantly pushed the development of faster and more sensitive instrumentation over the last decades (1Altelaar A.F.M. Munoz J. Heck A.J.R. Next-generation proteomics: towards an integrative view of proteome dynamics.Nat. Rev. Genet. 2012; 14: 35-48Crossref PubMed Scopus (521) Google Scholar, 3Aebersold R. Mann M. Mass-spectrometric exploration of proteome structure and function.Nature. 2016; 537: 347-355Crossref PubMed Scopus (1113) Google Scholar, 6Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5596) Google Scholar, 7Eliuk S. Makarov A. Evolution of Orbitrap Mass Spectrometry Instrumentation.Annu. Rev. Anal. Chem. 2015; 8: 61-80Crossref PubMed Scopus (252) Google Scholar). liquid chromatography collisional cross section ion mobility spectrometry parallel accumulation–serial fragmentation trapped ion mobility spectrometry. liquid chromatography collisional cross section ion mobility spectrometry parallel accumulation–serial fragmentation trapped ion mobility spectrometry. Time-of-flight (TOF) instruments have several very desirable properties for the analysis of complex peptide mixtures and have consequently been employed in shotgun proteomics for a long time (8Domon B. Aebersold R. Mass spectrometry and protein analysis.Science. 2006; 312: 212-217Crossref PubMed Scopus (1619) Google Scholar, 9Han X. Aslanian A. Yates J.R. Mass spectrometry for proteomics.Curr. Opin. Chem. Biol. 2008; 12: 483-490Crossref PubMed Scopus (504) Google Scholar). Instrumental performance has steadily improved over the years, and our groups have described shotgun proteome measurements at a mass resolution of more than 35,000 within about 100 μs on the impact II (10Beck S. Michalski A. Raether O. Lubeck M. Kaspar S. Goedecke N. Baessmann C. Hornburg D. Meier F. Paron I. Kulak N.A. Cox J. Mann M. The Impact II, a very high-resolution quadrupole time-of-flight instrument (QTOF) for deep shotgun proteomics.Mol. Cell. Proteomics. 2015; 14: 2014-2029Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), the predecessor of the instrument that is the subject of this paper. The high acquisition rate of TOF instruments allows coupling them with very fast separation techniques, such as ion mobility spectrometry (IMS) (11Kanu A.B. Dwivedi P. Tam M. Matz L. Hill H.H. Ion mobility-mass spectrometry.J. Mass Spectrom. 2008; 43: 1-22Crossref PubMed Scopus (885) Google Scholar, 12Cumeras R. Figueras E. Davis C.E. Baumbach J.I. Gràcia I. Review on Ion Mobility Spectrometry. Part 2: hyphenated methods and effects of experimental parameters.Analyst. 2015; 140: 1391-1410Crossref PubMed Google Scholar, 13May J.C. McLean J.A. Ion mobility-mass spectrometry: time-dispersive instrumentation.Anal. Chem. 2015; 87: 1422-1436Crossref PubMed Scopus (279) Google Scholar). IMS separates ions in the gas phase based on their size and shape, or more precisely their collisional cross section (CCS, Ω), typically within 10s to 100s of milliseconds (14Eiceman G.A. Karpas Z. Hill H.H.J. Ion Mobility Spectrometry. 3rd Ed. CRC Press, 2013Crossref Google Scholar). As the ions emerge from the IMS device, they can be efficiently sampled in the ms or sub-ms time frame with TOF analyzers. Nested between LC and MS, the technology provides an additional dimension of separation (15Valentine S.J. Counterman A.E. Hoaglund C.S. Reilly J.P. Clemmer D.E. Gas-phase separations of protease digests.J. Am. Soc. Mass Spectrom. 1998; 9: 1213-1216Crossref PubMed Scopus (97) Google Scholar, 16Srebalus Barnes C.A. Hilderbrand A.E. Valentine S.J. Clemmer D.E. Resolving isomeric peptide mixtures: A combined HPLC/ion mobility-TOFMS analysis of a 4000-component combinatorial library.Anal. Chem. 2002; 74: 26-36Crossref PubMed Scopus (90) Google Scholar, 17Ewing M.A. Glover M.S. Clemmer D.E. Hybrid ion mobility and mass spectrometry as a separation tool.J. Chromatogr. A. 2015; : 27-29Google Scholar) and can increase analysis speed and selectivity (18Lanucara F. Holman S.W. Gray C.J. Eyers C.E. The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics.Nat. Chem. 2014; 6: 281-294Crossref PubMed Scopus (659) Google Scholar), also with highly complex proteomics samples (19Valentine S.J. Plasencia M.D. Liu X. Krishnan M. Naylor S. Udseth H.R. Smith R.D. Clemmer D.E. Toward plasma proteome profiling with ion mobility-mass spectrometry.J. Proteome Res. 2006; 5: 2977-2984Crossref PubMed Scopus (128) Google Scholar, 20Baker E.S. Livesay E.A. Orton D.J. Moore R.J. Danielson W.F. Prior D.C. Ibrahim Y.M. LaMarche B.L. Mayampurath A.M. Schepmoes A.A. Hopkins D.F. Tang K. Smith R.D. Belov M.E. An LC-IMS-MS platform providing increased dynamic range for high-throughput proteomic studies.J. Proteome Res. 2010; 9: 997-1006Crossref PubMed Scopus (106) Google Scholar, 21Geromanos S.J. Hughes C. Ciavarini S. Vissers J.P.C. Langridge J.I. Using ion purity scores for enhancing quantitative accuracy and precision in complex proteomics samples.Anal. Bioanal. Chem. 2012; 404: 1127-1139Crossref PubMed Scopus (42) Google Scholar, 22Helm D. Vissers J.P.C. Hughes C.J. Hahne H. Ruprecht B. Pachl F. Grzyb A. Richardson K. Wildgoose J. Maier S.K. Marx H. Wilhelm M. Becher I. Lemeer S. Bantscheff M. Langridge J.I. Kuster B. Ion mobility tandem mass spectrometry enhances performance of bottom-up proteomics.Mol. Cell. Proteomics. 2014; 13: 3709-3715Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 23Distler U. Kuharev J. Navarro P. Levin Y. Schild H. Tenzer S. Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics.Nat. Methods. 2014; 11: 167-170Crossref PubMed Scopus (306) Google Scholar). However, many implementations of IMS, such as drift tubes, are challenging because of the device sizes and high voltages involved and may also limit the proportion of the continuous incoming beam that can be utilized (12Cumeras R. Figueras E. Davis C.E. Baumbach J.I. Gràcia I. Review on Ion Mobility Spectrometry. Part 2: hyphenated methods and effects of experimental parameters.Analyst. 2015; 140: 1391-1410Crossref PubMed Google Scholar, 13May J.C. McLean J.A. Ion mobility-mass spectrometry: time-dispersive instrumentation.Anal. Chem. 2015; 87: 1422-1436Crossref PubMed Scopus (279) Google Scholar, 24Cumeras R. Figueras E. Davis C.E. Baumbach J.I. Gràcia I. Review on Ion Mobility Spectrometry. Part 1: current instrumentation.Analyst. 2015; 140: 1376-1390Crossref PubMed Google Scholar). Trapped ion mobility spectrometry (TIMS) (25Fernandez-Lima F.A. Kaplan D.A. Park M.A. Note: Integration of trapped ion mobility spectrometry with mass spectrometry.Rev. Sci. Instrum. 2011; 82: 126106Crossref PubMed Scopus (163) Google Scholar, 26Fernandez-Lima F. Kaplan D.A. Suetering J. Park M.A. Gas-phase separation using a trapped ion mobility spectrometer.Int. J. Ion Mobil. Spectrom. 2011; 14: 93-98Crossref Scopus (214) Google Scholar) reverses the concept of traditional drift tube ion mobility by bringing ions to a rest at different positions in an ion tunnel device, balanced in an electrical field against a constant gas stream (27Ridgeway M.E. Lubeck M. Jordens J. Mann M. Park M.A. Trapped ion mobility spectrometry: A short review.Int. J. Mass Spectrom. 2018; 425: 22-35Crossref Scopus (159) Google Scholar). Once enough ions have been trapped and separated, lowering the electrical potential releases time-resolved ions from the TIMS device into the downstream mass analyzer. This design reduces the IMS analyzer dimensions to about 10 centimeters in length—allowing two of them to be implemented in series for 100% duty cycle operation (28Silveira J.A. Ridgeway M.E. Laukien F.H. Mann M. Park M.A. Parallel accumulation for 100% duty cycle trapped ion mobility-mass spectrometry.Int. J. Mass Spectrom. 2017; 413: 168-175Crossref Scopus (44) Google Scholar). TIMS furthermore offers high flexibility in that users can tune the ion mobility resolving power (Ω/ΔFWHMΩ) up to 200 or higher by simply lowering the TIMS scan speed (29Silveira J.A. Ridgeway M.E. Park M.A. High resolution trapped ion mobility spectrometry of peptides.Anal. Chem. 2014; 86: 5624-5627Crossref PubMed Scopus (132) Google Scholar, 30Ridgeway M.E. Silveira J.A. Meier J.E. Park M.A. Microheterogeneity within conformational states of ubiquitin revealed by high resolution trapped ion mobility spectrometry.Analyst. 2015; 140: 6964-6972Crossref PubMed Google Scholar). We have recently introduced "Parallel Accumulation - SErial Fragmentation" (PASEF) (31Meier F. Beck S. Grassl N. Lubeck M. Park M.A. Raether O. Mann M. Parallel accumulation–serial fragmentation (PASEF): multiplying sequencing speed and sensitivity by synchronized scans in a trapped ion mobility device.J. Proteome Res. 2015; 14: 5378-5387Crossref PubMed Scopus (179) Google Scholar), which synchronizes MS/MS precursor selection with TIMS separation. This acquisition scheme allows fragmentation of more than one precursor per TIMS scan and we demonstrated that PASEF increases the sequencing speed severalfold without loss of sensitivity. As precursor ions are accumulated in parallel, PASEF overcomes the diminishing returns of increasingly fast MS/MS acquisition, which otherwise necessarily implied less and less ions per spectrum. Our first iteration was implemented on a laboratory prototype, which required manual precursor programming and was limited by the speed of the electronics involved. Here, we describe the construction and investigate the proteomics performance of the first mass spectrometer that fully integrates the PASEF concept, the Bruker timsTOF Pro. Human cervical cancer cells (HeLa S3, ATCC, Manassas, VA) were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 20 mm glutamine and 1% penicillin-streptomycin (all Life Technologies Ltd., Paisley, UK). Escherichia coli (strain: XL1 blue) was cultured at 37 °C in LB medium until logarithmic phase (optical density = 0.5, λ = 600 nm). Cells were collected by centrifugation. Following a washing step with cold phosphate buffered saline, they were pelleted and flash frozen in liquid nitrogen and stored at −80 °C. One-device cell lysis, reduction, and alkylation was performed in sodium deoxycholate (SDC) buffer with chloroacetamide (PreOmics GmbH, Martinsried, Germany) according to our previously published protocol (32Kulak N.A. Pichler G. Paron I. Nagaraj N. Mann M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.Nat. Methods. 2014; 11: 319-324Crossref PubMed Scopus (1002) Google Scholar). Briefly, the cell suspension was twice boiled for 10 min at 95 °C and subsequently sonicated for 15 min at maximum energy (Bioruptor, Diagenode, Seraing, Belgium). Proteins were enzymatically hydrolyzed overnight at 37 °C by LysC and trypsin (1:100 enzyme:protein (wt/wt) for both). To stop the digestion, the reaction mixture was acidified with five volumes of isopropanol with 1% trifluoroacetic acid (TFA). Peptides were de-salted and purified in two steps, first on styrenedivinylbenzene-reversed phase sulfonate (SDB-RPS), and second on C18 sorbent. The dried eluates were re-constituted in water with 2% acetonitrile (ACN) and 0.1% TFA for direct LC-MS analysis or high pH reversed-phase fractionation. For the experiments with the Evosep One (see below), HeLa cell pellets were re-suspended and lysed in water/trifluoroethanol. Disulfide bonds were reduced with dithiothreitol and alkylated with iodoacetamide in ammonium bicarbonate buffer. Following tryptic digestion, the peptide mixture was de-salted and purified on C18 sorbent. High pH reversed-phase fractionation was performed on an EASY-nLC 1000 (Thermo Fisher Scientific, Bremen, Germany) coupled to a "spider fractionator" (PreOmics) as detailed in ref (33Kulak N.A. Geyer P.E. Mann M. Loss-less nano-fractionator for high sensitivity, high coverage proteomics.Mol. Cell. Proteomics. 2017; (mcp.O116.065136)Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Purified peptides were separated on a 30 cm × 250 μm reversed-phase column (PreOmics) at a flow rate of 2 μl/min at pH 10. The binary gradient started from 3% buffer B (PreOmics), followed by linear increases to first 30% B within 45 min, to 60% B within 17 min, and finally to 95% B within 5 min. Each sample was automatically concatenated into 48 fractions in 90 s time intervals. The fractions were dried in a vacuum-centrifuge and reconstituted in water with 2% ACN and 0.1% TFA for LC-MS analysis. An EASY-nLC 1200 (Thermo Fisher Scientific) ultra-high pressure nano-flow chromatography system was coupled online to a hybrid trapped ion mobility spectrometry - quadrupole time of flight mass spectrometer (timsTOF Pro, Bruker Daltonics, Bremen, Germany) with a modified nano-electrospray ion source (10Beck S. Michalski A. Raether O. Lubeck M. Kaspar S. Goedecke N. Baessmann C. Hornburg D. Meier F. Paron I. Kulak N.A. Cox J. Mann M. The Impact II, a very high-resolution quadrupole time-of-flight instrument (QTOF) for deep shotgun proteomics.Mol. Cell. Proteomics. 2015; 14: 2014-2029Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) (CaptiveSpray, Bruker Daltonics). Liquid chromatography was performed at 60 °C and with a constant flow of 400 nL/min on a reversed-phase column (50 cm × 75 μm i.d.) with a pulled emitter tip, packed with 1.9 μm C18-coated porous silica beads (Dr. Maisch, Ammerbuch-Entringen, Germany). Mobile phases A and B were water with 0.1% formic acid (v/v) and 80/20/0.1% ACN/water/formic acid (v/v/vol), respectively. In 120-min experiments, peptides were separated with a linear gradient from 7.5 to 27.5% B within 60 min, followed by an increase to 37.5% B within 30 min and further to 55% within 10 min, followed by a washing step at 95% B and re-equilibration. In 60 min separations, the gradient increased from 10 to 30% B within 30 min, followed by an increase to 40% B within 15 min and further to 57.5% B within 5 min before washing and re-equilibration. In 30 min separations, the initial 10–30% B step was 15 min, followed by a linear increase to 40% B (7.5 min) and 57.5% B (2.5 min) before washing and re-equilibration. For some experiments we used the Evosep One (Evosep, Odense, Denmark), a new HPLC instrument employing an embedded gradient and capable of fast turnaround between analyses (34Bache N. Geyer P.E. Bekker-Jensen D.B. Hoerning O. Falkenby L. Treit P.V. Doll S. Paron I. Müller J.B. Meier F. Olsen J.V. Vorm O. Mann M. A novel LC system embeds analytes in pre-formed gradients for rapid, ultra-robust proteomics.Mol. Cell. Proteomics. 2018; (mcp.TIR118.000853)Abstract Full Text Full Text PDF Scopus (140) Google Scholar). Samples were eluted from Evotips at low pressure into the storage loop with a gradient offset to lower the percentage of organic buffer. Separation was performed on a customized 5.6 min gradient (200 samples/day method) at a flow rate of 2.0 μl/min on a 4 cm x 150 μm i.d. reversed-phase column packed with 3 μm C18-coated porous silica beads (PepSep, Odense, Denmark). The timsTOF Pro is the successor to the impact II instrument, compared with which it features an additional ion mobility analyzer. However, the timsTOF Pro is a complete redesign in hardware and firmware. Apart from incorporating TIMS, the design goals included the achievement of similar or better mass resolution (>35,000) and improved robustness through a modified ion path. In the experiments described here, the mass spectrometer was operated in PASEF mode. Desolvated ions entered the vacuum region through the glass capillary and were deflected by 90°, focused in an electrodynamic funnel, and trapped in the front region of the TIMS tunnel consisting of stacked printed circuit boards (PCBs) with an inner diameter of 8 mm and a total length of 100 mm. The PCB electrodes form a stacked multipole in the direction of ion transfer, in which an applied RF potential of 350 Vpp confined the trapped ions radially. The TIMS tunnel is electrically separated into two parts (dual TIMS), where the first region is operated as an ion accumulation trap that primarily stores all ions entering the mass spectrometer, while the second part performs trapped ion mobility analysis (28Silveira J.A. Ridgeway M.E. Laukien F.H. Mann M. Park M.A. Parallel accumulation for 100% duty cycle trapped ion mobility-mass spectrometry.Int. J. Mass Spectrom. 2017; 413: 168-175Crossref Scopus (44) Google Scholar). As soon as the TIMS analysis is finished, all stored ions are transferred to the analyzer part and the storage region is filled again. If equal accumulation and analysis times are used in both TIMS regions, this enables operation at duty cycles close to 100%. Ion transfer between the two regions takes 2 ms and therefore does not affect the overall ion utilization for typical ramp and accumulation times around 25 to 200 ms. In both TIMS regions, the RF field is superimposed (from entrance to exit) by an increasing longitudinal electrical field gradient, such that ions in the tunnel simultaneously experience a drag from the incoming gas flow through the capillary and a repulsion from the electrical field. Here, we used a flow of ambient laboratory air for ion mobility separation. Depending on their collisional cross sections and charge states, ions come to rest closer to the entrance of the tunnel (high ion mobility) or closer to its exit (low ion mobility). Trapped ion mobility separation was achieved by ramping the entrance potential of the second TIMS region from −207 V to −77 V. A single TIMS-MS scan is composed of many individual TOF scans of about 110 μs each. In the experiments reported here, we first systematically varied the ramp times from 25, 50, 100, 150, to 200 ms while keeping the duty cycle fixed at 100%. All further experiments were acquired with a 100 ms ramp and 10 PASEF MS/MS scans per topN acquisition cycle, except for the Evosep One experiments, which were performed with four PASEF MS/MS scans per cycle. In TOF mass spectrometry, signal-to-noise ratios can conveniently be increased by summation of individual TOF scans. Here, low-abundance precursors with an intensity below a 'target value' were repeatedly scheduled for PASEF-MS/MS scans until the summed ion count reached the target value (e.g. four times for a precursor with the intensity 5000 arbitrary units (a.u.) and a target value of 20,000 a.u.). We set the target value to 20,000 a.u. for all methods, except for the Evosep One experiments where it was set to 24,000 a.u. MS and MS/MS spectra were recorded from m/z 100 to 1700. Suitable precursor ions for PASEF-MS/MS were selected in real time from TIMS-MS survey scans by a sophisticated PASEF scheduling algorithm (see also Results). A polygon filter was applied to the m/z and ion mobility plane to select features most likely representing peptide precursors rather than singly charged background ions. The quadrupole isolation width was set to 2 Th for m/z 700, and the collision energy was ramped stepwise as a function of increasing ion mobility: 52 eV for 0–19% of the ramp time; 47 eV from 19–38%; 42 eV from 38–57%; 37 eV from 57–76%; and 32 eV for the remainder. The TIMS elution voltage was calibrated linearly to obtain reduced ion mobility coefficients (1/K0) using three selected ions of the Agilent ESI-L Tuning Mix (m/z 622, 922, 1222) (35Stow S.M. Causon T.J. Zheng X. Kurulugama R.T. Mairinger T. May J.C. Rennie E.E. Baker E.S. Smith R.D. McLean J.A. Hann S. Fjeldsted J.C. An interlaboratory evaluation of drift tube ion mobility-mass spectrometry collision cross section measurements.Anal. Chem. 2017; 89: 9048-9055Crossref PubMed Scopus (282) Google Scholar). Collisional cross sections were calculated from the Mason Schamp equation (36Mason E.A. McDaniel E.W. Transport Properties of Ions in Gases. John Wiley & Sons, Inc., New York, NY1988Crossref Google Scholar). CCS=3ze161K02πμkbT where z is the charge of the ion, e is the elemental charge, kb is Boltzman's constant, μ is the reduced mass, and T the temperature (305 K). For all calculations, we assumed pure N2 as the drift gas. Mass spectrometry raw files were processed with MaxQuant (37Cox 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 (9214) Google Scholar) version 1.6.1.13, which has been extended to incorporate the additional ion mobility dimension and adapted to handle the TIMS data format. This new version of MaxQuant is publicly available and will be described in detail separately (Cox and co-workers, in preparation). Briefly, it assembles four-dimensional isotope clusters - defined by m/z, retention time, ion mobility and intensity - from the TIMS-MS spectra and extracts ion mobility separated MS/MS spectra from the PASEF scans. Each MS/MS spectrum is assigned to its respective precursor ion by quadrupole isolation m/z and ion mobility values, and in case a precursor has been fragmented multiple times in one acquisition cycle, the respective spectra are collapsed to a single spectrum with increased signal-to-noise. For the four-dimensional feature detection in MaxQuant, only every third data point (TOF scan) in the TIMS dimension was considered ("TIMS step width" = 3), and the "TIMS half width" parameter was set to 4 TOF scans (equivalent to about 440 μs). The "TIMS mass resolution" parameter was set to 32,000 and MS/MS peaks with an intensity below 1.5 units were discarded. The MS/MS spectra were matched to in silico derived fragment mass values of tryptic peptides from a reference proteome (Uniprot, 2016/05, HeLa: 91,618 entries including isoforms, E. coli: 4313 entries including isoforms) and 245 potential contaminants by the built-in Andromeda search engine (38Cox 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; 10: 1794-1805Crossref PubMed Scopus (3469) Google Scholar). A maximum of two missing cleavages were allowed, the required minimum peptide sequence length was 7 amino acids, and the peptide mass was limited to a maximum of 4600 Da. Carbamidomethylation of cysteine residues was set as a fixed modification, and methionine oxidation and acetylation of protein N termini as variable modifications. The initial maximum mass tolerances were 70 ppm for precursor ions and 35 ppm for fragment ions. After re-calibration, precursor mass tolerances were individually adjusted by MaxQuant to less or equal 20 ppm for each isotope pattern based on the local precision of the mass measurement. We employed a reversed sequence library to control the false discovery rate (FDR) at less than 1% for peptide spectrum matches and protein group identifications. Decoy database hits, proteins identified as potential contaminants, and proteins