Advertisement

A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients*

Open AccessPublished:February 12, 2020DOI:https://doi.org/10.1074/mcp.TIR119.001906
      State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 min LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument, we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.

      Graphical Abstract

      Deep proteome profiling of human cells can now be routinely achieved by shotgun proteomics using the latest advances in nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) (
      • Kim M.S.
      • Pinto S.M.
      • Getnet D.
      • Nirujogi R.S.
      • Manda S.S.
      • Chaerkady R.
      • Madugundu A.K.
      • Kelkar D.S.
      • Isserlin R.
      • Jain S.
      • Thomas J.K.
      • Muthusamy B.
      • Leal-Rojas P.
      • Kumar P.
      • Sahasrabuddhe N.A.
      • Balakrishnan L.
      • Advani J.
      • George B.
      • Renuse S.
      • Selvan L.D.
      • Patil A.H.
      • Nanjappa V.
      • Radhakrishnan A.
      • Prasad S.
      • Subbannayya T.
      • Raju R.
      • Kumar M.
      • Sreenivasamurthy S.K.
      • Marimuthu A.
      • Sathe G.J.
      • Chavan S.
      • Datta K.K.
      • Subbannayya Y.
      • Sahu A.
      • Yelamanchi S.D.
      • Jayaram S.
      • Rajagopalan P.
      • Sharma J.
      • Murthy K.R.
      • Syed N.
      • Goel R.
      • Khan A.A.
      • Ahmad S.
      • Dey G.
      • Mudgal K.
      • Chatterjee A.
      • Huang T.C.
      • Zhong J.
      • Wu X.
      • Shaw P.G.
      • Freed D.
      • Zahari M.S.
      • Mukherjee K.K.
      • Shankar S.
      • Mahadevan A.
      • Lam H.
      • Mitchell C.J.
      • Shankar S.K.
      • Satishchandra P.
      • Schroeder J.T.
      • Sirdeshmukh R.
      • Maitra A.
      • Leach S.D.
      • Drake C.G.
      • Halushka M.K.
      • Prasad T.S.
      • Hruban R.H.
      • Kerr C.L.
      • Bader G.D.
      • Iacobuzio-Donahue C.A.
      • Gowda H.
      • Pandey A.
      A Draft Map of the human proteome.
      ,
      • Wilhelm M.
      • Schlegl J.
      • Hahne H.
      • Gholami A.M.
      • Lieberenz M.
      • Savitski M.M.
      • Ziegler E.
      • Butzmann L.
      • Gessulat S.
      • Marx H.
      • Mathieson T.
      • Lemeer S.
      • Schnatbaum K.
      • Reimer U.
      • Wenschuh H.
      • Mollenhauer M.
      • Slotta-Huspenina J.
      • Boese J.H.
      • Bantscheff M.
      • Gerstmair A.
      • Faerber F.
      • Kuster B.
      Mass-spectrometry-based draft of the human proteome.
      ). However, comprehensive analysis of a human proteome typically requires days of mass spectrometric measurement time (
      • Bekker-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.
      • et al.
      An optimized shotgun strategy for the rapid generation of comprehensive human proteomes.
      ), making high-throughput analysis challenging and time-consuming. Therefore, new improved mass spectrometric technology and acquisition strategies are needed to overcome these limitations in current large-scale proteomics. The Orbitrap analyzer has become one of the major players in mass spectrometry-based proteomics (
      • Makarov A.
      Orbitrap journey: taming the ion rings.
      ). We have previously described the performance enhancement achieved over the different generations of the popular benchtop quadrupole-Orbitrap instruments, the Q Exactive™ series, for proteomics applications (
      • Kelstrup C.D.
      • Young C.
      • Lavallee R.
      • Nielsen M.L.
      • Olsen J.V.
      Optimized fast and sensitive acquisition methods for shotgun proteomics on a quadrupole orbitrap mass spectrometer.
      ,
      • Kelstrup C.D.
      • Jersie-Christensen R.R.
      • Batth T.S.
      • Arrey T.N.
      • Kuehn A.
      • Kellmann M.
      • Olsen J.V.
      Rapid and deep proteomes by faster sequencing on a benchtop quadrupole ultra-high-field orbitrap mass spectrometer.
      ,
      • Kelstrup C.D.
      • Bekker-Jensen D.B.
      • Arrey T.N.
      • Hogrebe A.
      • Harder A.
      • Olsen J.V.
      Performance Evaluation of the Q Exactive HF-X for Shotgun Proteomics.
      ). The previous generation Q Exactive HF-X™ hybrid quadrupole-Orbitrap instrument incorporated a new peak-picking algorithm, a brighter ion source, and optimized ion transfers enabling productive HCD-MS/MS (
      • Olsen J.V
      • Macek B.
      • Lange O.
      • Makarov A.
      • Horning S.
      • Mann M.
      Higher-energy C-trap dissociation for peptide modification analysis.
      ) acquisition above 40 Hz. These improvements collectively resulted in increased peptide and protein identification rates of up to fifty percent at short LC-MS gradients with more than 1000 unique peptides identified per minute (
      • Kelstrup C.D.
      • Bekker-Jensen D.B.
      • Arrey T.N.
      • Hogrebe A.
      • Harder A.
      • Olsen J.V.
      Performance Evaluation of the Q Exactive HF-X for Shotgun Proteomics.
      ). However, instrument performance after extended periods of continuous sample analyses could be comprised by contaminants entering the quadrupole and resulting in local charging and lower MS/MS sensitivity. This meant that regular preventative maintenance could be necessary to preserve high performance of the Q Exactive HF-X instrument. Moreover, the semi-stochastic nature of the data-dependent acquisition (DDA)
      The abbreviations used are:
      DDA
      data dependent acquisition
      ACN
      acetonitrile
      APD
      advanced peak detection
      DIA
      data independent acquisition
      FAIMS
      high-field asymmetric waveform ion mobility spectrometry
      FA
      formic acid
      HCD
      higher-energy collisional dissociation
      HCTT
      high capacity transfer tube
      IT
      maximum injection time
      LC
      liquid chromatography
      m/z
      mass-to-charge
      MS
      mass spectrometry
      MS/MS
      tandem mass spectrometry
      PSMs
      peptide spectrum matches
      PTMs
      post translational modifications
      TFA
      trifluoroacetic acid
      TMT
      tandem mass tags.
      1The abbreviations used are:DDA
      data dependent acquisition
      ACN
      acetonitrile
      APD
      advanced peak detection
      DIA
      data independent acquisition
      FAIMS
      high-field asymmetric waveform ion mobility spectrometry
      FA
      formic acid
      HCD
      higher-energy collisional dissociation
      HCTT
      high capacity transfer tube
      IT
      maximum injection time
      LC
      liquid chromatography
      m/z
      mass-to-charge
      MS
      mass spectrometry
      MS/MS
      tandem mass spectrometry
      PSMs
      peptide spectrum matches
      PTMs
      post translational modifications
      TFA
      trifluoroacetic acid
      TMT
      tandem mass tags.
      —the most popular tandem mass spectrometric acquisition strategy may create issues with reproducibility of peptide measurements between samples leading to the so-called missing value problem. To overcome the precursor selection problem inherent to DDA, data-independent acquisition (DIA) offers systematic measurement of all peptide ions regardless of their intensity by co-fragmenting all co-eluting peptides in broader precursor isolation windows. This provides wider dynamic range of the proteomes analyzed, improved reproducibility for identification and enabled better sensitivity and accuracy for quantification. The highly multiplexed fragment ion spectra in DIA require more elaborate processing algorithms for identification and quantification. These typically rely on spectral libraries previously recorded by data-dependent acquisition of similar sample types.
      The front-end high-field asymmetric waveform ion mobility spectrometry (FAIMS) (
      • Barnett D.A.
      • Ells B.
      • Guevremont R.
      • Purves R.W.
      Application of ESI-FAIMS-MS to the analysis of tryptic peptides.
      ,
      • Saba J.
      • Bonneil E.
      • Pomie's C.
      • Eng K.
      • Thibault P.
      Enhanced sensitivity in proteomics experiments using FAIMS coupled with a hybrid linear ion trap/orbitrap mass spectrometer.
      ,
      • Hebert A.S.
      • Prasad S.
      • Belford M.W.
      • Bailey D.J.
      • McAlister G.C.
      • Abbatiello S.E.
      • Huguet R.
      • Wouters E.R.
      • Dunyach J.J.
      • Brademan D.R.
      • et al.
      Comprehensive single-shot proteomics with FAIMS on a hybrid orbitrap mass spectrometer.
      ) interface functions as an ion selection device and an electrospray filter that prevents neutrals from entering the orifice of the mass spectrometer while reducing chemical background noise. This “purification” of the electrosprayed ions typically results in improved robustness and sensitivity for proteomics experiments. The FAIMS Pro interface continuously selects and focuses ions at atmospheric pressure based on their differential mobilities in a high field versus a low electric field established by applying an asymmetric (e.g. bi-sinusoidal) voltage to at least one of the inner and outer electrodes. To prevent an ion species from being discharged, the FAIMS Pro interface applies a small DC potential, called the compensation voltage (CV), to the inner electrode to compensate for the ion drift resulting from the applied asymmetric waveform. Selection of an ion species is based on a combination of its gas-phase charge-state and collisional cross-section. CVs can be tuned such that only selected subsets of ions are transmitted through the electrodes thereby favoring multiply-charged peptide species and filtering out singly-charged chemical background ions. The CV is typically negative for positive ions and positive for negative ions. The advantage of FAIMS in the context of large-scale proteomics on the Tribrid™ Orbitrap systems (i.e. those incorporating a quadrupole mass filter and dual-pressure linear quadrupole ion trap in addition to the Orbitrap mass analyzer) has previously demonstrated to improve proteome coverage for single-shot proteomics with long gradients by CV sweeping (
      • Hebert A.S.
      • Prasad S.
      • Belford M.W.
      • Bailey D.J.
      • McAlister G.C.
      • Abbatiello S.E.
      • Huguet R.
      • Wouters E.R.
      • Dunyach J.J.
      • Brademan D.R.
      • et al.
      Comprehensive single-shot proteomics with FAIMS on a hybrid orbitrap mass spectrometer.
      ) and reduce ratio compression in TMT experiment (
      • Schweppe D.K.
      • Prasad S.
      • Belford M.W.
      • Navarrete-Perea J.
      • Bailey D.J.
      • Huguet R.
      • Jedrychowski M.P.
      • Rad R.
      • McAlister G.
      • Abbatiello S.E.
      • et al.
      Characterization and optimization of multiplexed quantitative analyses using high-field asymmetric-waveform ion mobility mass spectrometry.
      ,
      • Pfammatter S.
      • Bonneil E.
      • McManus F.P.
      • Prasad S.
      • Bailey D.J.
      • Belford M.
      • Dunyach J.-J.
      • Thibault P.
      A novel differential ion mobility device expands the depth of proteome coverage and the sensitivity of multiplex proteomic measurements.
      ,
      • Pfammatter S.
      • Bonneil E.
      • Thibault P.
      Improvement of quantitative measurements in multiplex proteomics using high-field asymmetric waveform spectrometry.
      ). However, potential advantages of FAIMS for short gradients using single CVs have not been explored. Likewise, FAIMS in combination with DIA has not yet been investigated. Here we describe a thorough performance evaluation of the hybrid quadrupole-Orbitrap Exploris 480TM MS in combination with short LC gradients using the Evosep One system. Standard DDA in combination with FAIMS identifies more proteins but fewer peptides resulting in a compromise between sequence coverage and proteome depth. This is not the case for DIA, where FAIMS results in identification of more proteins while maintaining the number of peptide identifications.

      CONCLUSION

      The Orbitrap Exploris 480 MS is a compact hybrid quadrupole-Orbitrap mass spectrometer that provides high-quality HCD tandem mass spectra at acquisition rates of up to 40 Hz, which in combination with the Evosep One system, enables routine identification of more than 1,000 unique peptides per LC gradient minute. The application of FAIMS with a single CV increased proteome coverage in short gradients by close to 50% in data-dependent acquisition-based shotgun proteomics experiments of human cells at the expense of lower peptide coverage. Importantly, FAIMS enables analysis of minute sample amounts by effectively removing interfering singly-charged background ions. We demonstrate that it is possible to quantify more than 1,000 proteins from less than 50 HeLa cells with DIA-FAIMS, highlighting its potential for applications such as laser microdissected clinical samples and single-cell proteome analysis. The fast sequencing speed, high-sensitivity and robustness of the Orbitrap Exploris 480 MS makes it ideally suited for data-independent acquisition. The combination of FAIMS and data-independent acquisition provides more peptides identified and up to 5,100 mammalian proteins quantified in single-shot analysis using short LC gradients, allowing up to 60 samples to be measured in 24 h. This acquisition strategy enables high-throughput and large-scale profiling of hundreds of samples, as demonstrated by the quantitative organ tissue atlas we provide here, which includes a high-quality spectral library consisting of more than two hundred thousand unique peptides covering 12,900 protein-coding genes. This dataset comprises, to our knowledge, the deepest rat proteome to date. This spectral library was recorded by analyzing more than 1,000 LC-MS/MS runs of offline high pH reversed-phase peptide fractions back-to-back, which also demonstrates the robustness of the system throughout large-scale applications. In addition to label-free quantification the Orbitrap Exploris 480 MS is also optimized for tandem mass tag-based quantification enabled by the fast scanning turboTMT method. Additionally, we have presented a comprehensive benchmark of DIA-FAIMS against the well-established TMT-based quantification, showing that DIA-FAIMS provides equivalent proteome coverage as isobaric labeling. Interestingly, the observed biological reproducibility in TMT-based quantification was higher than for DIA-FAIMS, which likely reflects that all tissues from the same animal were combined in one TMT set. The limitation of analyzing 11 samples within a single TMT experiment is now ameliorated with the newly available TMT 16-plex reagents. Finally, the instrument is also a good match for quantitative analysis of post-translational modifications. Sensitive and large-scale analysis of cell line and organ phospho-proteomes is feasible in less than 1 day of analysis. Consequently, we envision that it will be find widespread use in many proteomics laboratories.

      DATA AVAILABILITY

      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository. Data are available via ProteomeXchange with identifier PXD016662.

      Acknowledgments

      We thank our colleagues at Thermo Fisher Scientific, especially Jan-Peter Hauschild, Amelia Peterson, Erik Couzijn, Eduard Denisov, Denis Chernyshev, Christian Hock, Hamish Stewart, Ralf Hartmer, Christian Thoeing, Oliver Lange, Mathias Mueller, Arne Kreutzmann, Wilko Balschun, Aivaras Venckus, Alexander Kholomeev, Gregor Quiring, Frank Czemper, Andreas Wieghaus, Michael Belford, Julia Kraegenbring, Alexander Harder, Kerstin Strupat, Markus Kellmann. We would like to thank Stoyan Stoychev and Justin Jordaan for great help and input for optimizing our phospho-enrichment workflow and establishing automated platform for sample preparation. We would also like to thank Nicolai Bache for help and input on the chromatographic methods as well as Anna Secher for providing rat tissues and all members of the Olsen Group for critical input on the manuscript.

      REFERENCES

        • Kim M.S.
        • Pinto S.M.
        • Getnet D.
        • Nirujogi R.S.
        • Manda S.S.
        • Chaerkady R.
        • Madugundu A.K.
        • Kelkar D.S.
        • Isserlin R.
        • Jain S.
        • Thomas J.K.
        • Muthusamy B.
        • Leal-Rojas P.
        • Kumar P.
        • Sahasrabuddhe N.A.
        • Balakrishnan L.
        • Advani J.
        • George B.
        • Renuse S.
        • Selvan L.D.
        • Patil A.H.
        • Nanjappa V.
        • Radhakrishnan A.
        • Prasad S.
        • Subbannayya T.
        • Raju R.
        • Kumar M.
        • Sreenivasamurthy S.K.
        • Marimuthu A.
        • Sathe G.J.
        • Chavan S.
        • Datta K.K.
        • Subbannayya Y.
        • Sahu A.
        • Yelamanchi S.D.
        • Jayaram S.
        • Rajagopalan P.
        • Sharma J.
        • Murthy K.R.
        • Syed N.
        • Goel R.
        • Khan A.A.
        • Ahmad S.
        • Dey G.
        • Mudgal K.
        • Chatterjee A.
        • Huang T.C.
        • Zhong J.
        • Wu X.
        • Shaw P.G.
        • Freed D.
        • Zahari M.S.
        • Mukherjee K.K.
        • Shankar S.
        • Mahadevan A.
        • Lam H.
        • Mitchell C.J.
        • Shankar S.K.
        • Satishchandra P.
        • Schroeder J.T.
        • Sirdeshmukh R.
        • Maitra A.
        • Leach S.D.
        • Drake C.G.
        • Halushka M.K.
        • Prasad T.S.
        • Hruban R.H.
        • Kerr C.L.
        • Bader G.D.
        • Iacobuzio-Donahue C.A.
        • Gowda H.
        • Pandey A.
        A Draft Map of the human proteome.
        Nature. 2014; 509: 575-581
        • Wilhelm M.
        • Schlegl J.
        • Hahne H.
        • Gholami A.M.
        • Lieberenz M.
        • Savitski M.M.
        • Ziegler E.
        • Butzmann L.
        • Gessulat S.
        • Marx H.
        • Mathieson T.
        • Lemeer S.
        • Schnatbaum K.
        • Reimer U.
        • Wenschuh H.
        • Mollenhauer M.
        • Slotta-Huspenina J.
        • Boese J.H.
        • Bantscheff M.
        • Gerstmair A.
        • Faerber F.
        • Kuster B.
        Mass-spectrometry-based draft of the human proteome.
        Nature. 2014; 509: 582-587
        • Bekker-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.
        • et al.
        An optimized shotgun strategy for the rapid generation of comprehensive human proteomes.
        Cell Syst. 2017; 4: 587-599
        • Makarov A.
        Orbitrap journey: taming the ion rings.
        Nat. Commun. 2019; 10: 3743
        • Kelstrup C.D.
        • Young C.
        • Lavallee R.
        • Nielsen M.L.
        • Olsen J.V.
        Optimized fast and sensitive acquisition methods for shotgun proteomics on a quadrupole orbitrap mass spectrometer.
        J. Proteome Res. 2012; 11: 3487-3497
        • Kelstrup C.D.
        • Jersie-Christensen R.R.
        • Batth T.S.
        • Arrey T.N.
        • Kuehn A.
        • Kellmann M.
        • Olsen J.V.
        Rapid and deep proteomes by faster sequencing on a benchtop quadrupole ultra-high-field orbitrap mass spectrometer.
        J. Proteome Res. 2014; 13: 6187-6195
        • Kelstrup C.D.
        • Bekker-Jensen D.B.
        • Arrey T.N.
        • Hogrebe A.
        • Harder A.
        • Olsen J.V.
        Performance Evaluation of the Q Exactive HF-X for Shotgun Proteomics.
        J. Proteome Res. 2018; 17: 727-738
        • Olsen 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: 709-712
        • Barnett D.A.
        • Ells B.
        • Guevremont R.
        • Purves R.W.
        Application of ESI-FAIMS-MS to the analysis of tryptic peptides.
        J. Am. Soc. Mass Spectrom. 2002; 13: 1282-1291
        • Saba J.
        • Bonneil E.
        • Pomie's C.
        • Eng K.
        • Thibault P.
        Enhanced sensitivity in proteomics experiments using FAIMS coupled with a hybrid linear ion trap/orbitrap mass spectrometer.
        J. Proteome Res. 2009; 8: 3355-3366
        • Hebert A.S.
        • Prasad S.
        • Belford M.W.
        • Bailey D.J.
        • McAlister G.C.
        • Abbatiello S.E.
        • Huguet R.
        • Wouters E.R.
        • Dunyach J.J.
        • Brademan D.R.
        • et al.
        Comprehensive single-shot proteomics with FAIMS on a hybrid orbitrap mass spectrometer.
        Anal. Chem. 2018; 90: 9529-9537
        • Schweppe D.K.
        • Prasad S.
        • Belford M.W.
        • Navarrete-Perea J.
        • Bailey D.J.
        • Huguet R.
        • Jedrychowski M.P.
        • Rad R.
        • McAlister G.
        • Abbatiello S.E.
        • et al.
        Characterization and optimization of multiplexed quantitative analyses using high-field asymmetric-waveform ion mobility mass spectrometry.
        Anal. Chem. 2019; 91: 4010-4016
        • Pfammatter S.
        • Bonneil E.
        • McManus F.P.
        • Prasad S.
        • Bailey D.J.
        • Belford M.
        • Dunyach J.-J.
        • Thibault P.
        A novel differential ion mobility device expands the depth of proteome coverage and the sensitivity of multiplex proteomic measurements.
        Mol. Cell. Proteomics. 2018; 17: 2051-2067
        • Pfammatter S.
        • Bonneil E.
        • Thibault P.
        Improvement of quantitative measurements in multiplex proteomics using high-field asymmetric waveform spectrometry.
        J. Proteome Res. 2016; 15: 4653-4665
        • Martins C.P.B.
        • Bromirski M.
        • Prieto Conaway M.C.
        • Makarov A.
        Orbitrap mass spectrometry: evolution and applicability.
        in: Perez S. Eichhorn P. Barcelo D.E. Applications of Time-of-Flight and Orbitrap Mass Spectrometry in Environmental, Food, Doping, and Forensic Analysis. Elsevier, Amsterdam, the Netherlands2016: 3-18
        • Scheltema R.A.
        • Hauschild J.P.
        • Lange O.
        • Hornburg D.
        • Denisov E.
        • Damoc E.
        • Kuehn A.
        • Makarov A.
        • Mann M.
        The Q Exactive HF, a Benchtop Mass Spectrometer with a Pre-Filter, High-Performance Quadrupole and an Ultra-High-Field Orbitrap Analyzer.
        Mol. Cell. Proteomics. 2014; 13: 3698-3708
        • Michalski A.
        • Damoc E.
        • Hauschild J.P.
        • Lange O.
        • Wieghaus A.
        • Makarov A.
        • Nagaraj N.
        • Cox J.
        • Mann M.
        • Horning S.
        Mass spectrometry-based proteomics using Q exactive, a high-performance benchtop quadrupole orbitrap mass spectrometer.
        Mol. Cell. Proteomics. 2011; 10
        • Senko M.W.
        • Remes P.M.
        • Canterbury J.D.
        • Mathur R.
        • Song Q.
        • Eliuk S.M.
        • Mullen C.
        • Earley L.
        • Hardman M.
        • Blethrow J.D.
        • et al.
        Novel parallelized quadrupole/linear ion trap/orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates.
        Anal. Chem. 2013; 85: 11710-11714
        • Lange O.
        • Damoc E.
        • Wieghaus A.
        • Makarov A.
        Enhanced fourier transform for orbitrap mass spectrometry.
        Int. J. Mass Spectrom. 2014; 369: 16-22
        • Werner T.
        • Becher I.
        • Sweetman G.
        • Doce C.
        • Savitski M.M.
        • Bantscheff M.
        High-resolution enabled TMT 8-plexing.
        Anal. Chem. 2012; 84: 7188-7194
        • McAlister G.C.
        • Huttlin E.L.
        • Haas W.
        • Ting L.
        • Jedrychowski M.P.
        • Rogers J.C.
        • Kuhn K.
        • Pike I.
        • Grothe R.A.
        • Blethrow J.D.
        • et al.
        Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses.
        Anal. Chem. 2012; 84: 7469-7478
        • Kelstrup C.D.
        • Aizikov K.
        • Batth T.S.
        • Kreutzman A.
        • Grinfeld D.
        • Lange O.
        • Mourad D.
        • Makarov A.A.
        • Olsen J.V.
        Limits for resolving isobaric tandem mass tag reporter ions using phase-constrained spectrum deconvolution.
        J. Proteome Res. 2018; 17: 4008-4016
        • Tape C.J.
        • Worboys J.D.
        • Sinclair J.
        • Gourlay R.
        • Vogt J.
        • McMahon K.M.
        • Trost M.
        • Lauffenburger D.A.
        • Lamont D.J.
        • Jørgensen C.
        Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC.
        Anal. Chem. 2014; 86: 10296-10302
        • Leutert M.
        • Rodríguez-Mias R.A.
        • Fukuda N.K.
        • Villén J.
        R2-P2 rapid-robotic phosphoproteomics enables multidimensional cell signaling studies.
        Mol. Syst. Biol. 2019; 15: e9021
        • Cox 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: 1367-1372
        • Cox 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: 1794-1805
        • Bruderer R.
        • Bernhardt O.M.
        • Gandhi T.
        • Xuan Y.
        • Sondermann J.
        • Schmidt M.
        • Gomez-Varela D.
        • Reiter L.
        Optimization of experimental parameters in data-independent mass spectrometry significantly increases depth and reproducibility of results.
        Mol. Cell. Proteomics. 2017; 16: 2296-2309
        • Bekker-Jensen D.B.
        • Bernhardt O.M.
        • Hogrebe A.
        • Val A.M. del
        • Verbeke L.
        • Gandhi T.
        • Kelstrup C.D.
        • Reiter L.
        • Olsen J.V.
        Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition without the need for spectral libraries.
        Nat. Comun. 2020; 11: 787
        • Leek J.T.
        • Johnson W.E.
        • Parker H.S.
        • Jaffe A.E.
        • Storey J.D.
        The Sva package for removing batch effects and other unwanted variation in high-throughput experiments.
        Bioinformatics. 2012; 28: 882-883
        • Batth T.S.
        • Tollenaere M.A.X.
        • Rüther P.
        • Gonzalez-Franquesa A.
        • Prabhakar B.S.
        • Bekker-Jensen S.
        • Deshmukh A.S.
        • Olsen J.V.
        Protein aggregation capture on microparticles enables multipurpose proteomics sample preparation.
        Mol. Cell. Proteomics. 2019; 18: 1027-1035
        • Bache N.
        • Geyer P.E.
        • Bekker-Jensen D.B.
        • Hoerning O.
        • Falkenby L.
        • Treit P.V.
        • Doll S.
        • Paron I.
        • Müller J.B.
        • Meier F.
        • et al.
        A novel LC system embeds analytes in pre-formed gradients for rapid, ultra-robust proteomics.
        Mol. Cell. Proteomics. 2018; 17: 2284-2296
        • Deshmukh A.S.
        Proteomics of skeletal muscle: focus on insulin resistance and exercise biology.
        Proteomes. 2016; 4 (pii): E6
        • Hogrebe A.
        • von Stechow L.
        • Bekker-Jensen D.B.
        • Weinert B.T.
        • Kelstrup C.D.
        • Olsen J.V.
        Benchmarking common quantification strategies for large-scale phosphoproteomics.
        Nat. Commun. 2018; 9: 1045
        • Fagerberg L.
        • Hallstrom B.M.
        • Oksvold P.
        • Kampf C.
        • Djureinovic D.
        • Odeberg J.
        • Habuka M.
        • Tahmasebpoor S.
        • Danielsson A.
        • Edlund K.
        • et al.
        Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics.
        Mol. Cell. Proteomics. 2014; 13: 397-406
        • Lambert S.A.
        • Jolma A.
        • Campitelli L.F.
        • Das P.K.
        • Yin Y.
        • Albu M.
        • Chen X.
        • Taipale J.
        • Hughes T.R.
        • Weirauch M.T.
        The human transcription factors.
        Cell. 2018; 172: 650-665
        • Zhou Q.
        • Liu M.
        • Xia X.
        • Gong T.
        • Feng J.
        • Liu W.
        • Liu Y.
        • Zhen B.
        • Wang Y.
        • Ding C.
        • et al.
        A mouse tissue transcription factor atlas.
        Nat. Commun. 2017; 8: 15089
        • Aebersold R.
        • Mann M.
        Mass-spectrometric exploration of proteome structure and function.
        Nature. 2016; 537: 347-355
        • Lundby A.
        • Franciosa G.
        • Emdal K.B.
        • Refsgaard J.C.
        • Gnosa S.P.
        • Bekker-Jensen D.B.
        • Secher A.
        • Maurya S.R.
        • Paul I.
        • Mendez B.L.
        • et al.
        Oncogenic mutations rewire signaling pathways by switching protein recruitment to phosphotyrosine sites.
        Cell. 2019; 179: 543-560
        • Olsen J.V.
        • Blagoev B.
        • Gnad F.
        • Macek B.
        • Kumar C.
        • Mortensen P.
        • Mann M.
        Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.
        Cell. 2006; 127: 635-648
        • Hornbeck P.V.
        • Zhang B.
        • Murray B.
        • Kornhauser J.M.
        • Latham V.
        • Skrzypek E.
        PhosphoSitePlus, 2014: mutations, ptms and recalibrations.
        Nucleic Acids Res. 2015; 43: D512-D520
        • Santalla M.
        • Valverde C.A.
        • Harnichar E.
        • Lacunza E.
        • Aguilar-Fuentes J.
        • Mattiazzi A.
        • Ferrero P.
        Aging and CaMKII alter intracellular Ca2+ transients and heart rhythm in Drosophila Melanogaster.
        PLoS ONE. 2014; 9: e101871