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      Online Parallel Accumulation–Serial Fragmentation (PASEF) with a Novel Trapped Ion Mobility Mass Spectrometer*


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          PASEF multiplies the sequencing speed without any loss in sensitivity and is implemented in the timsTOF Pro instrument introduced here. Sequencing speeds above 100 Hz enable single run proteome analysis at a depth of 6000 proteins, making the instrument particularly attractive for rapid and highly sensitive proteomics. Collisional cross sections can be determined with up to 0.1% precision and acquired on a scale of 100,000s, which opens exciting areas for proteomics exploration.

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          • Online PASEF achieves greater than 100 MS/MS per second at full sensitivity.

          • Accurate label-free quantification of over 6000 proteins in 2 h.

          • High throughput demonstrated on 50 ng digests measured in 5 min.

          • High-precision determination of 100,000 peptide collisional cross sections.


          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.

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          Most cited references37

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          The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics.

          Mass spectrometry is a vital tool for molecular characterization, and the allied technique of ion mobility is enhancing many areas of (bio)chemical analysis. Strong synergy arises between these two techniques because of their ability to ascertain complementary information about gas-phase ions. Ion mobility separates ions (from small molecules up to megadalton protein complexes) based on their differential mobility through a buffer gas. Ion mobility-mass spectrometry (IM-MS) can thus act as a tool to separate complex mixtures, to resolve ions that may be indistinguishable by mass spectrometry alone, or to determine structural information (for example rotationally averaged cross-sectional area), complementary to more traditional structural approaches. Finally, IM-MS can be used to gain insights into the conformational dynamics of a system, offering a unique means of characterizing flexibility and folding mechanisms. This Review critically describes how IM-MS has been used to enhance various areas of chemical and biophysical analysis.
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            Ion mobility-mass spectrometry.

            This review article compares and contrasts various types of ion mobility-mass spectrometers available today and describes their advantages for application to a wide range of analytes. Ion mobility spectrometry (IMS), when coupled with mass spectrometry, offers value-added data not possible from mass spectra alone. Separation of isomers, isobars, and conformers; reduction of chemical noise; and measurement of ion size are possible with the addition of ion mobility cells to mass spectrometers. In addition, structurally similar ions and ions of the same charge state can be separated into families of ions which appear along a unique mass-mobility correlation line. This review describes the four methods of ion mobility separation currently used with mass spectrometry. They are (1) drift-time ion mobility spectrometry (DTIMS), (2) aspiration ion mobility spectrometry (AIMS), (3) differential-mobility spectrometry (DMS) which is also called field-asymmetric waveform ion mobility spectrometry (FAIMS) and (4) traveling-wave ion mobility spectrometry (TWIMS). DTIMS provides the highest IMS resolving power and is the only IMS method which can directly measure collision cross-sections. AIMS is a low resolution mobility separation method but can monitor ions in a continuous manner. DMS and FAIMS offer continuous-ion monitoring capability as well as orthogonal ion mobility separation in which high-separation selectivity can be achieved. TWIMS is a novel method of IMS with a low resolving power but has good sensitivity and is well intergrated into a commercial mass spectrometer. One hundred and sixty references on ion mobility-mass spectrometry (IMMS) are provided. 2008 John Wiley & Sons, Ltd
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              Mass spectrometry for proteomics.

              Mass spectrometry has been widely used to analyze biological samples and has evolved into an indispensable tool for proteomics research. Our desire to understand the proteome has led to new technologies that push the boundary of mass spectrometry capabilities, which in return has allowed mass spectrometry to address an ever-increasing array of biological questions. The recent development of a novel mass spectrometer (Orbitrap) and new dissociation methods such as electron-transfer dissociation has made possible the exciting new areas of proteomic application. Although bottom-up proteomics (analysis of proteolytic peptide mixtures) remains the workhorse for proteomic analysis, middle-down and top-down strategies (analysis of longer peptides and intact proteins, respectively) should allow more complete characterization of protein isoforms and post-translational modifications. Finally, stable isotope labeling strategies have transformed mass spectrometry from merely descriptive to a tool for measuring dynamic changes in protein expression, interaction, and modification.

                Author and article information

                Mol Cell Proteomics
                Mol. Cell Proteomics
                Molecular & Cellular Proteomics : MCP
                The American Society for Biochemistry and Molecular Biology
                December 2018
                1 November 2018
                1 November 2018
                : 17
                : 12
                : 2533-2545
                [1]From the ‡Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
                [2]§Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany;
                [3]¶Bruker Daltonics Inc., Manning Road, Billerica, Massachusetts 01821;
                [4]‖Evosep Biosystems, Thriges Pl. 6, 5000 Odense, Denmark;
                [5]**Computational Systems Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
                [6]‡‡NNF Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
                Author notes
                §§ To whom correspondence should be addressed: Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. Tel.: +49 89 8578-2557; Fax: +49 89 8578-2412; E-mail: mmann@ 123456biochem.mpg.de .

                Author contributions: F.M., M.A.P., J.C., O.R., and M.M. designed research; F.M., A.-D.B., S.K., H.K., M.L., M.K., N.G., J.D., T.K., N.B., O.H., J.C., and O.R. performed research; F.M., A.-D.B., S.K., H.K., M.L., O.R., and M.M. analyzed data; F.M. and M.M. wrote the paper; N.B., O.H., and J.C. contributed new reagents/analytic tools.

                Author information
                © 2018 Meier et al.

                Published by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version open access under the terms of the Creative Commons CC-BY license.

                : 4 June 2018
                : 30 October 2018
                Funded by: Deutsche Forschungsgemeinschaft (DFG) , open-funder-registry 10.13039/501100001659;
                Award ID: Gottfried Wilhelm Leibniz Prize
                Funded by: Max-Planck-Gesellschaft (MPG) , open-funder-registry 10.13039/501100004189;
                Award ID: core funding
                Technological Innovation Resources

                Molecular biology
                hplc,label-free quantification,mass spectrometry,protein identification,quantification,sequencing ms,tandem mass spectrometry,collisional cross section,ion mobility,pasef,tims


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