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Cross-linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics

Christian H. Ihling, View ORCID ProfileLolita Piersimoni, Marc Kipping, View ORCID ProfileAndrea Sinz
doi: https://doi.org/10.1101/2021.03.26.437136
Christian H. Ihling
1Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany
2Center for Structural Mass Spectrometry, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany
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Lolita Piersimoni
1Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany
2Center for Structural Mass Spectrometry, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany
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  • ORCID record for Lolita Piersimoni
Marc Kipping
1Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany
2Center for Structural Mass Spectrometry, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany
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Andrea Sinz
1Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany
2Center for Structural Mass Spectrometry, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany
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  • ORCID record for Andrea Sinz
  • For correspondence: andrea.sinz@pharmazie.uni-halle.de
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Abstract

The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) Bovine serum albumin, (ii) E. coli ribosome, and (iii) HEK293T cell nuclear lysates. We identified a total of 508 unique cross-linking sites for BSA, 868 for the E. coli ribosome, and 1,623 unique cross-links for nuclear lysates, corresponding to 1,088 intra- and 535 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving 3D-structures of single proteins, but also for performing system-wide protein interaction studies.

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Competing Interest Statement

The authors have declared no competing interest.

  • Abbreviations

    ACN
    Acetonitrile
    BSA
    Bovine serum albumin
    Caps
    Collisional cross-section assisted precursor selection
    CCS
    Collisional cross section
    CSM
    Cross-link spectral match
    DSAU
    Disuccinimidyl diacetic urea
    DSBU
    Disuccinimidyl dibutyric urea
    DSPU
    Disuccinimidyl dipropionic urea
    DTT
    Dithiothreitol
    EM
    Electron microscopy
    FDR
    False discovery rate
    HEPES
    2-[4-(2-Hydroxyethyl)-piperazine-1-yl]ethanesulfonic acid
    IM
    Ion mobility
    IMAC
    Immobilized metal ion chromatography
    LC
    Liquid chromatography
    MS
    Mass spectrometry
    MS/MS
    Tandem mass spectrometry
    NHS
    N-Hydroxysuccinimide
    PASEF
    Parallel accumulation serial fragmentation
    PBS
    Phosphate buffered saline
    RT
    Room temperature
    SAXS
    Small-angle X-ray scattering
    SDS
    Sodium dodecyl sulfate
    SEC
    Size exclusion chromatography
    TIMS
    Trapped ion mobility spectrometry
    TOF
    Time-of-flight
    XL
    Cross-linking
    XL-MS
    Cross-linking/mass spectrometry
  • Copyright 
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    Posted March 26, 2021.
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    Cross-linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics
    Christian H. Ihling, Lolita Piersimoni, Marc Kipping, Andrea Sinz
    bioRxiv 2021.03.26.437136; doi: https://doi.org/10.1101/2021.03.26.437136
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    Cross-linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics
    Christian H. Ihling, Lolita Piersimoni, Marc Kipping, Andrea Sinz
    bioRxiv 2021.03.26.437136; doi: https://doi.org/10.1101/2021.03.26.437136

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