RT Journal Article
SR Electronic
T1 Cross-linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.03.26.437136
DO 10.1101/2021.03.26.437136
A1 Christian H. Ihling
A1 Lolita Piersimoni
A1 Marc Kipping
A1 Andrea Sinz
YR 2021
UL http://biorxiv.org/content/early/2021/03/26/2021.03.26.437136.abstract
AB 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.Competing Interest StatementThe authors have declared no competing interest.ACNAcetonitrileBSABovine serum albuminCapsCollisional cross-section assisted precursor selectionCCSCollisional cross sectionCSMCross-link spectral matchDSAUDisuccinimidyl diacetic ureaDSBUDisuccinimidyl dibutyric ureaDSPUDisuccinimidyl dipropionic ureaDTTDithiothreitolEMElectron microscopyFDRFalse discovery rateHEPES2-[4-(2-Hydroxyethyl)-piperazine-1-yl]ethanesulfonic acidIMIon mobilityIMACImmobilized metal ion chromatographyLCLiquid chromatographyMSMass spectrometryMS/MSTandem mass spectrometryNHSN-HydroxysuccinimidePASEFParallel accumulation serial fragmentationPBSPhosphate buffered salineRTRoom temperatureSAXSSmall-angle X-ray scatteringSDSSodium dodecyl sulfateSECSize exclusion chromatographyTIMSTrapped ion mobility spectrometryTOFTime-of-flightXLCross-linkingXL-MSCross-linking/mass spectrometry