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

2021 ◽  
Author(s):  
Christian H Ihling ◽  
Lolita Piersimoni ◽  
Marc Kipping ◽  
Andrea Sinz
Keyword(s):  
Mass Spectrometry ◽  
Cross Sections ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Ion Mobility ◽  
Hek293t Cell ◽  
Structural Proteomics ◽  
Cross Linking ◽  
E Coli ◽  
Interaction Studies

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.

scholarly journals Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

2020 ◽  
Author(s):  
Barbara Steigenberger ◽  
Henk W.P. van den Toorn ◽  
Emiel Bijl ◽  
Jean-François Greisch ◽  
Oliver Räther ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Cross Sections ◽  
Protein Interactions ◽  
Ion Mobility ◽  
Chemical Properties ◽  
Cross Linking ◽  
Mass Analyzer ◽  
Quadrupole Collision Cell ◽  
Collisional Cross Section

AbstractIon mobility separates molecules in the gas-phase on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a time-of-flight mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking mass spectrometry (XL-MS). Cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent – termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Å2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50 - 70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

scholarly journals Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

2020 ◽  
Vol 19 (10) ◽  
pp. 1677-1687 ◽  
Author(s):  
Barbara Steigenberger ◽  
Henk W. P. van den Toorn ◽  
Emiel Bijl ◽  
Jean-François Greisch ◽  
Oliver Räther ◽  
...  
Keyword(s):  
Gas Phase ◽  
Cross Sections ◽  
Protein Interactions ◽  
Ion Mobility ◽  
Chemical Properties ◽  
Cross Linking ◽  
Mass Analyzer ◽  
Protein Protein Interactions ◽  
Quadrupole Collision Cell ◽  
Collisional Cross Section

Ion mobility separates molecules in the gas-phase based on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a TOF mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking MS (XL-MS). Cross-linking reagents covalently link amino acids in proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent – termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Å2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50-70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

scholarly journals A Simple Cross-Linking/Mass Spectrometry Workflow to Study System-Wide Protein Interactions

2019 ◽  
Author(s):  
Michael Götze ◽  
Claudio Iacobucci ◽  
Christian Ihling ◽  
Andrea Sinz
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Interaction Networks ◽  
Interaction Networks ◽  
Cross Linking ◽  
Protein Protein Interaction ◽  
Cellular Environment ◽  
Protein Protein Interaction Networks

ABSTRACTWe present a cross-linking/mass spectrometry (XLMS) workflow for performing proteome-wide cross-linking analyses within one week. The workflow is based on the commercially available MS-cleavable cross-linker disuccinimidyl dibutyric urea (DSBU) and can be employed by every lab having access to a mass spectrometer with tandem MS capabilities. We provide an updated version 2.0 of the freeware software tool MeroX, available at www.StavroX.com, that allows conducting fully automated and reliable studies delivering insights into protein-protein interaction networks and protein conformations at the proteome level. We exemplify our optimized workflow for mapping protein-protein interaction networks in Drosophila melanogaster embryos on a system-wide level. From cross-linked Drosophila embryo extracts, we detected 18,037 cross-link spectrum matches corresponding to 5,129 unique cross-linked residues in biological triplicate experiments at 5% FDR (3,098 at 1% FDR). Among these, 1,237 interprotein cross-linking sites were identified that contain valuable information on protein-protein interactions. The remaining 3,892 intra-protein cross-links yield information on conformational changes of proteins in their cellular environment.

scholarly journals Optimized cross-linking mass spectrometry for in situ interaction proteomics

2018 ◽  
Author(s):  
Zheng Ser ◽  
Paolo Cifani ◽  
Alex Kentsis
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Large Scale ◽  
Electron Transfer Dissociation ◽  
Live Cells ◽  
Cross Linking ◽  
Native Protein ◽  
Wide Range

AbstractRecent development of mass spectrometer cleavable protein cross-linkers and algorithms for their spectral identification now permits large-scale cross-linking mass spectrometry (XL-MS). Here, we optimized the use of cleavable disuccinimidyl sulfoxide (DSSO) cross-linker for labeling native protein complexes in live human cells. We applied a generalized linear mixture model to calibrate cross-link peptide-spectra matching (CSM) scores to control the sensitivity and specificity of large-scale XL-MS. Using specific CSM score thresholds to control the false discovery rate, we found that higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) can both be effective for large-scale XL-MS protein interaction mapping. We found that the density and coverage of protein-protein interaction maps can be significantly improved through the use of multiple proteases. In addition, the use of sample-specific search databases can be used to improve the specificity of cross-linked peptide spectral matching. Application of this approach to human chromatin labeled in live cells recapitulated known and revealed new protein interactions of nucleosomes and other chromatin-associated complexes in situ. This optimized approach for mapping native protein interactions should be useful for a wide range of biological problems.

scholarly journals Protein interaction landscapes revealed by advanced in vivo cross-linking–mass spectrometry

2021 ◽  
Vol 118 (32) ◽  
pp. e2023360118
Author(s):  
Andrew Wheat ◽  
Clinton Yu ◽  
Xiaorong Wang ◽  
Anthony M. Burke ◽  
Ilan E. Chemmama ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Cross Linking ◽  
Technological Advancement ◽  
Protein Protein Interactions ◽  
Hek 293 ◽  
293 Cells ◽  
And Function

Defining protein–protein interactions (PPIs) in their native environment is crucial to understanding protein structure and function. Cross-linking–mass spectrometry (XL-MS) has proven effective in capturing PPIs in living cells; however, the proteome coverage remains limited. Here, we have developed a robust in vivo XL-MS platform to facilitate in-depth PPI mapping by integrating a multifunctional MS-cleavable cross-linker with sample preparation strategies and high-resolution MS. The advancement of click chemistry–based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform enabled the identification of 13,904 unique lysine–lysine linkages from in vivo cross-linked HEK 293 cells, permitting construction of the largest in vivo PPI network to date, comprising 6,439 interactions among 2,484 proteins. These results allowed us to generate a highly detailed yet panoramic portrait of human interactomes associated with diverse cellular pathways. The strategy presented here signifies a technological advancement for in vivo PPI mapping at the systems level and can be generalized for charting protein interaction landscapes in any organisms.

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