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 ◽  
Ion Mobility ◽  
Structural Proteomics ◽  
Cross Linking

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 Cross-linking and other structural proteomics techniques: how chemistry is enabling mass spectrometry applications in structural biology

2016 ◽  
Vol 7 (8) ◽  
pp. 4792-4803 ◽  
Author(s):  
Alexander Leitner
Keyword(s):  
Mass Spectrometry ◽  
Structural Biology ◽  
Higher Order ◽  
Structural Proteomics ◽  
Cross Linking ◽  
Protein Assemblies ◽  
Chemical Methods

In this perspective, I highlight the contribution of chemical methods to the field of structural proteomics, where mass spectrometry is used to probe the structures of proteins and higher-order protein assemblies.

scholarly journals Interfaces with Structure Dynamics of the Workhorses from Cells Revealed through Cross-Linking Mass Spectrometry (CLMS)

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 382 ◽  
Author(s):  
Umesh Kalathiya ◽  
Monikaben Padariya ◽  
Jakub Faktor ◽  
Etienne Coyaud ◽  
Javier A. Alfaro ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Structural Proteomics ◽  
Cross Linking ◽  
Diverse Range ◽  
Interaction Sites ◽  
Structural Systems Biology ◽  
Rna Complexes

The fundamentals of how protein–protein/RNA/DNA interactions influence the structures and functions of the workhorses from the cells have been well documented in the 20th century. A diverse set of methods exist to determine such interactions between different components, particularly, the mass spectrometry (MS) methods, with its advanced instrumentation, has become a significant approach to analyze a diverse range of biomolecules, as well as bring insights to their biomolecular processes. This review highlights the principal role of chemistry in MS-based structural proteomics approaches, with a particular focus on the chemical cross-linking of protein–protein/DNA/RNA complexes. In addition, we discuss different methods to prepare the cross-linked samples for MS analysis and tools to identify cross-linked peptides. Cross-linking mass spectrometry (CLMS) holds promise to identify interaction sites in larger and more complex biological systems. The typical CLMS workflow allows for the measurement of the proximity in three-dimensional space of amino acids, identifying proteins in direct contact with DNA or RNA, and it provides information on the folds of proteins as well as their topology in the complexes. Principal CLMS applications, its notable successes, as well as common pipelines that bridge proteomics, molecular biology, structural systems biology, and interactomics are outlined.

Integrated Workflow for Structural Proteomics Studies Based on Cross-Linking/Mass Spectrometry with an MS/MS Cleavable Cross-Linker

2016 ◽  
Vol 88 (16) ◽  
pp. 7930-7937 ◽  
Author(s):  
Christian Arlt ◽  
Michael Götze ◽  
Christian H. Ihling ◽  
Christoph Hage ◽  
Mathias Schäfer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Proteomics ◽  
Cross Linking ◽  
Cross Linker

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 Ion mobility–mass spectrometry for structural proteomics

2012 ◽  
Vol 9 (1) ◽  
pp. 47-58 ◽  
Author(s):  
Yueyang Zhong ◽  
Suk-Joon Hyung ◽  
Brandon T Ruotolo
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Structural Proteomics ◽  
Ion Mobility Mass Spectrometry
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