eXL-MS: An Enhanced Cross-Linking Mass Spectrometry Workflow To Study Protein Complexes
Martial Rey1, Mathieu Dupré1, Isabel Lopez-Neira1
1Mass Spectrometry for Biology Unit, CNRS USR 2000 , Institut Pasteur , Paris , 75015 , France.
Analytical Chemistry
|August 21, 2018
Summary
A new workflow enhances cross-linking mass spectrometry (XL-MS) efficiency and sensitivity. This method uses NNP9 cross-linker and click chemistry for improved protein complex analysis, even with limited sample amounts.
Area of Science:
- Biochemistry
- Proteomics
- Analytical Chemistry
Background:
- Cross-linking mass spectrometry (XL-MS) is crucial for studying protein and protein complex structures.
- Existing XL-MS methods often face limitations in efficiency and sensitivity, hindering comprehensive analysis.
- Analyzing large membrane proteins in detergents presents significant challenges.
Purpose of the Study:
- To introduce a novel, highly efficient, and sensitive XL-MS workflow.
- To enable the analysis of challenging samples, including large membrane proteins.
- To improve the identification and localization of cross-linking sites in proteins.
Main Methods:
- Utilized a trifunctional cross-linker (NNP9) with an azido group for biotinylation via click chemistry.
- Adapted the enhanced filter-aided sample preparation (eFASP) protocol for high peptide recovery and contaminant removal.
- Employed monoavidin bead enrichment followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
Main Results:
- The enhanced cross-linking mass spectrometry (eXL-MS) workflow identified significantly more cross-linked peptides (407 vs. 81) compared to conventional methods.
- The workflow demonstrated high efficiency, enabling analysis with as little as 15 μg of material.
- Higher-energy collision dissociation (HCD) yielded more identified cross-linked peptides, while electron-transfer/higher-energy collision dissociation (EThcD) provided better sequence coverage for site localization.
Conclusions:
- The developed eXL-MS workflow offers a substantial improvement in efficiency and sensitivity for protein complex analysis.
- This method is suitable for analyzing large membrane proteins and requires minimal sample input.
- Both HCD and EThcD fragmentation methods have complementary strengths for XL-MS data analysis.
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