eXL-MS: An Enhanced Cross-Linking Mass Spectrometry Workflow To Study Protein Complexes
| Autor: | Martial Rey, Julia Chamot-Rooke, Isabel Lopez-Neira, Mathieu Dupré, Magalie Duchateau |
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| Přispěvatelé: | Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), This work has been partially supported by Institut Pasteur and CNRS. Financial support was from l’Agence Nationale de la Recherche (CLICKMASSLINK-ANR 09-PIRI-0006) and from the 'Investissement d’Avenir' Bioinformatique program (grant BIP:BIP, ANR-10-BINF-03-13), Synthesis and characterization of NNP9 were performed by the COBRA lab (Mont-Saint-Aignan, University of Rouen)., The authors are grateful to Q. Giai-Gianetto for the statistical analysis of cross-linked peptides distances and to Jonathan Dhenin for his help on Figure 6., ANR-09-PIRI-0006,CLICKMASSLINK,Pontage covalent ' clickable ' optimisé pour la spectrométrie de masse, application à l'analyse structurale des pili de type IV de Neisseria meningitidis(2009), ANR-10-BINF-0003,Bip:Bip,Paradigme d'inference bayesienne pour la Biologie structurale in silico(2010), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
MESH: Cross-Linking Reagents [SDV]Life Sciences [q-bio] MESH: Limit of Detection Mass spectrometry 01 natural sciences Analytical Chemistry Turn (biochemistry) 03 medical and health sciences chemistry.chemical_compound Biotin Limit of Detection Tandem Mass Spectrometry [CHIM.ANAL]Chemical Sciences/Analytical chemistry Nanotechnology Sample preparation MESH: Proteins [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Polyacrylamide gel electrophoresis MESH: Click Chemistry MESH: Nanotechnology Chromatography Chemistry 010401 analytical chemistry Proteins MESH: Tandem Mass Spectrometry MESH: Multiprotein Complexes 0104 chemical sciences Cross-Linking Reagents 030104 developmental biology Membrane protein Multiprotein Complexes Biotinylation Click chemistry Click Chemistry Electrophoresis Polyacrylamide Gel MESH: Chromatography Liquid Chromatography Liquid MESH: Electrophoresis Polyacrylamide Gel |
| Zdroj: | Analytical Chemistry Analytical Chemistry, American Chemical Society, 2018, 90 (18), pp.10707-10714. ⟨10.1021/acs.analchem.8b00737⟩ Analytical Chemistry, 2018, 90 (18), pp.10707-10714. ⟨10.1021/acs.analchem.8b00737⟩ |
| ISSN: | 0003-2700 1520-6882 |
| DOI: | 10.1021/acs.analchem.8b00737⟩ |
| Popis: | International audience; The analysis of proteins and protein complexes by cross-linking mass spectrometry (XL-MS) has expanded in the past decade. However, mostly used approaches suffer important limitations in term of efficiency and sensitivity. We describe here a new workflow based on the advanced use of the trifunctional cross-linker NNP9. NNP9 carries an azido group allowing the quantitative and selective introduction of a biotin molecule into cross-linked proteins. The incorporation is performed by click-chemistry using an adapted version of the enhanced filter-aided sample preparation (eFASP) protocol. This protocol, based on the use of a molecular filter, allows a very high recovery of peptides after enzymatic digestion and complete removal of contaminants. This in turn offers the possibility for one to analyze very large membrane proteins solubilized in detergent. After trypsin digestion, biotinylated peptides can be easily enriched on monoavidin beads and analyzed by LC-MS/MS. The whole workflow was developed on creatine kinase in the presence of detergent. It led to a drastic improvement in the number of identified cross-linked peptides (407 vs 81), compared to the conventional approach using a gel-based separation. One great advantage of our enhanced cross-linking mass spectrometry (eXL-MS) workflow is its high efficiency, allowing the analysis of a very low amount of material (15 μg). We also demonstrate that higher-energy collision dissociation (HCD) outperforms electron-transfer/higher-energy collision dissociation (EThcD) in terms of number of cross-linked peptides identified, but EThcD leads to better sequence coverage than HCD and thus easier localization of cross-linking sites. |
| Databáze: | OpenAIRE |
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