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Joining forces: integrating proteomics and cross-linking with the mass spectrometry of intact complexes.

Stengel F, Aebersold R, Robinson CV - Mol. Cell Proteomics (2011)

Bottom Line: There is therefore a growing demand for hybrid technologies that are able to complement classical structural biology methods and thereby broaden our arsenal for the study of these important complexes.Exciting new developments in the field of mass spectrometry and proteomics have added a new dimension to the study of protein-protein interactions and protein complex architecture.In this review, we focus on how complementary mass spectrometry-based techniques can greatly facilitate structural understanding of protein assemblies.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford, OX1 3TA United Kingdom.

ABSTRACT
Protein assemblies are critical for cellular function and understanding their physical organization is the key aim of structural biology. However, applying conventional structural biology approaches is challenging for transient, dynamic, or polydisperse assemblies. There is therefore a growing demand for hybrid technologies that are able to complement classical structural biology methods and thereby broaden our arsenal for the study of these important complexes. Exciting new developments in the field of mass spectrometry and proteomics have added a new dimension to the study of protein-protein interactions and protein complex architecture. In this review, we focus on how complementary mass spectrometry-based techniques can greatly facilitate structural understanding of protein assemblies.

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Combining multiple MS-based methods for the analysis of protein complexes.A, after purification of the complex/assembly, usually by affinity purification in combination with one or more biochemical fractionation techniques, the sample is analyzed using the following methods. B–H, quantitative proteomics (B) to assess the subunit content assists the process in C, the assignment of the absolute stoichiometry of the intact complex and subcomplexes by MS of intact assemblies. Additional protein-protein interaction data are generated by AP-MS (D) and CXMS (E). Combining the data will give a complete and validated map of the subunit interactome (F). Topological information from CXMS and IM-MS (G) can be used to provide additional local and global restraints that can be used to together with the protein-protein interaction data to guide molecular modeling of the complex (H).
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Figure 5: Combining multiple MS-based methods for the analysis of protein complexes.A, after purification of the complex/assembly, usually by affinity purification in combination with one or more biochemical fractionation techniques, the sample is analyzed using the following methods. B–H, quantitative proteomics (B) to assess the subunit content assists the process in C, the assignment of the absolute stoichiometry of the intact complex and subcomplexes by MS of intact assemblies. Additional protein-protein interaction data are generated by AP-MS (D) and CXMS (E). Combining the data will give a complete and validated map of the subunit interactome (F). Topological information from CXMS and IM-MS (G) can be used to provide additional local and global restraints that can be used to together with the protein-protein interaction data to guide molecular modeling of the complex (H).

Mentions: These examples demonstrate that modern MS-based technologies, especially when used in conjunction with modeling and EM, represent a powerful hybrid approach for generating structural information. A potential work flow is depicted in Fig. 5. After purification of the sample, involving multiple biochemical and affinity purification steps, subunit interaction maps can be generated following analysis by CXMS, quantitative AP-MS, and MS of intact complexes. Each of these technologies has different strengths and weaknesses. AP-MS is able to detect subtle changes in interaction partners and dynamics and is able to deal with lowly expressed complexes. MS of intact assemblies is able to assess the absolute stoichiometry of the intact complex and multiple subcomplexes. CXMS is capable of providing many interactions for a single protein-protein interaction and at peptide resolution. Combining the different data sets could generate complete interaction maps for subsequent merging with topological information from IM-MS and CXMS.


Joining forces: integrating proteomics and cross-linking with the mass spectrometry of intact complexes.

Stengel F, Aebersold R, Robinson CV - Mol. Cell Proteomics (2011)

Combining multiple MS-based methods for the analysis of protein complexes.A, after purification of the complex/assembly, usually by affinity purification in combination with one or more biochemical fractionation techniques, the sample is analyzed using the following methods. B–H, quantitative proteomics (B) to assess the subunit content assists the process in C, the assignment of the absolute stoichiometry of the intact complex and subcomplexes by MS of intact assemblies. Additional protein-protein interaction data are generated by AP-MS (D) and CXMS (E). Combining the data will give a complete and validated map of the subunit interactome (F). Topological information from CXMS and IM-MS (G) can be used to provide additional local and global restraints that can be used to together with the protein-protein interaction data to guide molecular modeling of the complex (H).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3316738&req=5

Figure 5: Combining multiple MS-based methods for the analysis of protein complexes.A, after purification of the complex/assembly, usually by affinity purification in combination with one or more biochemical fractionation techniques, the sample is analyzed using the following methods. B–H, quantitative proteomics (B) to assess the subunit content assists the process in C, the assignment of the absolute stoichiometry of the intact complex and subcomplexes by MS of intact assemblies. Additional protein-protein interaction data are generated by AP-MS (D) and CXMS (E). Combining the data will give a complete and validated map of the subunit interactome (F). Topological information from CXMS and IM-MS (G) can be used to provide additional local and global restraints that can be used to together with the protein-protein interaction data to guide molecular modeling of the complex (H).
Mentions: These examples demonstrate that modern MS-based technologies, especially when used in conjunction with modeling and EM, represent a powerful hybrid approach for generating structural information. A potential work flow is depicted in Fig. 5. After purification of the sample, involving multiple biochemical and affinity purification steps, subunit interaction maps can be generated following analysis by CXMS, quantitative AP-MS, and MS of intact complexes. Each of these technologies has different strengths and weaknesses. AP-MS is able to detect subtle changes in interaction partners and dynamics and is able to deal with lowly expressed complexes. MS of intact assemblies is able to assess the absolute stoichiometry of the intact complex and multiple subcomplexes. CXMS is capable of providing many interactions for a single protein-protein interaction and at peptide resolution. Combining the different data sets could generate complete interaction maps for subsequent merging with topological information from IM-MS and CXMS.

Bottom Line: There is therefore a growing demand for hybrid technologies that are able to complement classical structural biology methods and thereby broaden our arsenal for the study of these important complexes.Exciting new developments in the field of mass spectrometry and proteomics have added a new dimension to the study of protein-protein interactions and protein complex architecture.In this review, we focus on how complementary mass spectrometry-based techniques can greatly facilitate structural understanding of protein assemblies.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford, OX1 3TA United Kingdom.

ABSTRACT
Protein assemblies are critical for cellular function and understanding their physical organization is the key aim of structural biology. However, applying conventional structural biology approaches is challenging for transient, dynamic, or polydisperse assemblies. There is therefore a growing demand for hybrid technologies that are able to complement classical structural biology methods and thereby broaden our arsenal for the study of these important complexes. Exciting new developments in the field of mass spectrometry and proteomics have added a new dimension to the study of protein-protein interactions and protein complex architecture. In this review, we focus on how complementary mass spectrometry-based techniques can greatly facilitate structural understanding of protein assemblies.

Show MeSH