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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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Binding of tubulin inside the central cavity of bovine Tric complex revealed by mass spectrometry. Comparison of nondenaturing MS spectra of intact bovine Tric complex before (upper panel) and after (lower panel) ATP incubation and SEC purification shows that the complex has one molecule of substrate bound. The two series are measured as 947.5 (blue) and 997.7 kDa (orange), respectively. Insets across the cryo-EM three-dimensional reconstitution at 30A of the purified complex (blue) also show additional EM density (yellow), in line with the MS data. These figures are reproduced from Ref. 118 with permission.
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Figure 4: Binding of tubulin inside the central cavity of bovine Tric complex revealed by mass spectrometry. Comparison of nondenaturing MS spectra of intact bovine Tric complex before (upper panel) and after (lower panel) ATP incubation and SEC purification shows that the complex has one molecule of substrate bound. The two series are measured as 947.5 (blue) and 997.7 kDa (orange), respectively. Insets across the cryo-EM three-dimensional reconstitution at 30A of the purified complex (blue) also show additional EM density (yellow), in line with the MS data. These figures are reproduced from Ref. 118 with permission.

Mentions: A further illustration of the power of CXMS and the MS of intact complexes in combination with EM is shown in Fig. 4, where these techniques were applied to study the binding of tubulin by bovine Tric complex (118). Comparisons of nondenaturing MS spectra of intact bovine Tric complex before and after ATP incubation were used to show that the Tric complex is able to bind one molecule of substrate. Additional details of this Tric-tubulin interaction were subsequently confirmed by CXMS, again using the xQuest algorithm (95). This information could be used to interpret densities in the cryo-EM three-dimensional reconstitution of the purified complex and helped to confirm substrate binding in the central cavity of the Tric complex.


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

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

Binding of tubulin inside the central cavity of bovine Tric complex revealed by mass spectrometry. Comparison of nondenaturing MS spectra of intact bovine Tric complex before (upper panel) and after (lower panel) ATP incubation and SEC purification shows that the complex has one molecule of substrate bound. The two series are measured as 947.5 (blue) and 997.7 kDa (orange), respectively. Insets across the cryo-EM three-dimensional reconstitution at 30A of the purified complex (blue) also show additional EM density (yellow), in line with the MS data. These figures are reproduced from Ref. 118 with permission.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Binding of tubulin inside the central cavity of bovine Tric complex revealed by mass spectrometry. Comparison of nondenaturing MS spectra of intact bovine Tric complex before (upper panel) and after (lower panel) ATP incubation and SEC purification shows that the complex has one molecule of substrate bound. The two series are measured as 947.5 (blue) and 997.7 kDa (orange), respectively. Insets across the cryo-EM three-dimensional reconstitution at 30A of the purified complex (blue) also show additional EM density (yellow), in line with the MS data. These figures are reproduced from Ref. 118 with permission.
Mentions: A further illustration of the power of CXMS and the MS of intact complexes in combination with EM is shown in Fig. 4, where these techniques were applied to study the binding of tubulin by bovine Tric complex (118). Comparisons of nondenaturing MS spectra of intact bovine Tric complex before and after ATP incubation were used to show that the Tric complex is able to bind one molecule of substrate. Additional details of this Tric-tubulin interaction were subsequently confirmed by CXMS, again using the xQuest algorithm (95). This information could be used to interpret densities in the cryo-EM three-dimensional reconstitution of the purified complex and helped to confirm substrate binding in the central cavity of the Tric complex.

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