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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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Related in: MedlinePlus

Combining MS of protein assemblies with CXMS.A, mass spectrometry of intact complexes reveals the stoichiometry of the intact AKAP complex: 2 × [AKAP79 + PP2B (A+B) + 2RII D/D + CaM] and demonstrates the dimerization of AKAP 79. B and C, confirmation of the AKAP dimer by chemical cross-linking and MS also suggests that the dimer is aligned in parallel. D, a proposed model for the architecture and dynamics of the core AKAP79 signaling complex, in conditions of both high and low [Ca2+]. The second AKAP79 monomer and associated proteins are in the background. These figures are reproduced from Ref. 104 with permission.
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Figure 3: Combining MS of protein assemblies with CXMS.A, mass spectrometry of intact complexes reveals the stoichiometry of the intact AKAP complex: 2 × [AKAP79 + PP2B (A+B) + 2RII D/D + CaM] and demonstrates the dimerization of AKAP 79. B and C, confirmation of the AKAP dimer by chemical cross-linking and MS also suggests that the dimer is aligned in parallel. D, a proposed model for the architecture and dynamics of the core AKAP79 signaling complex, in conditions of both high and low [Ca2+]. The second AKAP79 monomer and associated proteins are in the background. These figures are reproduced from Ref. 104 with permission.

Mentions: In cases where the identity of potential interactors is limited, and in principle known, as it is for many protein complexes, this problem is simplified. Only those proteins present in the complex need to be considered (101). This was the case in a recent study where the MS of intact assemblies was combined with CXMS to study the architecture and dynamics of a major signaling complex (104) (Fig. 3). Here, MS of the intact AKAP79 assemblies, expressed recombinantly, established the dimerization of AKAP79. It was also possible to demonstrate that one AKAP79 molecule binds to two fragments of its regulatory subunits. Using a CMXS strategy that employs the lysine linker disuccinimidylsuberate, dimeric AKAP79 cross-linking sites were identified with xQuest after selecting for homodimeric peptides. This procedure was used to verify the dimerization properties of AKAP79 identified in the MS of the intact complex. The detection of symmetrical cross-linked pairs by CXMS suggested that the AKAP79 dimer is aligned in parallel. Additional validation was carried out using in silico digestion of AKAP79 and consideration of all possible cross-linked peptides within a certain threshold. This confirmed the cross-linked peptides indentified by xQuest and provided further evidence for this unexpected dimerization of AKAP79. With the stoichiometry of this subcomplex firmly established, it was possible to assign the quaternary structure to the mass determined for the intact AKAP79 signaling complex. This study serves to highlight how combining MS of intact assemblies with CXMS not only adds an internal validation to protein-protein interactions at the subunit level but also helps to integrate the precise location of an interaction within the landscape of a multiprotein assembly.


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 MS of protein assemblies with CXMS.A, mass spectrometry of intact complexes reveals the stoichiometry of the intact AKAP complex: 2 × [AKAP79 + PP2B (A+B) + 2RII D/D + CaM] and demonstrates the dimerization of AKAP 79. B and C, confirmation of the AKAP dimer by chemical cross-linking and MS also suggests that the dimer is aligned in parallel. D, a proposed model for the architecture and dynamics of the core AKAP79 signaling complex, in conditions of both high and low [Ca2+]. The second AKAP79 monomer and associated proteins are in the background. These figures are reproduced from Ref. 104 with permission.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Combining MS of protein assemblies with CXMS.A, mass spectrometry of intact complexes reveals the stoichiometry of the intact AKAP complex: 2 × [AKAP79 + PP2B (A+B) + 2RII D/D + CaM] and demonstrates the dimerization of AKAP 79. B and C, confirmation of the AKAP dimer by chemical cross-linking and MS also suggests that the dimer is aligned in parallel. D, a proposed model for the architecture and dynamics of the core AKAP79 signaling complex, in conditions of both high and low [Ca2+]. The second AKAP79 monomer and associated proteins are in the background. These figures are reproduced from Ref. 104 with permission.
Mentions: In cases where the identity of potential interactors is limited, and in principle known, as it is for many protein complexes, this problem is simplified. Only those proteins present in the complex need to be considered (101). This was the case in a recent study where the MS of intact assemblies was combined with CXMS to study the architecture and dynamics of a major signaling complex (104) (Fig. 3). Here, MS of the intact AKAP79 assemblies, expressed recombinantly, established the dimerization of AKAP79. It was also possible to demonstrate that one AKAP79 molecule binds to two fragments of its regulatory subunits. Using a CMXS strategy that employs the lysine linker disuccinimidylsuberate, dimeric AKAP79 cross-linking sites were identified with xQuest after selecting for homodimeric peptides. This procedure was used to verify the dimerization properties of AKAP79 identified in the MS of the intact complex. The detection of symmetrical cross-linked pairs by CXMS suggested that the AKAP79 dimer is aligned in parallel. Additional validation was carried out using in silico digestion of AKAP79 and consideration of all possible cross-linked peptides within a certain threshold. This confirmed the cross-linked peptides indentified by xQuest and provided further evidence for this unexpected dimerization of AKAP79. With the stoichiometry of this subcomplex firmly established, it was possible to assign the quaternary structure to the mass determined for the intact AKAP79 signaling complex. This study serves to highlight how combining MS of intact assemblies with CXMS not only adds an internal validation to protein-protein interactions at the subunit level but also helps to integrate the precise location of an interaction within the landscape of a multiprotein assembly.

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
Related in: MedlinePlus