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MS of Intact Complexes and Hybrid Technologies

MS of Intact Complexes and Hybrid Technologies. Elise Cai Bi/Ch132 11-27-12. Protein Complexes. Subunit composition, stoichiometry Protein-protein interactions Architectural organization Dynamic changes. Native MS. MS of whole protein assemblies Examine large, heterogeneous complexes

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MS of Intact Complexes and Hybrid Technologies

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  1. MS of Intact Complexes and Hybrid Technologies Elise Cai Bi/Ch132 11-27-12

  2. Protein Complexes • Subunit composition, stoichiometry • Protein-protein interactions • Architectural organization • Dynamic changes

  3. Native MS • MS of whole protein assemblies • Examine large, heterogeneous complexes • Determine subunit stoichiometry and dynamic changes

  4. Native MS

  5. Native MS Limitations: • Cannot distinguish 3-D packing arrangements • Challenges to identifying subunit components • Transient and weak interactions • Gentle conditions introduce uncertainty • Heterogeneity of samples

  6. Hybrid Technologies Integrating native MS with: • Quantitative proteomics • Quantitative interaction proteomics • Cross-linking MS • Ion mobility-MS

  7. Quantitative Proteomics • Identify and quantify proteins in a sample • MS not inherently quantitative

  8. Native MS and Quantitative Proteomics • Native MS challenges: • Transient interactors at substoichiometric levels • Many possible subunits of similar masses • Quantitative proteomics creates reference library of all potential candidates for native MS  Reduced ambiguity of stoichiometric assignments

  9. Quantitative Interaction Proteomics • Identify and quantify protein complex components from affinity purification (AP-MS) • Various quantification strategies • isotope-labeled reference peptides • label-free quantification

  10. Native MS with Quantitative Interaction Proteomics • Both methods assess stoichiometry of protein complexes • --Quantitative interaction proteomics detects subtle changes in interaction partners • --Native MS determines absolute subunit • stoichiometry

  11. Cross-linking MS (CXMS) • Identify protein contacts at peptide level • Problem: possible cross-links for n peptides= (n2+n)/2 • Analyzing CXMS datasets computationally expensive

  12. Native MS with CXMS

  13. Native MS with CXMS • CXMS precisely locates protein-protein interactions detected by native MS • Both techniques can be applied to heterogeneous environments • Both techniques analyze large protein assemblies in native state

  14. Ion Mobility-MS • Separate ions by m/z and mobility in a carrier buffer gas

  15. Potential Workflow

  16. Thanks!Questions?

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