1. MS of Intact Complexes and Hybrid Technologies
Elise Cai
Bi/Ch132

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

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

5. Native MS

6. 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

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

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

9. 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

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

11. 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

12. 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

13. Native MS with CXMS

14. 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

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

16. Potential Workflow

17. Thanks! Questions?