MS of Intact Complexes and
Hybrid Technologies
Elise Cai
Bi/Ch132
11-27-12
Protein Complexes
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Subunit composition, stoichiometry
Protein-protein interactions
Architectural organization
Dynamic changes
Native MS
• MS of whole protein assemblies
• Examine large, heterogeneous complexes
• Determine subunit stoichiometry and dynamic changes
Native MS
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
Hybrid Technologies
Integrating native MS with:
• Quantitative proteomics
• Quantitative interaction proteomics
• Cross-linking MS
• Ion mobility-MS
Quantitative Proteomics
• Identify and quantify proteins in a sample
• MS not inherently quantitative
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
Quantitative Interaction Proteomics
• Identify and quantify
protein complex
components from affinity
purification (AP-MS)
• Various quantification
strategies
– isotope-labeled reference
peptides
– label-free quantification
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
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
Native MS with CXMS
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
Ion Mobility-MS
•
Separate ions by m/z and mobility in a carrier buffer gas
Potential Workflow
Thanks!
Questions?