MS of Intact Complexes and Hybrid Technologies

Elise CaiBi/Ch13211-27-12

Protein Complexes

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

Native MS• MS of whole protein assemblies• Examine large, heterogeneous complexes• 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?