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 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

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