ms of intact complexes and hybrid technologies elise cai bi/ch132 11-27-12
TRANSCRIPT
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
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
• 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