title: Quantification of protein-protein interactions with chemical cross-linking and mass spectrometry.
abstract: Chemical cross-linking in combination with mass spectrometry has largely been used to study protein structures and protein-protein interactions. Typically, it is used in a qualitative manner to identify cross-linked sites and provide a low-resolution topological map of the interacting regions of proteins. Here, we investigate the capability of chemical cross-linking to quantify protein-protein interactions using a model system of calmodulin and substrates melittin and mastoparan. Calmodulin is a well-characterized protein which has many substrates. Melittin and mastoparan are two such substrates which bind to calmodulin in 1:1 ratios in the presence of calcium. Both the calmodulin-melittin and calmodulin-mastoparan complexes have had chemical cross-linking strategies successfully applied in the past to investigate topological properties. We utilized an excess of immobilized calmodulin on agarose beads and formed complexes with varying quantities of mastoparan and melittin. Then, we applied disuccinimidyl suberate (DSS) chemical cross-linker, digested and detected cross-links through an LC-MS analytical method. We identified five interpeptide cross-links for calmodulin-melittin and three interpeptide cross-links for calmodulin-mastoparan. Using cross-linking sites of calmodulin-mastoparan, we demonstrated that mastoparan also binds in two orientations to calmodulin. We quantitatively demonstrated that both melittin and mastoparan preferentially bind to calmodulin in a parallel fashion, which is opposite to the preferred binding mode of the majority of known calmodulin binding peptides. We also demonstrated that the relative abundances of cross-linked peptide products quantitatively reflected the abundances of the calmodulin peptide complexes formed.
authors.affiliation: Department of Genome Sciences, University of Washington, PO Box 358050, Seattle, Washington 98195, United States.
authors.forename: Juan D
authors.initials: JD
authors.lastname: Chavez
authors.forename: Neal L
authors.initials: NL
authors.lastname: Liu
authors.forename: James E
authors.initials: JE
authors.lastname: Bruce
mesh_terms: D000595:Amino Acid Sequence; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D036341:Intercellular Signaling Peptides and Proteins; D013058:Mass Spectrometry / Q000379:methods*; D008555:Melitten / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods*; D013388:Succinimides / Q000737:chemistry; D014862:Wasp Venoms / Q000737:chemistry / Q000378:metabolism
chemical_list: D002147:Calmodulin; D003432:Cross-Linking Reagents; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D013388:Succinimides; D014862:Wasp Venoms; D008555:Melitten; C037500:mastoparan; C019358:disuccinimidyl suberate