title: Low temperature aqueous electrospray ionization mass spectrometry of noncovalent complexes.
abstract: In the present study we describe conditions that permit the characterization of noncovalent protein-substrate complexes in aqueous solution by microspray electrospray ionization-mass spectrometry (ESI-MS), using a heated transfer capillary at low temperature (45 degrees C). Specifically, we examined the binding of calmodulin to two polypeptides; the calmodulin-binding domain of calmodulin-dependent protein kinase II (CamK-II) and melittin. Calmodulin, a well known calcium-binding protein, binds to a number of small amphipathic peptides in a calcium-dependent manner. Our results directly show that both peptides form equimolar complexes with calmodulin only in the presence of calcium. The stoichiometry necessary for the formation of each complex was 1:1:4 for calmodulin:peptide (melittin or CamK-II):Ca2+, respectively. Furthermore, it is demonstrated that the detection of the complex in ESI-MS is source temperature dependent.
authors.affiliation: Nephrology Research Unit, Mayo Clinic Foundation, Rochester, Minnesota 55905, USA.
authors.forename: T D
authors.initials: TD
authors.lastname: Veenstra
authors.forename: A J
authors.initials: AJ
authors.lastname: Tomlinson
authors.forename: L
authors.initials: L
authors.lastname: Benson
authors.forename: R
authors.initials: R
authors.lastname: Kumar
authors.forename: S
authors.initials: S
authors.lastname: Naylor
mesh_terms: D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis; D002147:Calmodulin / Q000032:analysis; D013058:Mass Spectrometry / Q000379:methods*; D008555:Melitten / Q000032:analysis; D013696:Temperature
chemical_list: D002147:Calmodulin; D008555:Melitten; D017871:Calcium-Calmodulin-Dependent Protein Kinases