title: Protein-metal ion interactions, stoichiometries and relative affinities determined by on-line size exclusion gel filtration mass spectrometry.
abstract: The modulation of metal ions on protein function is well recognized and of paramount importance in protein biochemistry. To date, very few methods allow direct determination of protein-metal ion interactions, as well as exact stoichiometric binding ratios. In this work we demonstrate the usefulness of two on-line size exclusion gel filtration mass spectrometry approaches to directly detect protein-metal ion adducts, as well as determine exact protein-metal ion stoichiometries. We show that on-line size exclusion column chromatography (SEC) and rapid in-line desalting (RILED) coupled to microelectrospray mass spectrometry (microESI-MS) can be used for such analyses. The SEC approach can be effectively used to both separate proteins in a complex mixture and exchange buffers prior to the electrospray process. While RILED does not allow for protein separation, it provides a much faster high-throughput desalting procedure than the conventional SEC technique. Specifically, we show that SEC/microESI-MS and RILED/MS can be used to determine calcium ion binding stoichiometries to a high-affinity, metal ion binding protein, calbindin D(28K). Furthermore, the same approaches can also be used to determine metal ion binding stoichiometries of low-affinity metal-binding proteins such as Spo0F.
authors.affiliation: Biomedical Mass Spectrometry and Functional Proteomics Facility, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Linda M
authors.initials: LM
authors.lastname: Benson
authors.forename: Rajiv
authors.initials: R
authors.lastname: Kumar
authors.forename: John
authors.initials: J
authors.lastname: Cavanagh
authors.forename: Stephen
authors.initials: S
authors.lastname: Naylor
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D064026:Calbindins; D002118:Calcium / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry; D002850:Chromatography, Gel / Q000379:methods*; D008274:Magnesium / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008670:Metals / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000737:chemistry
chemical_list: D064026:Calbindins; D002352:Carrier Proteins; D008670:Metals; D011506:Proteins; D064030:S100 Calcium Binding Protein G; D008274:Magnesium; D002118:Calcium