Metal displacement and stoichiometry of protein-metal complexes under native conditions using capillary electrophoresis/mass spectrometry.
Increases in the study of protein-metal complexes, as well as in metal displacement in protein-metal complexes under native conditions for optimum catalytic properties in drug research and catalyst design, demands a separation/detection technology that can accurately measure metal displacement and stoichiometry in protein-metal complexes. Both nuclear magnetic resonance (NMR) and X-ray diffraction techniques have been used for this purpose; however, these techniques lack sensitivity. Electrospray ionization mass spectrometry (ESI-MS) using direct infusion offers higher sensitivity than the former techniques and provides molecular distribution of various protein-metal complexes. However, since protein-metal complexes under native conditions usually are dissolved in salt solutions, their direct ESI-MS analysis requires off-line sample clean-up prior to MS analysis to avoid sample suppression during ESI. Moreover, direct infusion of the salty solution promotes non-specific salt adduct formation by the protein-metal complexes under ESI-MS, which complicates the identification and stoichiometry measurements of the protein-metal complexes. Because of the high mass of protein-metal complexes and lack of sufficient resolution by most mass spectrometers to separate non-specific from specific metal-protein complexes, accurate protein-metal stoichiometry measurements require some form of sample clean up prior to ESI-MS analysis. In this study, we demonstrate that capillary electrophoresis/electrospray ionization in conjunction with a medium-resolution (approximately 10,000) mass spectrometer is an efficient and fast method for the measurement of the stoichiometry of the protein-metal complexes under physiological conditions (pH approximately 7). The metal displacement of Co(2+) to Cd(2+), two metal ions necessary for activation in the monomeric AHL lactonase produced by B. thuringiensis, has been used as a proof of concept.
Garza
Selynda
S
Cedra Corp., Austin, TX, USA. [corrected]
Thomas
Pei W
PW
Fast
Walter
W
Moini
Mehdi
M
D000595:Amino Acid Sequence; D001413:Bacillus thuringiensis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002104:Cadmium / Q000737:chemistry; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics; D003035:Cobalt / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics; D008667:Metalloproteins / Q000737:chemistry*; D019216:Metals, Heavy / Q000737:chemistry*; D008969:Molecular Sequence Data; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D001426:Bacterial Proteins; D008667:Metalloproteins; D019216:Metals, Heavy; D002104:Cadmium; D003035:Cobalt; D002265:Carboxylic Ester Hydrolases; C432003:N-acyl homoserine lactonase; C406735:AiiA protein, Bacillus; D008666:Metalloendopeptidases