title: Influence of one- and two-dimensional gel electrophoresis procedure on metal-protein bindings examined by electrospray ionization mass spectrometry, inductively coupled plasma mass spectrometry, and ultrafiltration.
abstract: Three independent methods, (i) electrospray ionization mass spectrometry (ESI-MS), (ii) carrying out the complete protein preparation procedure required for protein gel electrophoresis (GE) including extraction, precipitation, washing, and desalting with subsequent microwave digestion of the produced protein fractions for metal content quantification, and (iii) ultrafiltration for separating protein-bound and unbound metal fractions, were employed to elucidate the influences of protein sample preparation and GE running conditions on metal-protein bindings. A treatment of the protein solution with acetone instead of trichloroacetic acid or ammonium sulfate for precipitate formation led to a strongly enhanced metal binding capacity. The desalting step of the resolubilized protein sample caused a metal loss between 10 and 35%. The omission of some extraction buffer additives led to a diminished metal binding capacity of protein fractions obtained from the sample preparation procedure for GE, whereas a tenside addition to the protein solution inhibited metal-protein bindings. The binding stoichiometry of Cu and Zn-protein complexes determined by ESI-MS was influenced by the type of the metal salt which was applied to the protein solution. A higher pH value of the sample solution promoted the metal ion complexation by the proteins. Ultrafiltration experiments revealed a higher Cu- and Zn-binding capacity of the model protein lysozyme in both resolubilization buffers for 1D- and 2D-GE compared to the protein extraction buffer. Strongly diminished metal binding capacities of lysozyme were recorded in the running buffer of 1D-GE and in the gel staining solutions.
authors: Technical University Bergakademie Freiberg, Institute of Analytical Chemistry, Leipziger Strasse 29, D-09599 Freiberg, Germany. Anne-Christine.Schmidt@chemie.tu-freiberg.de
authors: Anne-Christine
authors: AC
authors: Schmidt
authors: Bianca
authors: B
authors: Störr
authors: Nicolai-Alexeji
authors: NA
authors: Kummer
mesh_terms: D000818:Animals; D002021:Buffers; D002417:Cattle; D003300:Copper / Q000378:metabolism*; D003956:Dialysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014462:Ultrafiltration / Q000379:methods*; D015032:Zinc / Q000378:metabolism*
chemical_list: D002021:Buffers; D011506:Proteins; D003300:Copper; D015032:Zinc