Capillary electrophoresis/mass spectrometry for the separation and characterization of bovine Cu,Zn-superoxide dismutase.
The native form of Cu,Zn-superoxide dismutase (SOD-1) is a homodimer that coordinates one Cu(2+) and one Zn(2+) per monomer. Cu(2+) and Zn(2+) ions play crucial roles in enzyme activity and structural stability, respectively. In addition, dimer formation is essential for SOD-1 functionality, and in humans several SOD-1 mutant isoforms have been associated with certain types of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder. In this paper we used capillary electrophoresis and mass spectrometry to study the different structures of bovine SOD-1. The metal ions of the native enzyme (Cu(2),Zn(2)-dimer SOD-1) were released in acidic medium in order to obtain apo-SOD-1, which is a monomer. Both substances were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis with ultraviolet and electrospray ionization mass spectrometry detection (CE/UV and CE/ESI-MS, respectively). With MALDI-TOF-MS, using matrices of sinapinic acid (SA) or 2,5-dihydroxybenzoic acid (DHB) with or without trifluoroacetic acid (TFA), similar mass spectra were obtained for the metalated and non-metalated samples. In both cases, an average molecular mass corresponding to the apo-monomer SOD-1 was calculated. This finding indicated that the metals were released from the Cu(2),Zn(2)-dimer SOD-1 during sample preparation or ionization. For CE/UV and CE/ESI-MS, two background electrolytes (BGEs) potentially compatible with ESI-MS detection were used, namely 1 M of acetic acid (pH 2.3) and 10 mM of ammonium acetate (pH 7.3). Using a sheath liquid of 2-propanol/water (60:40 v/v), with or without 0.1% v/v of formic acid, CE/ESI-MS sensitivity was enhanced when the acidic BGE and the acidic sheath liquid were used. However, the electrophoretic profiles and the mass spectra obtained suggested that the metals of Cu(2),Zn(2)-dimer SOD-1 were released, which generated the apo-monomer during the electrophoretic separation. The neutral BGE provided enhanced conditions for the detection of the native enzyme. The differences between the mass spectra obtained for the Cu(2),Zn(2)-dimer and the apo-monomer forms were significant and the presence of formic acid in the sheath liquid affected only sensitivity. Our results highlight the importance of selecting appropriate non-denaturing separation and detection conditions to obtain reliable structural information about non-covalent protein complexes by CE/ESI-MS.
Borges-Alvarez
Marta
M
Department of Analytical Chemistry, University of Barcelona, Diagonal 647, 08028 Barcelona, Spain.
Benavente
Fernando
F
Barbosa
José
J
Sanz-Nebot
Victoria
V
D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D009994:Osmolar Concentration; D055503:Protein Multimerization; D013482:Superoxide Dismutase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000072105:Superoxide Dismutase-1
D001059:Apoproteins; C000606290:SOD1 protein, human; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1