Separation and characterization of superoxide dismutase 1 (SOD-1) from human erythrocytes by capillary electrophoresis time-of-flight mass spectrometry.
Cu, Zn-superoxide dismutase (SOD-1) is a homodimeric metalloenzyme that has been related to ALS (amyotrophic lateral sclerosis). The majority of ALS cases are sporadic while approximately 10% are inherited (familial ALS, FALS). Mutations in the amino acid sequence of human SOD-1 cause only 25% of the FALS cases, while the explanation for the rest is not clear yet. In this way, several authors have suggested the importance of posttranslational modifications or dimer dissociation on formation of the characteristic fatal intraneuronal SOD-1 aggregates. In this paper, we used capillary electrophoresis-electrospray mass spectrometry with an accurate mass and high-resolution time-of-flight mass spectrometer (CE-TOF-MS) for separation and characterization of standard bovine SOD-1 and human SOD-1 purified from erythrocytes. Two background electrolytes (BGEs) were used for CE-TOF-MS experiments in positive ion mode. An acidic BGE allowed detection of apo-monomer SOD-1, because the metal ions were completely released during the electrophoretic separation. The better sensitivity at acidic pH was especially interesting to detect different isoforms of human SOD-1. In contrast, a neutral BGE provided enhanced conditions for detection of the fully metalated dimeric and monomeric enzyme, but selecting an appropriate fragmentor voltage value in the TOF analyzer was critical to obtain reliable quantitative information. Anyway, only the metalated forms involving the main isoform of human SOD-1 were detected due to the lower sensitivity. Hence, the combination of both methodologies resulted necessary to obtain detailed structural information from the enzyme.
Department of Analytical Chemistry, University of Barcelona, Barcelona, Spain.
Marta
M
Borges-Alvarez
Fernando
F
Benavente
José
J
Barbosa
Victoria
V
Sanz-Nebot
D000085:Acetates / Q000737:chemistry; D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D013482:Superoxide Dismutase / Q000737:chemistry / Q000302:isolation & purification*; D000072105:Superoxide Dismutase-1
D000085:Acetates; C000606290:SOD1 protein, human; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; C018824:ammonium acetate