Separation and characterization of superoxide dismutase 1 (SOD‐1) from human erythrocytes by capillary electrophoresis time‐of‐flight mass spectrometry

Abstract

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.

Characterization of superoxide dismutase 1 (SOD‐1) by electrospray ionization‐ion mobility mass spectrometry

“…A comparison of the m/z values of the ions of both forms shows that only the ions of the dimer with an odd charge are specific to the dimeric form (‘odd ions’). Therefore, careful analysis of the overlapped mass spectra obtained by CE‐MS at neutral pH allowed a first approximation to the monomer‐dimer composition of SOD‐1 . Furthermore, we observed that dimer dissociation was promoted by low pH of the separation buffer or by certain parameters of the MS detector, especially the fragmentor voltage of the oa‐TOF mass spectrometer .…”

“…In previous studies, we demonstrated the suitability of CE‐MS at neutral pH to detect the presence of monomeric and dimeric forms of bovine and human SOD‐1 in solution . However, these forms were not baseline resolved by CE, and their mass spectra overlapped greatly.…”

“…The prevention of ALS and the development of effective treatments for this disease call for greater knowledge of the mechanisms behind SOD‐1 aggregation. In general, the MS techniques used to analyse SOD‐1 require analytical conditions such as low pH, which promote metal release from the enzyme structure, thus leading to metal‐deficient forms, mostly the apo‐monomer . Recently, we demonstrated that capillary electrophoresis‐MS (CE‐MS) at neutral pH can provide improved molecular mass information that is useful to estimate monomer‐dimer composition .…”

Mechanisms tagging senescent red blood cells for clearance in healthy humans

“…Interaction of O − 2 with NO ( k = 4–610 9 M −1 s −1 ) is four orders of magnitude faster than dismutation of O − 2 to H 2 O 2 catalyzed by SOD ( k = 210 5 M −1 s −1 ). Thus, formation of peroxynitrite (ONOO − ) from O − 2 dominates over the transformation of O − 2 to H 2 O 2 catalyzed by superoxide dismutase if NO is available (Borges-Alvarez et al, 2012). Peroxynitrite generated in this manner is believed to be a potent mediator of oxidative stress in RBC (Minetti et al, 2008; Rifkind and Nagababu, 2013).…”

Determination of metal content in superoxide dismutase enzymes by capillary electrophoresis†

“…Compared to these techniques, CE is a fast and simple method that has advantages such as high separation ability of mixtures of ions, small sample, and BGE consumption and ease of automation . Moreover, CE has been used for the general characterization of SODs .…”