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.

CE on-line coupled to orthogonal accelerated TOF-MS (CE-oa-TOF-MS) is an emerging technique offering efficient charge-to-mass-based separations, as well as accurate and high-resolution mass measurements. Here, we investigated the main factors influencing the analysis of low molecular mass peptides using a sheath-flow electrospray ionization interface and several neuropeptides as model compounds. Moderate fragmentor voltage values of the oa-TOF-MS were crucial to maximize the production of molecular ions for optimum sensitivity precluding molecular fragmentation. However, the major fragments provided specific information that may result valuable for confirmatory purposes. Advantages and disadvantages of adding internal mass references to the sheath liquid for continuous mass-to-charge recalibration during the electrophoretic separations were discussed with regard to mass accuracy and sensitivity. Furthermore, several instrumental modes related to mass resolution were also examined. Finally, the method was validated for quantitative analysis of the studied neuropeptides in terms of repeatability, linearity and LODs. The results obtained using CE-oa-TOF-MS were compared with those obtained using CE coupled to other mass spectrometers. In addition to the simplicity and reliability on the identification, CE-oa-TOF-MS allowed improved repeatability and an around 10-fold improvement in sensitivity with respect to conventional reflectron TOF and ion trap mass analyzers.

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.

In this paper, we report nano-electrospray ionization-ion mobility mass spectrometry (nano-ESI-IM-MS) characterization of bovine superoxide dismutase (SOD-1) and human SOD-1 purified from erythrocytes. SOD-1 aggregates are characteristic of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease in humans that could be triggered by dissociation of the native dimeric enzyme (Cu(2),Zn(2)-dimer SOD-1). In contrast to ESI-MS, nano-ESI-IM-MS allowed an extra dimension for ion separation, yielding three-way mass spectra (drift time, mass-to-charge ratio and intensity). Drift time provided valuable structural information related to ion size, which proved useful to differentiate between the dimeric and monomeric forms of SOD-1 under non denaturing conditions. In order to obtain detailed structural information, including the most relevant post-translational modifications, we evaluated several parameters of the IM method, such as sample composition (10 mM ammonium acetate, pH 7) and activation voltages (trap collision energy and cone voltage). Neutral pH and a careful selection of the most appropriate activation voltages were necessary to minimize dimer dissociation, although human enzyme resulted less prone to dissociation. Under optimum conditions, a comparison between monomer-to-dimer abundance ratios of two small sets of blood samples from healthy control and ALS patients demonstrated the presence of a higher relative abundance of Cu,Zn-monomer SOD-1 in patient samples.

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