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Capillary electrophoresis/mass spectrometry for the separation and characterization of bovine Cu,Zn-superoxide dismutase.
Rapid Commun Mass Spectrom. 2010 May 30; 24(10):1411-8.RC

Abstract

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.

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Publisher Full Text (DOI)

Authors+Show Affiliations

Borges-Alvarez M
Department of Analytical Chemistry, University of Barcelona, Diagonal 647, 08028 Barcelona, Spain.
Benavente F
No affiliation info available
Barbosa J
No affiliation info available
Sanz-Nebot V
No affiliation info available

MeSH

AnimalsApoproteinsCattleElectrophoresis, CapillaryHydrogen-Ion ConcentrationMass SpectrometryOsmolar ConcentrationProtein MultimerizationSuperoxide DismutaseSuperoxide Dismutase-1

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

20411580

Citation

Borges-Alvarez, Marta, et al. "Capillary Electrophoresis/mass Spectrometry for the Separation and Characterization of Bovine Cu,Zn-superoxide Dismutase." Rapid Communications in Mass Spectrometry : RCM, vol. 24, no. 10, 2010, pp. 1411-8.
Borges-Alvarez M, Benavente F, Barbosa J, et al. Capillary electrophoresis/mass spectrometry for the separation and characterization of bovine Cu,Zn-superoxide dismutase. Rapid Commun Mass Spectrom. 2010;24(10):1411-8.
Borges-Alvarez, M., Benavente, F., Barbosa, J., & Sanz-Nebot, V. (2010). Capillary electrophoresis/mass spectrometry for the separation and characterization of bovine Cu,Zn-superoxide dismutase. Rapid Communications in Mass Spectrometry : RCM, 24(10), 1411-8. https://doi.org/10.1002/rcm.4530
Borges-Alvarez M, et al. Capillary Electrophoresis/mass Spectrometry for the Separation and Characterization of Bovine Cu,Zn-superoxide Dismutase. Rapid Commun Mass Spectrom. 2010 May 30;24(10):1411-8. PubMed PMID: 20411580.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Capillary electrophoresis/mass spectrometry for the separation and characterization of bovine Cu,Zn-superoxide dismutase. AU - Borges-Alvarez,Marta, AU - Benavente,Fernando, AU - Barbosa,José, AU - Sanz-Nebot,Victoria, PY - 2010/4/23/entrez PY - 2010/4/23/pubmed PY - 2010/8/3/medline SP - 1411 EP - 8 JF - Rapid communications in mass spectrometry : RCM JO - Rapid Commun Mass Spectrom VL - 24 IS - 10 N2 - 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. SN - 1097-0231 UR - https://www.unboundmedicine.com/medline/citation/20411580/Capillary_electrophoresis/mass_spectrometry_for_the_separation_and_characterization_of_bovine_CuZn_superoxide_dismutase_ L2 - https://doi.org/10.1002/rcm.4530 DB - PRIME DP - Unbound Medicine ER -