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

Abstract

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.

Identification of Unfolding and Dissociation Pathways of Superoxide Dismutase in the Gas Phase by Ion-Mobility Separation and Tandem Mass Spectrometry

“…Consistent with previous works, − the fully metalated dimeric and monomeric species of SOD1 could be observed by ESI-MS using 10 mM ammonium acetate buffer (pH 6.8). The mass spectrum was dominated by dimer ions (charge states 10+ to 13+); monomer ions (carrying 6+ to 8+ charges) were produced in low abundance.…”

Elemental and molecular analysis of metal containing biomolecules using laser induced breakdown spectroscopy and sonic spray ionization mass spectrometry: A step towards full integration and simultaneous analysis

“…Metalloprotein standards: Superoxide dismutase (SOD) was purchased from Aldrich (S7571-75KU). The protein consists of two identical subunits and is known to bind two copper(II) ions per molecule one on each subunit [30]. SOD standards were prepared by dilution of an appropriate amount of the protein in water or 1:1 water methanol mixture.…”

“…The concentration of Cu 2+ ions in the analyzed SOD solution was measured by LIBS and was found to be 4.91x10 -5 M, which was also confirmed by ICP-MS analysis. On the basis of the molecular mass measured and the mass to volume concentration of the SOD solution, the molar concentration of the SOD (dimer) solution is calculated to be 5.3x10 -5 M. Taking into account that each SOD subunit can bind one Cu 2+ ion [30], the bound Cu concentration of the protein solution, provided that each SOD dimer contains 2 Cu atoms, would be expected to be 10.6x10 -5 M. This turns out to be approximately a factor of 2 higher compared to what was actually measured by LIBS and ICP-MS. This discrepancy may be due to a number of factors such as protein purity or loss of metal during protein isolation and purification.…”