Ubiquitinated-SOD1 (SOD1-Ub) ion extract of LC-ESI-MS chromatogram for DRP from ALS variant and hWT SOD1 mouse spinal cord. G93A, G37R, H46R/H48Q, and hWT SOD1 ion extracts for +20 molecular ion are shown for SOD1-Ub₁ and SOD1-Ub₂; +30 ion shown for SOD1-Ub₃. The ion extract for the +14 molecular ion of unmodified SOD1 is shown for each variant, with strong signals appearing between 39 and 41 min, where SOD1 elutes (note: signal marked with asterisk in each plot corresponds to injection peak and is due to salt clusters across a broad m/z range).

LC-ESI mass spectra of ALS variant SOD1 from DRPs prepared from symptomatic ALS mouse spinal cord. DRPs that were resistant to 10% Nonidet P-40 are denoted “P2”; pellets resistant to 10% Nonidet P-40 and an additional exposure to 0.5% Nonidet P-40, 0.25% SDS, and 0.5% deoxycholate are denoted “P3.” A, total ion current chromatograms of P2 DRPs (solubilized for LC-MS analysis with methanol/chloroform) from non-transgenic (nTg) and hWT, H46R/H48Q, G37R, and G93A mice are overlaid. B, expanded and overlaid view of SOD1 elution peak at 39–41 min shows that hWT SOD1 is not present in DRPs from transgenic mice expressing hWT SOD1; dashed and solid lines represent hWT and H46R/H48Q samples, respectively. C, raw mass spectra of ALS variant SOD1 recovered from P2 DRP. D and E, reconstructed mass spectra of ALS variant SOD1 deconvoluted from raw mass spectra from P2 (D) and P3 (E) DRPs, respectively. See Table 1 for mass values of SOD1.

LC-ESI-MS of soluble hWT and ALS variant SOD1 from ALS-mouse spinal cord. A, total ion current LC chromatogram from LC-ESI-MS analysis of supernatant solution (referred to as “S1”). ALS variant and hWT SOD1 elute at ∼39–41 min (peak corresponding to hSOD1 marked with red arrow). B, raw mass spectra of SOD1 species eluting between 38 and 41 min from hWT, G37R, H46R/H48Q, and G93A mice. C, reconstructed mass spectra that were deconvoluted from the corresponding raw spectra in B (see Table 1 for mass values). D–F, oxidative modifications to SOD1 do not significantly alter retention time on HPLC column. D, LC-ESI-MS total ion current chromatogram of recombinantly purified H48Q SOD1 that has been partially oxidized in vitro with H₂O₂. Peak at 39–41 min corresponds to H48Q SOD1 (peak at ∼9 min is eluted salt from sample injection). E and F, raw mass spectrum (E) and deconvoluted mass spectrum (F) of H48Q SOD1 species eluting in selected region of chromatogram in D. Two SOD1 species are present and have co-eluted, demonstrating that oxidatively modified SOD1 in mouse spinal cord would have eluted with unmodified SOD1 at ∼39–41 min. Species with mass of 15839.47 Da corresponds to unmodified H48Q SOD1; species with 15869.36 Da corresponds to oxidatively modified H48Q SOD1.

MALDI-TOF and ESI-MS/MS mass spectra of soluble and aggregated G37R SOD1 purified from symptomatic mouse spinal cord with size exclusion and anti-SOD1 immunoaffinity liquid chromatography (SE/IA-LC). A, SDS-PAGE and Western blot analysis of eluted SE fractions reveal SOD1 species with different molecular mass ranges. SOD1 in fractions 22–24 (dashed box on left) eluted in void volume indicating a molecular mass > 70 kDa; SOD1 species that eluted in fractions 31–37 (dashed box on right) have a molecular mass between 7 and 70 kDa (i.e. the resolving range of the G-75 media). Fraction numbers are listed at the bottom of the Western blot (see “Experimental Procedures” for chromatographic parameters), and “M” denotes the hWT SOD1 marker. Arrows on Western blot indicate SOD1 aggregate species that are heat- and SDS-stable. Right spectra: MALDI-TOF mass spectrum of SOD1 species that eluted in resolving region of G-75 column (i.e. soluble SOD1) and were pooled and further purified with anti-SOD1 immunoaffinity chromatography. The theoretical average mass of G37R SOD1 is 15945.8 Da. The 16952 m/z signal corresponds to myoglobin (co-spotted with protein samples as an internal calibrant). The 16153 m/z signal arises from a gas-phase SOD1-matrix adduct (matrix: sinapic acid; molecular mass 224.22 Da). Left spectra: MALDI-TOF spectrum of SOD1 species that eluted in the void volume of a G-75 column (i.e. aggregated SOD1) and were pooled and further purified with anti-SOD1 immunoaffinity chromatography. Insets for both spectra show expanded spectrum from 15,000 to 18,000 m/z. B, ESI-MS/MS spectra of tryptic fragment of G37R SOD1 purified from symptomatic mouse spinal cord with SE/IA-LC. The spectrum shown here was generated from the void volume (>70 kDa) fractions of the G-75 column. A ladder scheme numerates the C and N terminus daughter ions (e.g. y and b daughter ions, respectively) for the tryptic SOD1 peptide (residues 80–91). Daughter ions assigned “b_o”or“y_o” refer to ions that have undergone the loss of H₂O.