Coupling SEC and IEF to SXRF for metallation analysis of SOD1 enzyme isolated from the human central nervous system.

Copper-zinc superoxide dismutase enzyme (SOD1) is one of the 16,227 expressed proteins in the central nervous system (CNS). Abnormal metallation and aggregation of SOD1 are suspected to play a role in amyotrophic lateral sclerosis (ALS) and Parkinson's disease, but data describing SOD1 metal occupancy in human tissues have not previously been reported. The analysis of metals in metalloproteins under native conditions remains a difficult exercise, especially when the protein is present at physiological levels in a complex tissue. In this work, we developed a methodological workflow that allows synchrotron X-ray fluorescence (SXRF) analysis of metalloenzymes isolated from small amounts of tissue. This protocol allowed us to characterize the metallation of endogenous SOD1 in the human CNS from only 20 mg of post-mortem tissue. In a first 2D-chromatography step, a soluble protein extract is prepared from post-mortem CNS tissues and then separated according to molecular weight using Size Exclusion Chromatography (SEC), and then according to isoelectric point by native isoelectric focusing (IEF) in gel. The processed samples were analyzed by SXRF in the microprobe hutch of the Hard X-ray Micro/Nano-Probe beamline P06 at PETRA III (DESY) in Hamburg (Germany). In total, we analyzed post-mortem CNS tissue from 30 individuals (17 without neurological disease and 13 ALS cases). The Cu/Zn atomic ratio in active SOD1 from control cases was 1.12 ± 0.28 (mean±sd) and did not differ between CNS regions or individuals. To our knowledge, this is the first report of a ratio value close to the theoretical value of 1 in active SOD1 isolated from human tissue. In addition to its excellent detection limit (∼0.1 µg/g), SXRF also offers excellent repeatability (Cu/Zn ratio in SOD1 standard=0.93 +/-0.01, n=9). Furthermore, our protocol preserves both the metallation and the activity of SOD1, and reduces proteome complexity by 97.8%. 2D chromatography enriches SOD1 99-fold compared to IEF alone, and also improves the accuracy and precision of the measurements (mean Cu/Zn=1.12 vs 2.39, sd = 0.28 vs 1.48). Using this SXRF-based method we revealed that SOD1 metallation is altered in ALS. This method is applicable to other metalloproteins when only small amounts of tissue are available and excellent sensitivity and reproducibility are required. [ABSTRACT FROM AUTHOR]