Structural Analysis of SOD1 Fibrils with Mass Spectrometry, Limited Proteolysis, and Atomic Force Microscopy (AFM)

Abstract

This protocol describes a method to purify SOD1 in Saccharomyces cerevisiae to characterize using ICP-MS and AFM, to agitate and fibrillate for aggregation of SOD1. The human SOD1 (hSOD1) is a 32-kDa homodimer, with one copper- and one zinc-binding site per 153-amino acid subunit. Misfolded protein aggregates are often correlated with diseases known as amyloidosis, including ALS, Alzheimer’s, Parkinson’s, and prion disease (Valentine and Hart, Proc Natl Acad Sci USA 100: 3617–3622, 2003; Tanzi and Bertram, Cell 120: 545–555, 2005; Soto and Pritzkow, Nat Neurosci 21:1332–1340, 2018; Sarafian et al., J Neurosci Res 95:1871–1887, 2017). Proteinaceous aggregates containing hSOD1 have frequently been found in the spinal cords of ALS patients.