Cell type specific analysis of ALS associated proteins reveals immune-regulated targets

Many proteins associated with amyotrophic lateral sclerosis (ALS) remain poorly characterized, in part because validated reagents for protein-level studies are scarce. We previously established knockout (KO)-based antibody characterization workflows and showed that widely used antibodies against the ALS-associated protein C9orf72 lacked specificity (Laflamme et al., 2019), and subsequently scaled this framework to systematically benchmark research antibodies, revealing that up to 61% do not perform as recommended by manufacturers (Ayoubi et al., 2023). Here, we extend this approach by establishing the ALS-Reproducible Antibody Platform (ALS-RAP) to evaluate antibodies against proteins encoded by ALS risk genes. We characterized 303 antibodies targeting 33 ALS-associated proteins using KO-based antibody characterization workflows to identify high-quality reagents for common experimental applications. Using validated antibodies, we profiled protein levels across human induced pluripotent stem cell (iPSC)-derived and primary neurological cell types, revealing diverse cellular distributions and higher protein levels for several ALS-associated proteins in glial and immune populations. Together, ALS-RAP provides a validated antibody toolbox and protein expression resource for studying ALS-associated proteins, supporting the view that ALS genetics converges on multicellular disease mechanisms involving both neuronal and glial populations.