Label-free LC-MS/MS analysis of proteins associated with expanded G4C2 hexanucleotide repeat-containing transcripts in human induced pluripotent stem cells

Proteins co-localizing with RNA targets of interest in human induced pluripotent stem cells (iPSCs) were identified using our recently optimized hybridization-proximity labeling approach (HyPro2) combined with label-free mass spectrometry. Briefly, fixed and permeabilized iPSCs from an ALS patient (DN19V4) or a healthy donor (C0053) were hybridized with a digoxigenin-labeled antisense oligonucleotide probe targeting transcripts containing the G4C2 hexanucleotide repeat expansion, which is present in the C9orf72 gene in DN19V4 but not in C0053. Alternatively, a control probe set was used to target ACTB pre-mRNA, expressed in both DN19V4 and C0053. The purified HyPro2 enzyme, which contains a digoxigenin-binding domain and a modified ascorbate peroxidase domain, was then recruited to the RNA targets, enabling in vitro biotinylation of target-proximal proteins. Following streptavidin pull-down, biotinylated proteins were fragmented using a Trypsin/Lys-C protease mix and identified by label-free LC-MS/MS analysis. All experiments were performed in triplicate