Label-free LC-MS/MS analysis of proteins associated with specific RNAs in HeLa cells

Proteins co-localizing with RNA targets of interest in HeLa cells were identified using our recently optimized hybridization-proximity labeling approach (HyPro2) combined with label-free mass spectrometry. Briefly, fixed and permeabilized cells were hybridized with digoxigenin-labeled antisense oligonucleotide probes targeting the long noncoding RNA PNCTR or pre-mRNAs encoding ACTB and FUS proteins. A no-probe negative control was also included. 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.