RNase/SDS-PAGE mobility assay-based validation of protein-specific UV-crosslinking efficiency estimated by LC–MS/MS. (A) Estimation of protein-specific UV-crosslinking efficiencies by comparing serial dilutions of RNase-treated input and clRNP fractions with SRA and immunoblot. Protein-specific UV-crosslinking efficiencies estimated by LC–MS/MS represent the mean ± 1 SD of n = 3 biologically independent replicates. Full blots for PDIA3, RanBP2, and HNRNPD are provided in Supplemental Figure S1. Full blots for other targets assayed herein were reported previously (Supplemental Fig. S1A; Kristofich and Nicchitta 2023). (B) Scatterplot showing a correlation between LC–MS/MS and SRA %CL rank of proteins assayed herein (A), or previously (Kristofich and Nicchitta 2023), color overlay based on GO-annotation (GO: RBP) status. (C) Scatterplot showing a correlation between protein-specific UV-crosslinking efficiencies estimated by LC–MS/MS and crosslink rates estimated by easyCLIP (Porter et al. 2021); color overlay based on GO-annotation (GO: RBP) status. For additional information on samples, data analysis, and graphical representations, see Source Data and Methods.
