High-throughput quantitation of protein–RNA UV-crosslinking efficiencies as a predictive tool for high-confidence identification of RNA-binding proteins

Protein-specific UV-crosslinking efficiency values predict protein abundance and UV-enrichment efficiency (S/N) in LEAP-RBP fractions. (A–D) Analytical summary: protein-specific UV-crosslinking efficiencies were estimated using protein abundances estimated by LC–MS/MS analysis of input (total protein) and LEAP-RBP (clRNP) fractions isolated from HeLa cells and protein S/N ratios estimated by SILAC LC–MS/MS analysis of LEAP-RBP (clRNP) fractions isolated from pooled samples containing UV-crosslinked and non-crosslinked HeLa cells (Kristofich and Nicchitta 2023). For additional information on samples, data analysis, and graphical representations, see Source Data and Methods. (A) Scatterplot showing observed abundances (log₁₀[%TP]) of Gene-Ontology (GO)-annotated RBPs and other proteins lacking GO-annotated RNA-binding in LEAP-RBP (clRNP) fractions by LC–MS/MS as a function of their estimated UV-crosslinking efficiencies (log₁₀[%CL]). (B) Scatterplot showing estimated UV-crosslinking efficiencies (log₁₀[%CL]) of GO-annotated RBPs and other proteins as a function of their displayed log₁₀(S/N) ratios in clRNP fractions by SILAC LC–MS/MS. (C) Scatterplot showing observed abundances (log₁₀[%TP]) of GO-annotated RBPs and other proteins in input (total protein) samples by LC–MS/MS as a function of their estimated UV-crosslinking efficiencies (log₁₀[%CL]). (D) Scatterplot showing a correlation between ordinal RCS rank and ordinal %CL rank of GO-annotated RBPs and other proteins.