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

Quantitative determination of S:N-based RBP metrics. (A) Schematic illustration of the LEAP-RBP method, sample fractions, and fraction compositions. LEAP-RBP (RNP) fractions containing total RNA and total RNA-bound protein, or input (total protein) samples can be isolated from acidic guanidinium thiocyanate-phenol (AGP) input suspensions containing starting samples (e.g., cells or cell lysates) mixed with AGP. LEAP-RBP (clRNP) fractions isolated from final AGPC interphase samples contain total RNA-bound protein and/or protein-bound RNA. Efficient (near 100%) recovery of total protein, total RNA, and/or total RNA-bound protein allows accurate estimation of RNA and protein UV-crosslinking efficiencies. (B) Schematic illustration of S:N-based determination of protein-specific UV-crosslinking efficiencies by SILAC LC–MS/MS. From left to right: bar chart showing %TP contribution of hypothetical “RBP A” in clRNP fractions and corresponding S/N ratio of 4; line graph showing the estimated % of RBP A (yellow marker) that is RNA-bound based on its displayed S/N ratio of 4 (80% RNA-bound); stacked bar chart showing %TP_S (RNA-bound) and %TP_N (free) contributions of RBP A in clRNP fractions and stacked bar chart showing %TP_S contributions of RBP A in input (total protein) samples based on protein UV-crosslinking efficiency of 10%. UV-crosslinking efficiency of RBP A = (%TP_S)_input/(%TP_O)_input × 100; and (%TP_S)_input = (%TP_S)_clRNP × protein yield of clRNP fraction/protein yield of the input sample.