biostudies-literatureTan TCJWellcome Principal Research FellowshipWork in the Wellcome Centre for Cell BiologyWellcome Truste21https://www.ebi.ac.uk/biostudies/studies/S-EPMC10899761biostudies-literatureUnknown52(4)The RNA-interacting proteome is commonly characterized by UV-crosslinking followed by RNA purification, with protein recovery quantified using SILAC labeling followed by data-dependent acquisition (DDA) of proteomic data. However, the low efficiency of UV-crosslinking, combined with limited sensitivity of the DDA approach often restricts detection to relatively abundant proteins, necessitating multiple mass spec injections of fractionated peptides for each biological sample. Here we report an application of data-independent acquisition (DIA) with SILAC in a total RNA-associated protein purification (TRAPP) UV-crosslinking experiment. This gave 15% greater protein detection and lower inter-replicate variation relative to the same biological materials analyzed using DDA, while allowing singlNucleic acids researchImproved detection and consistency of RNA-interacting proteomes using DIA SILAC.PMC10899761222516203149Spanos CTan TCJTollervey DfalseImproved detection and consistency of RNA-interacting proteomes using DIA SILAC.The RNA-interacting proteome is commonly characterized by UV-crosslinking followed by RNA purification, with protein recovery quantified using SILAC labeling followed by data-dependent acquisition (DDA) of proteomic data. However, the low efficiency of UV-crosslinking, combined with limited sensitivity of the DDA approach often restricts detection to relatively abundant proteins, necessitating multiple mass spec injections of fractionated peptides for each biological sample. Here we report an application of data-independent acquisition (DIA) with SILAC in a total RNA-associated protein purification (TRAPP) UV-crosslinking experiment. This gave 15% greater protein detection and lower inter-replicate variation relative to the same biological materials analyzed using DDA, while allowing singl2024-01-01T00:00:00Z2024 Feb2025-04-18T14:45:58.261Z2025-04-07T01:03:10.608ZS-EPMC108997613819723710.1093/nar/gkad1249