Ltq orbitrap velos pro mass spectrometry

Total protein of each AbOMV was used to estimate OMV yield and the concentration of total protein was measured by the Lowry method. Protein composition was determined by 12% SDS-PAGE stained with Coomassie blue and Linear Trap Quadropole (LTQ) Orbitrap Velos Pro Mass Spectrometry (Thermo Fisher Scientific) (31 (link)). Residual lipopolysaccharide (LPS) concentration was determined with the KDO assay (32 (link)). DNA quantification assays were performed using Quant-iT™ PicoGreen® dsDNA reagent according to the manufacturer's recommendation.

Biotinylated RNA bound to streptavidin beads pulled down associated proteins^{87 (link)}. Biotin-labeled RNAs were in vitro transcribed using AmpliScribe T7-Flash Biotin-RNA transcription Kits (Lucigen, WI, USA). Nuclear lysates were prepared with Magna Nuclear RIP™ (Native) Nuclear RNA-Binding Protein Immunoprecipitation Kits (Sigma–Aldrich). RNA-protein complexes were recovered using Dynabeads MyOne Streptavidin T1 (Thermo Fisher Scientific). Pull-down protein samples were run on SDS-PAGE gels, followed by detection with Silver Stain KANTOIII (KANTO CHEMICAL, Japan) or Western blotting. The band around 140 kDa detected by the silver staining was excised and subjected to peptide digestion. Mass spectrometry (LC–MS/MS) analyzes of the digested peptides utilized LTQ Orbitrap Velos pro mass spectrometry (Thermo Scientific). Protein with an approximate molecular mass of 140 kDa detected in sense TUG1 pull-down product but not in antisense TUG1 pull-down product was identified as TUG1-interacting protein.