Orbitrap velos pro lc ms system

The biotin-labeled lncRNA with wild type full length, mutant full length as well as the antisense was used as previous report by using Thermo Scientific Pierce RNA 3′ Desthiobiotinylation Kit (Thermo Scientific Pierce, CA, USA). The RNA pull-down was conducted by using The Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific Pierce, CA, USA) according to the manufacturer’s instructions. In brief, labeled RNA was applied to bind to streptavidin magnetic beads and Incubate for 15–30 min at room temperature with agitation. The protein binding to RNA was conducted by using RNA–protein binding reaction mix buffer, then the RNA and protein complex was washed. The complex was resolved by SDS-PAGE, and specific bands were excised and analyzed by mass spectrometry. Proteins in bands were eluted and digested. Digests were analyzed by Orbitrap Velos Pro LC/MS system (Thermo Scientific, CA, USA). Data was analyzed by Proteome Discoverer and the resulting peak lists were used for searching the NCBI protein database with the Mascot search engine.

The biotin-labeled lncRNA was transcribed with a Biotin RNA Labeling Mix (Roche, CA, USA) and the T7 RNA polymerase (Roche, CA, USA), treated with RNase-free DNase I (Roche, CA, USA) and purified with an RNeasy Mini Kit (Qiagen, Hilden, Germany). Protein extracted from MGC803 was mixed with biotinylated RNA. 60 μL washed streptavidin agarose beads (Invitrogen Life Technologies, CA, USA) was then added to each binding reaction and washed. The associated proteins were resolved by SDS-PAGE, and specific bands were excised and analyzed by mass spectrometry. Proteins in bands were eluted and digested. Digests were analyzed by Orbitrap Velos Pro LC/MS system (Thermo Scientific, CA, USA). Data was analyzed by Proteome Discoverer and the resulting peak lists were used for searching the NCBI protein database with the Mascot search engine.
RIP assay was performed by using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) according to the manufacturer’s instructions. The EGFR antibody (ab2430) was used for RIP (Abcam, Cambridge, UK). The co-precipitated RNAs were detected by reverse transcription PCR and quantitative PCR. Total RNAs (input controls) and IgG were assayed simultaneously to demonstrate that the detected signals were the result of RNAs specifically binding to EGFR.

The related proteins were resolved by electrophoresis, and a specific band was excised. Digested proteins were subjected to the Orbitrap Velos Pro LC/MS system (Thermo Fisher Scientific, USA). The fragment spectra were used for checking the NCBI database with the Mascot search engine.

The biotin-labeled circRNA and the antisense were transcribed with a Biotin RNA Labeling Mix (Roche, CA, USA) and the T7 RNA polymerase (Roche, CA, USA), treated with RNase-free DNase I (Roche, CA, USA) and purified with a RNeasy Mini Kit (Qiagen, Hilden, Germany). Cells were incubated with biotinylated RNAs and 60 μL of streptavidin agarose beads (Invitrogen Life Technologies, CA, USA). The associated proteins were resolved by SDS-PAGE, and specific bands were excised. Proteins were eluted, digested and subjected to the Orbitrap Velos Pro LC/MS system (Thermo Scientific, CA, USA). Data were analyzed by Proteome Discoverer and the resulting peak lists were used for searching the NCBI protein database with the Mascot search engine.