Proteome discover v2

The TMT–liquid chromatography (LC)–mass spectrometry (MS)/MS experiment was conducted and analysed by Oebiotech (Shanghai, China). In brief, total protein in spinal cord tissue (three samples each for SCI‐WT and SCI‐TLR4‐KO groups, collected at 7 days post‐SCI) was extracted, followed by digestion and labelling using a Thermo Fisher Scientific TMT labelling kit (Waltham, MA, USA). Then, the above TMT‐labelled sample was fractionated by basic pH reverse‐phase LC using an Agilent 1100 high‐pressure LC system with fraction combing (Santa Clara, CA, USA). The samples were then loaded onto a trap column (350 nl/min) and analytical column (RP‐C18; New Objective, Littleton, MA, USA) before being analysed using a mass spectrometer (Q‐Exactive HF). Finally, the data were analysed with Proteome Discover v2.4 (Thermo Fisher Scientific).

To identify the unique a.a. sequence of circRNAs encoded peptides, the total protein was subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) sequencing and data analysis by Oebiotech Co., Ltd. (Shanghai, China). In brief, enzymolysis was performed using 0.02 μg/μl trypsin, and then the peptides were desalinated. The sample was analyzed using a Nano-HPLC liquid phase system EASY-NLC1200 and a Q-Exactive mass spectrometer. The acquired data were analyzed using ProteomeDiscover (V2.4, Thermo, United States) software.

Desalted peptides cleaved by LysC were analyzed by LC-MS/MS. Then peptides were separated by reverse-phase chromatography (2–90% ACN/0.1% formic acid gradient over 63 min at a rate of 300 nL min⁻¹) on a 75 μm ×  150 mm ReproSIL-Pur-120-C18-AQ column (Dr. Albin Maisch, Germany) using the nano-EasyLC 1200 system (Thermo). Eluting peptides were sprayed into an Orbitrap-Lumos_ETD mass spectrometer through a 1-μm emitter tip (New Objective) at 2.4 kV. Scans were acquired within 350–1600 Da m/z targeting the truncated reporter with 15 s dynamic exclusion. Precursor ions were individually isolated and were fragmented (MS/MS) using an HCD activation collision energy of 30. Precursor (fragment) ions were analyzed at a resolution of 200 Da of 120,000 with the following parameters: max injection time (IT), 100 ms (resolution of 30,000) in three cycles. The MS/MS spectra were processed with Proteome Discover v2.4 (Thermo Fisher) and were analyzed with Mascot v.2.8.0 (Matrix Science) using RefSeq2021_204_Bacillus.S and a database with peptides from the NanoLucBleR reporter protein. Peptide identifications from Mascot searches were processed within the Proteome Discoverer-Percolator to identify peptides with a confidence threshold of a 5% false discovery rate, as determined by an auto-concatenated decoy database search.