Rp c18

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).

The volatile dry peptide samples were redissolved in Nano‐HPLC Buffer A. Separation was performed using the Nano‐HPLC Liquid Phase System UltiMate 3000 RSLCnano (Thermo Fisher Scientific). Liquid phase A was a 0.1% formic acid–water solution, and liquid phase B was a 0.1% formic acid–acetonitrile solution. The chromatographic trap column (100 µm × 20 mm; RP‐C18, Agilent Technologies) was equilibrated with 100% A solution at 3 µL min⁻¹. Samples were loaded using an autosampler, combined on the trap column, and then separated by an analysis column that was 75 µm × 150 mm (RP‐C18, New Objective, Littleton, MA, USA), at a flow rate of 300 nL min⁻¹. Samples were washed once with a 30 min mobile phase gradient, with a blank solvent between samples. The enzymatic products were separated using capillary HPLC and analyzed using mass spectrometry with a Q‐Exactive Plus mass spectrometer (Thermo Fisher Scientific).

The dried polypeptide extracts were re-dissolved in Nano-HPLC Buffer A and separated by the Nano-HPLC liquid system on an UltiMate 3000 RSLCnano (Thermo Fisher Scientific, MA, USA). Solution A was 0.1% formic acid-water, and solution B was 0.1% formic acid-acetonitrile. The trap column was balanced with 100% solution A at 3 μL/min (RP-C18, Agilent). Then the samples were loaded by an automatic sampler, combined with the trap column, and separated on an analysis column at a flow rate of 300 nL/min on a 75 μm × 150 mm column (RP-C18, New Objective, USA). Peptides were separated by capillary high-performance liquid chromatography, and MS was performed with a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). The detection methods were as follows: after calibration with the standard calibration solution, the mother solution was scanned using data dependent acquisition (DDA) mode (350–2000 m/z). Then the 20 strongest fragment profiles (MS2 scan) were collected after high energy collision dissociation (NCE energy 28, dynamic exclusion time 25 s). We set the resolution of MS1 to 70 000 at M/Z 200, the AGC target to 3e6, and the maximum injection time to 100 ms. We also set the resolution of MS2 to 17 500, the AGC target to 1e5, and the maximum injection time to 50 ms.