Q exactive hf system

The protein bands were extracted from the gel for protein identification using a Q Exactive HF system coupled to an Easy-nLC 1000 (Thermo Fisher Scientific, Waltham, MA, USA) via a nanoelectrospray ion source. To identify the protein bands, the MS/MS data were searched against an expected protein sequence using the in-house PEAKS Studio software (version 10.5, Bioinformatics Solutions Inc, Waterloo, ON, Canada) [28 (link)].

SDS-PAGE–resolved, 4–12%-gradient gels were silver-stained with SilverQuest (Life Technologies), and appropriate bands were cut and subjected to in-gel digestion, followed by liquid chromatography–MS with a Q Exactive HF system (Thermo Fisher Scientific). Resulting peptides were identified by protein-sequence database searches using MaxQuant software.

To determine differences in protein expression between the model group and medium-concentration or high-concentration PRP groups (which had better recovery), three sciatic nerves were harvested from the model group for proteomics analysis. Two sciatic nerves were collected from the medium-concentration PRP group and one sciatic nerve was collected from the high-concentration PRP groups, which had satisfactory recovery. The tissues were removed rapidly and stored in liquid nitrogen. The method used for this analysis was the label-free relative quantification method, and the method of sample preparation is shown in Additional file 1. The samples (1–2 μg) were dissolved in the loading buffer (1% acetonitrile and 0.1% formic acid) for analysis. An Easy-nLC 1000 system (Thermo Fisher Scientific) was used with an analytical column (PepMap 100 C18, 2 μm, 75 μm × 250 mm with NanoViper fittings; Thermo Fisher Scientific, Stockbridge, GA, USA) and a flow rate of 0.2 mL/minute. The mass spectrometer was a Q-Exactive HF system (Thermo Fisher Scientific). Tandem mass spectrometry was performed in the data dependent acquisition mode. The full scan resolution was 60,000 (full width at half maximum), the scanning range of parent ions was 400–1600 m/z, and the collision energy was 32% higher energy collision-induced dissociation.