Materials

Protein kinase inhibitor was purchased from Calbiochem; trypsin was purchased from Promega; acetonitrile and ultrapure water (H2O) were purchased from Fisher Chemical; trifluoroacetic acid, iodoacetamide, dithiothreitol, urea, and triethylammonium bicarbonate (TEAB) were purchased from Sigma; formic acid purchased from Fluka. Zhishi Rhubarb Soup (ZRS), a Chinese herbal regimen, was purchased from the Department of Pharmacy, Nanjing Chinese Medicine Hospital Affiliated to Nanjing Universityof Traditional Chinese Medicine, decocted according to the conventional method, concentrated to 2.5 g/ml and stored at 4 °C for later use.

Ethics statement

All animal procedures and protocols were performed in accordance with The Guide for the Care and Use of Laboratory Animals (NIH publication, 85–23, revised 1996) and were reviewed and approved by the Animal Research Committee at the National Research Institute of Chinese Medicine: IACUC protocol no. P-99-11; IACUC Approval No. A-99-1. All surgeries were performed under anaesthesia, and all efforts were made to minimize suffering.

Animal model, grouping and administration

The MCAO (middle cerebral artery occlusion) model was established using the thread bolt method [24]. The method refers to the modified Longa suture method. Briefly, the experimental animals were anaesthetized with the administration of 10% chloral hydrate (0.3 ml/100 g) into the abdominal cavity, and they were fixed on the operating table in the supine position (the rectal temperature was controlled at 37.3 ± 0.5 °C). The common carotid artery and vagus nerve were quickly exposed and separated, and the proximal end of the common carotid artery and the external carotid artery were connected. The internal carotid artery was threaded for use, and a small opening was cut at the upper end of the common carotid artery ligation, extending from the bifurcation of the common carotid artery. Premeasured fishing line was then inserted into the internal carotid artery along the common carotid artery, and the internal carotid artery was ligated when the line reached the specified length (approximately 18 mm). Tethers of different diameters were chosen according to the animal’s weight and nutrient intake, and then, the incision was sutured. After the operation, the body temperature was maintained at 37 ± 0.5 °C with an irradiation lamp, and the rectal temperature, respiration and heart rate were monitored. Before further experimentation, the animals were maintained in a cage until they awakened.

The rats were randomized into the following three groups: the con group, con-operated rats were i.g. with an equal volume of sterile saline (Group A); the vehicle group, MCAO rats i.g. with an equal volume of sterile saline after surgery and once daily (Group B); the ZRS treatment group, for MCAO rats, the effective dose was determined to be 10 g crude drug/kg body weight, and gavage was started 3 h after model establishment and once per day for 7 days (Group C). Hippocampal tissue was collected after treatment on Day 7.

Nerve function score

After a rat was awake for 2 h, the behaviour and neurological symptoms were observed, and a score was given according to the Longa 5-level standard scoring method: 0, normal, without any neurological deficits; 1, the front paw cannot be straightened when lifted vertically; 2, leaning to the right and rotating to the right when walking; 3, the body falls to the right side while walking; and 4, not walking spontaneously or showing signs of a consciousness disorder. According to the first score, animals with no neurological deficit, 4 points, dyspnoea, early death, or subarachnoid haemorrhage found at the time of execution were discarded. Animals excluded from the group were replaced in subsequent experiments.

Protein extraction and trypsin treatment

An appropriate amount of tissue (rat hippocampus) was weighed into a mortar precooled with liquid nitrogen, and more liquid nitrogen were added to fully grind the tissue to powder. Then, samples of each group were added to 4-fold the volume of powdered lysis buffer (8 M urea, 1% protease inhibitor, 3 μM TSA, 50 mM NAM and 2 mM EDTA) and lysed by ultrasound. The cell debris was removed after centrifugation, the supernatant was transferred to a new centrifuge tube, and the protein concentration was determined using a BCA kit.

Dithiothreitol was added to the protein supernatant to a final concentration of 5 mM and was reduced to 56 °C for 30 min. Then, iodoacetamide was added to a final concentration of 11 mM, and the supernatant was incubated for 15 min at room temperature in the dark. The urea concentration of the sample was diluted to be less than 2 M. Pancreatin was added at a mass ratio of 1:50 (pancreatin:protein), and the protein was digested overnight at 37 °C. Finally, the protein was subjected to a second enzymatic hydrolysis for 4 h after pancreatin was added at a mass ratio of 1:100 (pancreatin:protein).

TMT labelling

The peptides digested by trypsin were desalted with Strata X C18 (Phenomenex), freeze-dried in vacuo, and then dissolved in 0.5 M TEAB and labelled according to the TMT kit operating instructions. Briefly, the labelling reagent was thawed, dissolved in acetonitrile, mixed with the peptide and incubated at room temperature for 2 h. The labelled peptide was mixed, the salt was removed, and the sample was freeze-dried under vacuum.

HPLC fractionation

The peptides were fractionated by high-pH reverse HPLC, and the column was an Agilent 300Extend C18 (5 μm particle size, 4.6 mm inner diameter, 250 mm length). The peptides were subjected to a step gradient of 8–32% acetonitrile, pH 9, and 60 components were separated in 60 min. Then, the peptides were combined into 9 component samples, and the combined components were vacuum freeze-dried for subsequent operations.

LC–MS analysis

The peptides were dissolved in mobile phase A for liquid chromatography (0.1% (v/v) formic acid aqueous solution) and separated using an EASY-nLC 1000 ultra-high-performance liquid-system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid gradient settings were as follows: 0–30 min, 12% ~ 26% B; 30–52 min, 26% ~ 40% B; 52–56 min, 40% ~ 80% B; 56–60 min, 80% B. The flow rate was maintained at 320 nL/min.

The peptides were separated by an ultrahigh-performance liquid system, injected into an NSI ion source for ionization and then analysed by Orbitrap Fusion Lumos mass spectrometry. The ion source voltage was set to 2.0 kV, and the peptide precursor ions and their secondary fragments were detected and analysed by high-resolution Orbitrap. The scanning range of the primary mass spectrum was set to 350–1550 m/z, and the scanning resolution was set to 60,000; the scanning range of the secondary mass spectrum was set to a fixed starting point of 100 m/z, and the secondary scanning resolution was set to 15,000. The data acquisition mode was based on the data-dependent scanning (DDA) program; that is, the first 20 peptide precursor ions with the highest signal intensity were selected to enter the HCD collision cell, and 32% fragmentation energy was used for fragmentation after the first scan. The mass spectrometry analysis was then graded. To improve the effective utilization of the mass spectrometer, the automatic gain control (AGC) was set to 5E4, the signal threshold was set to 50,000 ions/s, the maximum injection time was set to 70 ms, and the dynamic rejection time of the tandem mass spectrometry scan was set to 30 s to avoid precursor ions.

Database search

The resulting MS/MS data were processed using the MaxQuant search engine (v.1.5.2.8). Tandem mass spectra were searched against the human UniProt database concatenated with the reverse decoy database. Trypsin/P was specified as a cleavage enzyme allowing up to 4 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance for fragment ions was set as 0.02 Da. Cysteine alkylation was set as a fixed modification, and the variable modification was the oxidation of methionine, acetylation of the N-terminus of the protein, and deamidation (NQ). The quantitative method was set to TMT-6plex, and the FDR for protein identification and PSM identification was set to 1%. Carbamidomethyl on Cys was specified as a fixed modification, and acetylation modification and oxidation on Met were specified as variable modifications. The FDR was adjusted to < 1%, and the minimum score for modified peptides was set > 40.

Western blot analysis

Twenty micrograms of protein/well was loaded onto 10% gels for separation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). The gels were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (0.45 or 0.20 μm pore size; Millipore, Billerica, MA, USA). The blotted membranes were blocked with 5% nonfat dry milk in a Tris-buffered saline solution (25 mM Tris, pH 7.5, and 150 mM NaCl) containing 0.05% Tween 20 (TBST) for 2 h at room temperature, followed by incubation with the diluted primary antibody against the target protein for 4 h at room temperature. After washing for 10 min in TBST solution, the membranes were incubated with properly diluted secondary antibody conjugated with horseradish peroxidase for 2 h at room temperature. Western blots were developed using ECL chemiluminescent reagents obtained from Thermo Scientific (Waltham, MA, USA). The β-actin level was used as the loading control.

Statistical analysis

For protein difference analysis, the ratio of the average value of all biological replicated quantitative values of each protein in the control group is regarded as a fold change (FC). A fold change in differential expression exceeding 1.2 was used as the change threshold for significant upregulation, and the FC threshold for significant downregulation was less than 1/1.2. For biological or technical replicate samples, we used principal component analysis (PCA), relative standard deviation (RSD) and Pearson’s correlation coefficient to evaluate protein quantitative repeatability.

Other data set were illustrated using the mean ± SEM and carefully checked by SPSS 20.0 statistical analysis software (SPSS, Chicago, Illinois, USA). The MWM measurement datasets were investigated by two-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.