Forty-two male Wistar rats (200–250 g) were purchased from Charles River China (Beijing, China). All animals were maintained on a standard laboratory diet with a controlled indoor temperature (21±2℃), humidity (65–70%) and 12/12 h light–dark cycle conditions. The animal experiments were reviewed and approved by the Qingdao University Hospital Medical Ethics Committee (No.027-2019). All methods were carried out in accordance with relevant guidelines and regulations of the National Health Commission and the Ministry of Science and Technology and conformed to the guidelines for animal research.
The Wistar rats were randomly divided into two groups: a control group (n = 21) and an I/R group (n = 21). Ischemia was induced by Pulsinelli’s four-vessel occlusion method . Briefly, the bilateral common carotid arteries were surgically exposed and clamped shut with microclips for 10 min. The experiment was conducted in two phases; for details, see Figure 1. For the discovery phase, differentially abundant urinary proteins were identified by label-free DIA quantification in twenty-one independent samples from the control group (7 samples) and the I/R group at 12 and 48 h (7 samples per time point). For the validation phase, the 21 remaining urine samples (7 from the control group and 7 per time point from the I/R group at 12 and 48 h) were evaluated by targeted quantification with PRM.
2. Histological Analysis
For histopathology, three rats in the I/R group and three rats in the control group were randomly sacrificed at 12 h and 48 h after I/R. The hippocampus was harvested and then quickly fixed in 10% neutral buffered formalin. The formalin-fixed tissues were embedded in paraffin, sectioned (4 mm) and stained with hematoxylin and eosin (H&E) to reveal histopathological lesions.
3. Urine Collection and Sample Preparation
Urine samples were collected from the control and I/R groups at 12 and 48 h after I/R. Rats were individually placed in metabolic cages for six hours. During urine collection, food was withheld from the rats to prevent the urine from being contaminated. After collection, the urine samples were immediately centrifuged at 2 000g for 30 min at 4°C and then stored at −80°C.
Urinary protein extraction: Urine samples were centrifuged at 12 000g for 30 min at 4°C. Six volumes of prechilled acetone were added after the pellets were removed, and the samples were precipitated at 4°C overnight. Then, lysis buffer (8 mol/L urea, 2 mol/L thiourea, 50 mmol/L Tris, and 25 mmol/L DTT) was used to dissolve the pellets. The protein concentration of each sample was measured by a Bradford protein assay.
Tryptic digestion: The proteins were digested with trypsin (Promega, USA) using filter-aided sample preparation methods . Briefly, 100 µg of the protein sample was loaded onto a 10-kDa filter unit (Pall, USA). The protein solution was reduced with 4.5 mM DTT for 1 h at 37°C and then alkylated with 10 mM indoleacetic acid for 30 min at room temperature in the dark. The proteins were digested with trypsin (enzyme-to-protein ratio of 1:50) for 14 h at 37°C. The peptides were desalted on Oasis HLB cartridges (Waters, USA) and lyophilized for trap column fractionation and LC-MS/MS analysis.
4. Spin Column Separation
To generate a spectral library for DIA analysis, pooled peptide samples from all samples were fractionated using a high-pH reversed-phase peptide fractionation kit (Thermo Pierce, USA) according to the manufacturer’s instructions. Briefly, 60 µg of a pooled peptide sample was loaded onto the spin column. A step gradient of increasing acetonitrile concentrations was applied to the column to elute the bound peptides. Ten different fractions were collected by centrifugation, including the flow-through fraction, the wash fraction and eight step gradient sample fractions (5, 7.5, 10, 12.5, 15, 17.5, 20 and 50% acetonitrile). The fractionated samples were dried completely and resuspended in 20 μL of 0.1% formic acid. Three microliters of each of the fractions was loaded for LC–data-dependent acquisition (DDA)–MS/MS analysis.
5. LC-MS/MS Setup for DDA and DIA
An Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific, Germany) was coupled with an EASY-nLC 1000 HPLC system (Thermo Scientific, Germany). For DDA-MS and data-independent acquisition (DIA)–MS modes, the same LC settings were used for retention time stability. The digested peptides were dissolved in 0.1% formic acid and loaded onto a trap column (75 µm × 2 cm, 3 µm, C18, 100 A°). The eluent was transferred to a reversed-phase analytical column (50 µm × 250 mm, 2 µm, C18, 100 A°). The eluted gradient was 5–30% buffer B (0.1% formic acid in 80% acetonitrile; flow rate of 0.8 μL/min) for 90 min. To enable fully automated and sensitive signal processing, the calibration kit (iRT kit from Biognosys, Switzerland) reagent was spiked at a concentration of 1:20 v/v in all samples. The iRT kit reagent was spiked into the urinary peptides for spectral library generation. Additionally, before the real DIA runs, the iRT kit reagent was also spiked into all urinary samples.
For the generation of the spectral library, the ten fractions from the spin column were analyzed in DDA-MS mode. The parameters were set as follows: the full scan was acquired from 350 to 1 550 m/z at 60 000, the cycle time was set to 3 s (top speed mode), the automatic gain control (AGC) was set to 1E6, and the maximum injection time was set to 50 ms. MS/MS scans were acquired in the Orbitrap at a resolution of 15,000 with an isolation window of 2 Da and collision energy of 32% (higher-energy collisional dissociation, HCD); the AGC target was set to 5E4, and the maximum injection time was 30 ms.
For the DIA-MS method, forty individual samples were analyzed in DIA mode. For MS acquisition, the variable isolation window DIA method with 26 windows was developed (Table S1). The full scan was set at a resolution of 60,000 over an m/z range of 350 to 1,200, followed by DIA scans with a resolution of 30,000, HCD collision energy of 32%, AGC target of 1E6 and maximal injection time of 50 ms.
6. LC-MS/MS Setup for PRM
In the discovery phase, seventy-one differentially abundant urinary proteins were identified by the label-free DIA proteomic method. All of these proteins were evaluated by the PRM-MS method in the remaining twenty-one urine samples. LC-PRM-MS/MS data were acquired in an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific, Germany) coupled with an EASY-nLC 1200 HPLC system (Thermo Scientific, Germany).
For the generation of the PRM spectral library, pooled peptide samples were analyzed in DDA-MS mode 6 times. The peptides were loaded on a reversed-phase trap column (75 µm × 2 cm, 3 µm, C18, 100 Å, Thermo Scientific, Germany), and the eluent was then transferred to a reversed-phase analytical column (50 µm × 250 mm, 2 µm, C18, 100 Å, Thermo Scientific, Germany). The elution gradient consisted of 5–35% buffer B (0.1% formic acid in 80% acetonitrile; flow rate 0.8 μL/min) for 90 min. The MS parameters were set as follows: the full scan was acquired from 350 to 1 550 m/z at 60 000, the cycle time was set to 3 s (top speed mode), the AGC was set to 1E6, and the maximum injection time was set to 50 ms. MS/MS scans were acquired using the Orbitrap at a resolution of 30 000 with an isolation window of 1.6 Da and collision energy at 30% (HCD), the AGC target was set to 5E4, and the maximum injection time was 60 ms.
For the PRM-MS method, thirty-two individual samples were analyzed in PRM mode. Ultimately, 255 peptides were scheduled, and the retention time (RT) segment was set to 8 min for each targeted peptide (Table S2). The normalized collision energy was fixed ats 30%, and the quadrupole isolation window was fixed at 1.6 Da. The other parameters were the same as described in the last paragraph.
7. Label-Free DIA Quantification Analysis
To generate the spectral library, the raw data files acquired for the ten fractions in DDA mode were processed using Proteome Discoverer (version 2.3; Thermo Scientific, Germany) with SEQUEST HT against the SwissProt Rattus database (released in May 2019, containing 8086 sequences) appended with the iRT peptide sequences. The search parameters consisted of a parent ion mass tolerance of 10 ppm; fragment ion mass tolerance of 0.02 Da; fixed modification of carbamidomethylated cysteine (+58.00 Da); and variable modifications of oxidized methionine (+15.995 Da) and deamidated glutamine and asparagine (+0.984 Da). For other settings, the default parameters were used. A false discovery rate (FDR) cutoff of 0.01 was applied at the protein level. The results were then imported to Spectronaut™ Pulsar (Biognosys, Switzerland) software to generate the spectral library .
The raw DIA-MS files were imported into Spectronaut Pulsar with the default settings. In brief, a dynamic window for the XIC extraction window and a nonlinear iRT calibration strategy were used. Mass calibration was set to local mass calibration. Cross-run normalization was enabled to correct for systematic variance in LC-MS performance, and a local normalization strategy was used . Protein inference, which gave rise to the protein groups, was performed on the principle of parsimony using the ID picker algorithm as implemented in Spectronaut Pulsar . All results were filtered by a Q value cutoff of 0.01 (corresponding to an FDR of 1%). Peptide intensity was calculated by summing the peak areas of the respective fragment ions for MS2. Student’s t-test was applied with a significance criterion of <0.05. A minimum of two peptides matched to a protein and a fold change >1.5 were used as the criteria for the identification of differentially expressed proteins.
8. PRM-MS Quantification Analysis
Skyline (version 3.6.1 10279)  was used to build the spectrum library and filter peptides for PRM analysis. For each targeted protein, 2-6 associated peptides were selected using the following rules: (i) identification in the untargeted analysis with a q value <1%, (ii) complete digestion by trypsin, (iii) containing 8–18 amino acid residues, (iv) exclusion of the first 25 amino acids at the N-terminus of proteins, and (v) fixed carbamidomethylation of cysteine. Prior to individual sample analysis, pooled peptide samples were subjected to PRM experiments to refine the target list. Finally, forty-four proteins with 255 peptides (Table S2) were scheduled. The RT segment was set to 8 min for each targeted peptide with its expected RT in the center based on the pooled sample analysis.
All of the PRM-MS data were processed with Skyline. By comparing the same peptide across runs, the RT location and integration boundaries were adjusted manually to exclude interfering regions. Each protein’s intensity was quantitated using the summation of intensities from its corresponding transitions. The transition settings were as follows: precursor charges +2, +3; ion charge +1; ion type b, y, p; product ions from ion 3 to last ion -1; automatically select all matching transitions; ion match tolerance 0.02 m/z; select the 6 most intense product ions. The details of the transition are listed in supporting Table S2. Prior to the statistical analysis, the quantified protein intensities were normalized according to the summed intensity. The differentially abundant proteins were selected using one-way ANOVA, and p-values were adjusted by the Benjamini & Hochberg method. Significance was defined by a p-value of < 0.05 and a fold change of 1.5.
9. Bioinformatics Analysis
Bioinformatics analysis was carried out to better study the biological function of the dysregulated proteins. The Database for Annotation, Visualization and Integrated Discovery (DAVID) 6.8 (https://david.ncifcrf.gov/) was used to perform the functional annotation of the differentially abundant urinary proteins identified at 12 and 48 h. In this study, significant GO enrichment was defined as p<0.05. Protein-protein interaction networks were constructed using the STRING database (http://www.string-db.org), which is a database of known and predicted protein interactions, including direct (physical) and indirect (functional) associations.