Patients and sample collection
From the perspective of the clinical outcomes, this study examined three pairs of BALF samples from patients diagnosed with ARDS type I in the acute and recovery phases (Table 1). The BALF samples of acute inflammation-phase and the hyperplasia recovery-phase ARDS were obtained from clinically indicated bronchoscopies with excess material made available for the study. BAL (100 ml of normal saline) was performed using a standard protocol in either the right middle lobe or left upper lobe (lingual). For this study, the patients were grouped based on the timing of the bronchoscopy: conducted in either the acute inflammation phase of ARDS (days 1-3) or the recovery phase (days 4-8), referenced to the initiation of mechanical ventilation (designated day 1). The APACHE-II scores were calculated to assess the severity of illness on the day of bronchoscopy for patients with ARDS, as previously described[21].
Protein extraction and quality control
Figure 1 shows the framework of this study. The high abundance proteins were removed using the Proteominer kit (Bio-Rad, Hercules, CA, USA). The proteins were reduced and alkylated with 10 mM dithiothreitol (DTT) and 55 mM iodoacetamide (IAM) followed by precipitation with five volumes of cold acetone at -20°C for >2 h. After sonication, the samples were centrifuged at 25,000 ×g for 15 min at 4℃ to remove debris. The protein concentration was determined using the Bradford assay. Proteins (10 μg) were mixed with loading buffer, heated at 95°C for 5 min, centrifuged at 25,000 ×g for 5 min, and were separated by 12% SDS polyacrylamide gel. After electrophoresis, the gel was stained and de-stained by a protein staining instrument for 10 min. The images were scanned. Dyeing and stripping instrument: L00657C, Genscript, China; Gluesweeper: 2100XL, UMAX, China; Electrophoresis: P0WERPAC BASIC, BIO-RAD, USA
Protein digestion, iTRAQ labeling, and peptide fractionation
Total protein (100 μg) was added to a 1.5-mL centrifuge tube. Trypsin enzyme (Hualishi Scientific, Shanghai, China) was added 1:20, vortexed, centrifuged at a low speed 12,000 ×g for 1 min, and incubated at 37°C for 4 h. The peptide solution was frozen-dried after salt removal. The tryptic peptides for each pooled sample were labeled using one of the 8-plex iTRAQ reagents (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s protocol. In the 8-plex labeling, reporters 113, 115, and 117 were used for duplicates of the pooled acute ARDS samples, and reporters 114, 116, and 118 were used for duplicates of the pooled recovery ARDS samples. The Shimadzu LC-20AB liquid phase system (Prominence, Shimadzu, Japan) was used, and the separation column was a 5 µm 4.6×250 mm Gemini C18 column (150 µm inner diameter, 1.8 µm column particle size, 35 cm column length) for liquid phase separation of the sample. The elution peak was monitored at a wavelength of 214 nm, and one component was collected per minute. The samples were combined according to the chromatographic elution peak map to obtain 10 fractions, which were then frozen-dried.
HPLC, LC-MS/MS identification, and quantification of the labeled peptides
The dried peptide samples were reconstituted with mobile phase A (2% ACN, 0.1% FA), centrifuged at 20,000 ×g for 10 min, and the supernatant was taken for injection. Separation was performed using an Easy-nLC 1200 (Thermo Fisher Scientific, San Jose, CA, USA) and tandem self-packed C18 column (75 μm internal diameter, 1.9 μm column size, 25 cm column length) and separated at a flow rate of 200 nL/min by the effective gradient. The nanoliter liquid phase separation end was directly connected to a mass spectrometer. The peptides separated by liquid-phase chromatography were ionized by a nanoESI source and then passed to a tandem mass spectrometer Oritrap Exploris 480 (Thermo Fisher Scientific, San Jose, CA, USA) for data-dependent acquisition (DDA) mode detection.
Identification of proteins by quantitative proteomics analysis
The resulting data were searched in the UniProt database (https://www.uniprot.org/). The data were analyzed using the IQuant (BGI) software[22].
Gene ontology enrichment and pathway enrichment analyses
Gene ontology (GO) (http://www.geneontology.org) is a commonly used bioinformatic tool that provides comprehensive information on the gene function of individual genomic products based on defined features. The Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg) is an easy-to-use automated database resource for understanding high-level biological functions and utilities. GO and KEGG pathway analyses of co-upregulated and co-downregulated proteins were performed via The Database for Annotation, Visualization, and Integrated Discovery. The GO analysis consisted of biological processes (BP), cellular components (CC), and molecular functions (MF).
PPI network creation and hub gene identification
The PPI network of differentially expressed proteins (DEPs) was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING; https://string-db.org/)[23], with a combined score >0.4 as the cut-off point. Hub genes were identified using Cytohubba, a plug-in of the Cytoscape software (Cytoscape, 3.9.0)[24]. Significant modules in the PPI network were identified by molecular complex detection (MCODE)[25], another plug-in of Cytoscape software.
Cell culture and stimulation
A549 cells were purchased from CELLCOOK (http://www.cellcook.com/). A549 cells are human alveolar basal epithelial cells that grow adherently as a monolayer in vitro. The A549 cell line is widely used as a model of lung adenocarcinoma, as well as an in vitro model for type II pulmonary epithelial cells. The cells were cultured in 1640 medium with 10% fetal bovine serum (FBS). LPS (10 µg/ml, Sigma, St, Louis, MO, USA) was added to the culture medium to stimulate the cells for 48 h.
RNA extraction, reverse transcription, and quantitative real-time PCR
RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA synthesis was performed with 1 µg of total RNA and the 5× HiScript Ⅱ qRT SuperMix (Vazyme Biotech, Co., Ltd., Nanjing, China), according to the manufacturer’s instructions. Endogenous mRNA levels were measured by real-time PCR analysis based on AceQ qPCR SYBR Green Master Mix (Vazyme Biotech, Co., Ltd., Nanjing, China) detection with a Roche real-time PCR machine. In order to confirm the expression of RNAs, qPCR was performed under the following sequential conditions: 1) 95°C for 5 min, 2) 40 cycles at 95°C for 10 s and 60°C for 34 s. The primers are shown in Table 2. Samples were assayed in duplicate. The 2-ΔΔCT methodology was to obtain the ratios after normalizing to endogenous β-actin.
Western blotting and antibodies
Protein extracts were analyzed by western blotting according to standard protocols. Cells were harvested and boiled in a 4× loading buffer. Equal amounts of proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane, blocked with 8% skimmed milk. The membranes were incubated with primary antibodies against RPSA (1:1000 dilution, eBioscience, San Diego, CA, USA), and anti-GAPDH (#5174S, 1:1000 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA) followed by a secondary antibody (#PMS302, 1:1000 dilution, Proteinbiotechnology, Shanghai, China) bearing an oligonucleotide. After two washes, each membrane was read using a chemiluminescence developing solution (#180-506, Tanon™ Femto-sig ECL).
Enzyme-linked immunosorbent assay (ELISA) detection process
RPSA was detected in A549 cell supernatant using ELISA, according to the manufacturer’s instructions. 1) Adding standards and samples in duplicate to the microtiter plate. 2) Adding 50μl of standard or sample to the appropriate wells. 3) Adding 100μl of enzyme conjugate to standard wells and sample wells except the blank well, covered with an adhesive strip and incubate for 60 minutes at 37°C. 4) Washing the microtiter plate 4 times, remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well completely with wash solution (1X), then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure for a total of four times. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. 5) Aspirating all wells, then wash plates four times using wash buffer (1X). Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350 ul/well/wash. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. 6) Adding substrate A 50μl and substrate B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. 7) Adding 50μl stop solution to each well. The color in the wells should change from blue to yellow. 8) Reading the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
Statistical analysis
The identification and verification results were based upon statistical evaluation and were expressed as means ± SDs. Single sample t-tests were used to compare the two groups. P-values <0.05 were considered statistically significant. *P<0.05, **P<0.01, ***P<0.001.