Chemicals and reagents
Cellytic MT cell lysis reagent, ammonium hydroxide solution, formic acid (FA), triethylammonium bicarbonate (TEABC), and tween-20 were purchased from Sigma-Aldrich (Saint Louis, USA). Trifluoroacetic acid (TFA) were purchased from Wako (Osaka, Japan). Clarity™ Western ECL Substrate and nitrocellulose membranes were purchased from Bio-Rad (California, USA). Acetonitrile (ACN) were purchased from Spectrum (California, USA). Ethylenediaminetetraacetic acid (EDTA) and protease inhibitors were purchased from G-Biosciences (St. Louis, MO, USA). Trypsin was purchased from Promega (Madison, USA). Bicinchoninic acid (BCA) protein assay kit, and Tandem Mass Tag assay (TMT) were purchased from Thermo Fisher Scientific (Rockford, USA). 10% neutral buffered formalin were purchased from CHIN IPAO CO., LTD (Taipei, Taiwan), paraffin was from Leica Microsystems Pty Ltd (Macquarie Park, Australia), and hematoxylin and eosin (H&E) was from Roche Diagnostics (Indianapolis, USA)
Rat model with whole-body exposure to traffic related air pollution (TRAP)
The rat model was previously published in (16) to mimic the TRAP exposure in human. A whole-body exposure system was developed, and the ambient air was continuously sampled by an omnidirectional PM inlet located on the roof of the animal housing followed by exposure into the animal cages. All procedures were performed compliance with the animal and ethics review committee of the Laboratory Animal Center at Taipei Medical University (Taipei, Taiwan).
Each rat was randomly assigned into three groups for exposure within two different periods: (1) three and six months of exposures to whole air from TRAP (3M-PM1 and 6M-PM1 groups, respectively); (2) three and six months of exposures to high-efficiency particulate air (HEPA) filtered TRAP (traffic-related gaseous pollutants, shorted as 3M-GAS and 6M-GAS groups, respectively); and (3) three and six months of exposure to conditioned clean air (3M-CTL and 6M-CTL group, respectively). The PM1 and GAS groups were placed in an urban region nearby a major highway and expressway in New Taipei City, Taiwan. The CTL group was housed in a specific pathogen free I level of Laboratory Animal Center (Taipei, Taiwan).
As indicated in (16), the characteristics of ambient air exposure were continuously monitored. The daily distribution of the geometric mean diameter was 55.8±7.3 (40.3-74.5) nm, and particles were categorized as ultrafine-sized fractions (<100 nm; PM1) with mass concentration of 16.3±8.2 (4.7-68.8) µg/m3. The lung deposition surface area in the alveolar region was 55.1±21.7 (20.7-136.6) mm2/cm3. Black carbon and particle number concentrations were 1800±784 (219-4732) ng/m3 and 11257±4388 (2218-25733) #/m3, respectively.
Lung function examination
After the designated time period of exposure, each rat from every group was examined for the lung function by using a Forced Pulmonary Maneuver System (Buxco Research Systems, Wilmington, NC, USA) following the manufacturer’s protocols. Briefly, an endotracheal tube was attached to an airway port, and the distal end of the catheter was passed through a small opening located near the airway port. The catheter was then connected to the system. Forced expiratory flow at 25-75% of the pulmonary volume (FEF25-75) and forced expiratory volume at 20 ms (FEV20) were measured. For each test, at least three acceptable measurements were conducted to obtain a reliable mean for all numeric parameters. All procedures were performed under sufficient anesthesia.
Lung tissue were collected and washed with ice-cold PBS, followed by fixation with 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Histological examinations were conducted under light microscopy by a histopathologist in a blinded manner. The degree of lung injury was scored according to the four criteria: (1) alveolar congestion, (2) hemorrhage, (3) immune cell infiltration, and (4) thickness of the alveolar wall (56). Congestion and thickness of the alveolar wall were graded by a five-points scale as follows: 0 for minimal (little) damage, 1 for mild damage, 2 for moderate damage, 3 for severe damage, and 4 for maximal damage. Hemorrhage was graded as follows: 0 for no red blood cells (RBC) outside of blood vessels, 1 for few interstitial RBC, 2 for few RBC in some alveoli, 3 for moderate number of RBC in some alveoli, 4 for many RBC in most alveoli, 5 for large numbers of RBC in all alveoli. Infiltration of macrophage was graded as follows: 0 for none-rare, 1 for 1-10% of alveoli/saccules contain macrophages, 2 for 10-25%, 3 for 25-75%, 4 for >75% (57).
Tissue lysate collection
Lung tissue from each rat was grounded in liquid nitrogen, collected into an microcentrifuge tube, and lysed with Cellytic MT cell lysis reagent, protease inhibitors, and EDTA in the volume ratio of 98:1:1. The tissue lysate was homogenized by using Minilys® personal homogenizer (Bertin, Rockville, MD, USA) in high speed mode for 15 s twice and the homogenate was centrifuged at 13,000 rpm at 4°C for 10 min to collect the clear supernatant as lung tissue lysate. The lung tissue lysate from each rat was assayed by using BCA protein assay kit to determine the protein concentration.
Gel-assisted digestion, tandem mass tag (TMT) labeling and high pH reversed phage (RP) StageTip fractionation
Fifty micrograms of lung tissue proteins were aliquoted from each of the 5 rats from the 6 exposure groups for our reported gel-assisted digestion with trypsin individually (58). The resulting peptides were vacuum-dried and resuspended in 100 mM TEABC for BCA protein assay. Ten micrograms of peptides were aliquoted from each of the 5 rats in one exposure group to generate a pooled peptide sample. The pooled peptides from 3M-CTL, 3M-PM1 and 3M-GAS groups were labeled with TMT126, TMT127 and TMT128 respectively, whereas the 6M-CTL, 6M-PM1 and 6M-GAS were labeled with TMT129, TMT130 and TMT131 respectively. The TMT-labeled peptides from each group were pooled for RP StageTip fractionation following the protocol in (59). Peptides were eluted sequentially by using 11.1%, 14.5%, 17.4%, 19%, 23%, and 27-80% ACN in ammonium hydroxide solution (pH 11.5). Peptides from each fraction was vacuum-dried and resuspended in 0.1% FA for LC-MS/MS analysis.
NanoLC-nanoESI-MS/MS analysis was performed on a Thermo UltiMate 3000 RSLCnano system connected to a Orbitrap Fusion™ Tribrid™ Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a nanospray interface (New Objective, Woburn, MA). Peptide mixtures were loaded onto a 75-μm ID, 25-cm PepMap C18 column (Thermo Fisher Scientific) packed with 2 μm particles with a pore width of 100 Å. The peptides were eluted by a 103-min gradient using 5% to 45% mobile phase B (99.9% ACN, 0.1% FA in HPLC-grade water) at a flow rate of 0.4 μL/min. The gradients were slightly adjusted for each RP fraction.
The LC-MS/MS experiments were performed in a data-dependent acquisition mode to sequentially select the top 15 intense precursor ions for higher-energy collision dissociation with the normalized collision energy of 40%. Full MS scans were acquired in orbitrap from m/z 300-1,600 with resolution of 120,000 and automated gain control of 400,000 charges or maximum ion time of 50 msec. For MS/MS scans, fragment ions were acquired in Orbitrap with the resolution of 60,000 and automated gain control of 1E5 or max ion time of 100 msec. Precursors with assigned charge states from 2+ to 7+ were included. Previously targeted precursors were dynamically excluded from re-acquisition for 15 s.
Proteome identification and quantification
The LC-MS raw data were searched against SwissProt Rattus norvegicus database (version 2018_11, 8,054 entries) using Mascot implemented in Proteome Discoverer (version 22.214.171.1248, Thermo Fisher). The MS and MS/MS tolerances were set to 20 ppm and 0.1 Da, respectively. Tryptic peptides with a maximum of two missed cleavages were allowed. Methylthio (Cys) was set as fixed modification, whereas oxidation (Met), acetylation (protein N-terminal), deamidation (Asn and Gln), and TMT tags (N-terminal, Lys) were set as variable modifications. 1% false discovery rate (FDR) was applied in peptide spectral matches, peptide and protein levels for confident identification. Identified peptides in high confidence with at least 6 amino acids were accepted. For proteome quantification, only unique peptides were included to estimate the protein abundance which was further normalized by the total peptide abundance. Proteins with 1.3-fold changes in abundance (log2 ratio >0.38 or <-0.38) were considered as differentially expressed proteins (DEPs).
The DEPs from each exposure group were submitted to Ingenuity Pathway Analysis (IPA) (60) for pathway enrichment analysis and Cytoscape (version 3.7.1) with ClueGo (version 2.5.4) plugin (61, 62) for Gene Ontology (GO) analysis (rattus norvegicus database, v.20.05.2019). GO fusion and GO group were selected while other settings remained default. Only the GO terms from biological process, molecular function, and cellular component with lowest term p-value (corrected with Bonferroni step down) were included for analysis. For pathway enrichment analysis using IPA, z-scores were obtained for the enriched canonical pathway as well as the disease and biofunction annotations. Annotations with p<0.05 were considered significant. A z-score of >0 indicates activation, while <0 indicates inhibition of a cellular function or pathway.
Western blot analysis
Forty μg of protein was run in gel electrophoresis by using 4–20% Mini-PROTEAN® TGX™ Gel (Bio-Rad, CA, USA) followed by protein transfer onto nitrocellulose membranes. After blocking for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against Chp1, Serpina3 (1:1000, ABclonal technology, MA, USA), C3, or Vcl (1:1000, both from Santa Cruz, TX, USA), separately. The membranes were thoroughly washed with PBST and then incubated with anti-rabbit or anti-mouse IgG secondary antibody (1:10000, Bioss Antibodies, MA, USA) for 1 h at room temperature. Clarity™ Western ECL Substrate was used to detect the protein bands. Vinculin (Vcl) was used as the loading control and one rat sample from 6M-CTL group was included in each gel as an inter-sample variation control. Quantification of the detected protein bands were quantified by AzureSpot (Azure Biosystems, CA, USA).
Enzyme-linked immunosorbent assay (ELISA)
ELISA approach was adapted to measure 8-isoprostane (Cayman, USA) and IL-6 (R&D System, Minneapolis, MN, USA) in lung tissue lysate, each following the manufacturer’s instructions. Data are presented after normalization to the total protein amount.
The lung function and lung injury results were reported as the median with interquartile range. Mann-Whitney U test was used to evaluate the significance of difference. Only differences with p<0.05 were considered as significant. Statistical analyses were performed by using GraphPad (version 8).