2.1 Cells culture and chemical treatment
The human BEAS-2B cell lines were purchased from Food Industry Research and Development Institute（FIRDI, Taiwan）, which were derived from human bronchial epithelial cell. The BEAS-2B cells were grown in 100 mm dishes using LHC-9 medium (Biofluid, Rockville, MD), at 37℃ in a humidified mixture of air: carbon dioxide (95:5) atmosphere. At ~70% of confluency, the cells were treated with 10M BaP, NNK, and ttDDE for 48 hours, respectively. The control groups were treated with DMSO for 48 h. All treatment experiments repeated six times.
2.2 Cell harvesting
After chemical treatment, cells were harvested using NP-40 lysis buffer. The adherent cells were washed twice with cold 1 × PBS, and then softly scraped off with 1 mL of NP-40 lysis buffer (50mM Tris [pH 8.0], 1% NP-40, 150mM sodium chloride and protease inhibitor cocktail) by rubber policeman. Cells were lysed in lysis buffer for another 30 min with gentle mixing at 4℃, followed by centrifugation at 13000 rpm for 20 min. And the supernatant was collected and stored at -20℃ until used. The protein concentration was determined using a Micro BCA protein reagent.
2.3 Sample preparation- Precipitation with TCA in acetone
BEAS-2B cell extract was solubilized in 11% TCA in acetone containing 20mM DTT, and kept for 15min on ice for complete protein precipitation.
Following centrifugation (8500 rpm, 15 min), the supernatant was discarded and washed pellet twice with cold acetone containing 20mM DTT*. After washing, pellet was dried at room temperature to remove residual acetone. The pellet was
solubilized in 500L rehydration buffer (6M Urea, 2M Thiourea, 2% CHAPS, 0.5% IPG buffer, bromophenol blue, 20mM DTT*)for 90 min, and the n centrifuged (13000rpm, 2h, 4℃). The supernatant was stored in aliquots at -20
℃ until analyzed.
2.4 Two- dimensional gel electrophoresis
IEF was performed at 20℃ on the IPGphorTM Isoelectric Focusing system (Amersham Pharmacia Biotech, Uppsala; Sweden) according to the method described by manufacturer’s direction. The Immobiline Dry strips (18cm, pH 4-7; Amersham Pharmacia) were rehydrated in 350μL of the protein extract and focused according to the following protocol: (1) 30 V for 16 h (rehydration step), (2) 500 V for 1 h, (3) 1000 V for 1 h, and (4) 8000 V until 32000VHr were reached. Prior to the second- dimensional (SDS-PAGE on vertical), the IPG strips were equilibrated for 2× 15 min with gentle shaking in 10mL equilibrium
buffer (50 mM Tr is-HC l, p H 8 . 8 ; 6 M ure a , 3 0 % g l yc e ro l, 2 % S D S , bromophenol blue). DTT (1% w/v) was added to the first, and iodoacetamide (2.5% w/v) to the second equilibration step. The strips were then applied onto 12% SDS-PAGE gels and overlaid with 1% agarose. The second dimension electrophoresis were run on BioRad gel apparatus (Bio-Rad Laboratories, Sundbyberg, Sweden), running buffer was 25mM Tris (pH 8.8), 192 mM glycine, 0.1% SDS, and running condition was 15℃, 100 V constant voltage until the bromophenol blue dye front reached the bottom of the gel. Molecular weight markers were Broad Range, unstained markers (Bio-Rad, Hercules, CA, USA).
2.5 Protein visualization and image analysis
Proteins were visualized with silver staining. Gels were scanned (Umax, USA) and digitized at 300dpi using ImageMaster Labscan Version 3.0 software
(Amersham Pharamacia Biotech, USA). These spots were quantified and
compared by the ImageMaster 2D Elite software (Amersham Pharamacia Biotech, USA).
For spot detection, ImageMaster uses a specific detection algorithm where every pixel in the image is considered by constructing a matrix centered on each pixel. The algorithm compares the values on the outside of the matrix to those inside. If the average pixel value of the matrix is bigger than outside the matrix, the pixel at the center of the matrix is considered as part of spot. In the process of spot detection, four parameters need to be set, noise factor, operating size, sensitivity and background factor. For spot matching, we synthesized the master gel from 12 gels as reference gel. Spot intensity represented as normalized volume【(spot volume/ total spot volume)× 100】.
2.6 Data analysis
For ease of data processing, the intensities of undetected spots were assigned to silver-staining detection limit, which is the lowest value among the
12 gels. Because the between-run variations of protein expression were greater than the with-in run variations, we compared the ratio of protein expression in pairs (total: 6 pairs, 12 gels). Each protein spot had a set of six ratios, which were generated from six pairs (intensity of the chemical treated group/the
intensity of the control group). Following data transformation (ratio as logarithm r a t i o ) , a n e w t-distribution dataset symmetrical to zero was generated. Then we constructed the 95% confidence interval (95% C.I) of each dataset and compared to the genomic mean (GM) of the ratio value. When the GM is within the interval range, then the protein is not considered regulated with confidence. When the GM is above the interval range, the protein is considered down- regulated. When the GM is below the interval range, the protein is considered up- regulated.
2.7 In-gel digestion and protein identification by mass spectrometry
The protein spots of interest were excised, cut into small pieces, transferred into 1.5 mL polyethylene sample vial, and washed twice with deionizied water for 15 min. The gel pieces were destained by adding 1:1 solution of 30mM potassium ferricyanide and 100mM sodium thiosulfate, washed twice with deionizied water, dried under vacuum, rehydrated with 10 mM DTT/100 mM NH4HCO3 and incubated at 56℃ for 45 min. Once reduction was complete, the supernatant was discarded and 55 mM iodoacetamide/100 mM NH4HCO3 were
immediately added and incubated for 30 min at room temperature. After alkylation the supernatant was discarded and the gel pieces were washed twice
with 40% acetonitrile/0.1 M NH4HCO3 and twice with deionizied water. The gel pieces were again dried under vacuum and rehydrated in 20 μL of trypsin solution (Boehringer Mannheim, sequencing grade). Protein digestion was performed overnight at 37℃. The supernatant was removed and the gel pieces were extracted twice with 10 μL 50% acetonitrile. The extracts were combined, dried under vacuum centrifuge and stored at -20 °C until use.
The protein identity was determined using a API QSTAR Pulsar i instrument (MDS Sciex, Ontario, Canada) equipped with a nanoESI source. The peptide tandem-MS data were searched using PepSea software, against a h u ma n database (NCBI).