Environmental contaminants can affect many intracellular enzyme activities. The effects of enzyme inhibitors on the proteome can be determined using bidirectional electrophoresis. In neuroendocrine cells, precursor protein convertases 1 and 2 (proprotein convertase 1 and 2, P C l and PC2 ) mediate the hydrolysis of many protein precursors to peptide hormones and neuropeptides. The activity of these calcium-dependent proteases can be regulated by chelating agents and heavy metal ions in the environment. This inhibition can lead to potential pathological disruption of the peptidergic system. We wanted to know to what extent the specific inhibition of these enzymes can affect the proteome of neuroendocrine cells. To address this question, we used murine pituitary AtT2 0 cells as a model. We compared the proteomes of control cells and cells overexpressing the P C l-specific inhibitor proSAAS. The comparison process included bidirectional electrophoresis, mass spectrometry identification of differential proteins, and immunoblotting verification. Bidirectional electrophoresis analysis revealed many alterations in the proteome of proSAAS overexpressing cells. Mass spectrometry analysis of the digested peptides revealed that two proteins, proSAAS and EphrinA-like receptor 2, were increased in abundance in this cell type.
By Martin, this experiment is from "Environmental Genomics Lab Guide".
Operation method
Proteomic analysis of AtT20 pituitary cells as a model for neuroendocrine peptidergic system interference Move I. Materials 1 Cell culture and lysis (1) Mouse pituitary AtT20 and AtT20 (proSAAS) cell lines. The AtT20 . (proSAAS) cell line was obtained by transfecting AtT2 0 cells with a proSAAS expression vector containing a neomycin resistance gene and then selecting positive cells using this gene for resistance to the cytotoxic drug G4 1 8 (11). (2) DMEM medium (Gibco/BLR, Grandlsland, N Y) containing 1 0 % fetal bovine serum (Wisent Inc., Quebec, Canada) and 30 pg/m L bisacodyl sulfate with or without 500 pg/mL of G418 (Gibco/BLR). (3) Phosphate buffer solution (PBS): 1. 35 mol/L NaCU 28 mmol/L KC1, 80 mmol/L Na2 HPO4-7 H20, 15 mmol/L KH2PO4, pH 7.2. (4) Versine: PBS containing 0.I mmol/L EDTA. (5) Buffer B: 10 mmol/L hydroxyethylpiperazine ethylsulfonic acid (HEPES)-KOH, pH 7.4, 10 mmol/L KC1, I.5 mmol/L MgCl2, 0.5 mmol,』L sodium EGTA, I mmol/L dithiothreitol. (6) Protease inhibitor (PIC) tablets (Roche Diagnostics GmbH, Mannheim, Germany): one tablet dissolved in 10 mL of buffer B (7) Corning cell scraper (Sigma-Aldrich, St Louise, MO). (8) Injector (3 mL) and needle (2 6 eyes). (9) Protein concentration assay kit (Bio-Rad, Hercules, CA). (1) IPGphor gel tank, 18 cm (AmershamBiosciences, Piscataway, NJ). (2) IPGphor isoelectric focusing system (Amersham Biosciences). (3) Hoeffer DALT electrophoresis system (Amersham Biosciences). (4) pH 4-7L solid phase pH gradient (IPG) adhesive tape, 18 cm (Amersham Biosciences, Uppsala, Sweden). Store at 20°C. (5) Hydration Buffer (RB): 7 m o l/L urea, 2 m o l/L thiourea, 4% CHAPS, 1 % DTT, 4% CHAPS, 1 % DTT, 1 % DTT, 1 % DTT, 1 % DTT, 1 % DTT, 1 % DTT, 1 % DTT. (6) BiaLyte 3/10 amphoteric electrolyte (Bio^Rad). Store at 4°C. (7) Mineral Oil (Bio--Rad). (8) IPG Strip Equilibration Buffer (EBl): 50 mmol/L Tris-HCl, pH 8.8, 6 mol/L urea, 30% (V/V) glycerol, 2 % sodium dodecyl sulfate (SDS), 1 % DTT, 1X 10-4% bromophenol blue. (9) IPG Strip Equilibration Buffer (EB2): 50 mmol/L Tris-HCl, pH 8.8, 6 mol/L urea, 30 % (V/V) glycerol, 2 % SDS, 4 % iodoacetamide, 1X 10-4% bromophenol blue. (10) Methylated bisacrylamide solution (30_8 % T ) (neurotoxic, wear gloves!) : 3 0 % acrylamide, 0.8 (11) 10 % SDS. (12) 1 0 % amine persulfate. (13) Tetramethylethylenediamine (TEMED, BitRad). (14) SDS electrophoresis buffer: 25 mmol/L Tris, 192 mmol/L glycine, 0.1% SDS. (15) Low melting point agarose (LMT) (Gibco/BLR). Dissolve 1 % LMT in SDS electrophoresis buffer by microwave heating. Keep at 50°C until use. (16) Silver staining solution: a Fixing solution: 30% ethanol, 5% acetic acid; b Sensitizing solution: 0.02% sodium thiosulfate; c Silver staining reagent: 0.2% silver nitrate; d Developing solution: 4% potassium carbonate, 0.025% formaldehyde. 2 % silver nitrate. d Developing solution: 4 % potassium carbonate, 0.025 % formaldehyde (37%); e Stopping reagent: 4 % Tris base, 2 % acetic acid. (17) Umax Powerlook 1100 scanner (Umax Technologies, Dallas, TX). (1) Trypsin (cat. no. V5111, Promega, Madison, WI). (2) ZipTip (:18 pipette tip (Millipore, Bedford, MA). (3) Substrate solution: 10 m g/m L of a-cyano-4-hydroxycinnamic acid was dissolved in 50% acetonitrile containing 0.1% trifluoroacetic acid. (4) Voyager DETm-P R O matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF) (PerSeptive Biosystems, Framingham, MA). (5) MALDI plates (PerSeptive Biosystems, Framingham, MA). (1) Cellulose nitrate membrane (Bio-Rad). (2) Towbin buffer: 25 mmol/L Tris, 192 mmol/L glycine, 0.1% SDS, 2 0 % (V/V) methanol. (3) IX T ris Buffer Salt Solution (1XTBS): 10 mmol/L Tris-. HCl, pH 8_0, 150 mmol/L NaCl (4) TBS--T : I X TBS, 0.1% Tween-20. (5) 5 % skimmed milk solution : TBS^T , 5 % skimmed milk. (6) ECL™ anti-rabbit antibody, horseradish peroxidase-linked F Ub')2 fragment (from donkey) (AmershamBiosciences)" (7) Western Lightning chemiluminescent reagents (PerkinElmer, Boston, MA). (1) AtT20 and AtT20 (proSAAS) cells were cultured in 150 m m Petri dishes (3 dishes per cell type) with DMEM and grown at 37°C in a humidified environment of 5% C02-95% air until full of AtT20 (proSAAS) in medium supplemented with 500 fxg/mL of G418. (2) The monolayer was rinsed 3 times each time with 10 mL of PBS. (3) Cover the cells with Versine; scrape the cells off the plate with a cell scraper and transfer the cell suspension to a centrifuge tube. (4) Centrifuge at 1300 g at 4°C for 5 min to precipitate the cells. Remove and discard the supernatant. (5) Centrifuge again as described above to remove residual Versine. (6) Resuspend the cells with 0.5 mL of PIC-containing Buffer B and rupture the cells by pushing and pulling 30 times with a syringe with a 2.6-mesh needle. (7) Centrifuge the lysate at 4°C, IOOOg for 7 min. Transfer the supernatant to a new tube and discard the precipitate. (8) Centrifuge the supernatant for 30 min at 4°C, l0 V. Transfer the supernatant (cytoplasmic fraction) to a new tube and keep the supernatant in a clean place. (9) Add 3 times the volume of acetone to each cytoplasmic fraction and place at 20°C for lh to precipitate proteins. Centrifuge at 15,800 g at 4°C for 15 min. Discard the supernatant and retain the precipitate. (10) Dissolve each cytoplasmic precipitate or microsomal protein in 500 of R B by sonication (see Note 1). (11) Determine protein concentration using the Bio--Rad Protein Concentration Assay Kit, Bradford method. 3.1 Silver staining (see Note 7) The following steps are performed with oscillation. (2) Replace the fixative and oscillate for 30 min. (3) Rinse the gel with water 5 times for 5 min each time. (4) Incubate the gel in sensitizing solution for lmin. (5) Rinse the gel with water twice for lmin each time. (6) Incubate the gel in silver staining reagent for 30 min. (7) Rinse the gel with water twice for lmin each time. (8) Incubate the gel in developing solution until protein spots appear. (9) Terminate development for 30 min with stopping reagent. (10) Rinse the gel with water 3 times for 5 min each time. 3.2 2D image acquisition Silver-stained gels were scanned on an Umax Powerlook 1100 scanner (Fig. 1) and digital images were saved. (1) Cut the gel spot of interest. (2) Mix 30 mmol/L potassium ferricyanide and 100 mmol/L thiosulfate in a 1 : 1 ratio. (3) Cover the gel sheet with 100 uL of the above solution. (4) Shake at room temperature until the silver stain is removed. (5) Rinse the gel piece with 500 uL of water at a time and change the water several times until the yellow reagent is removed. (6) Incubate the gel pieces in 200 uL of 200 mmol/L ammonia bicarbonate for 20 min with shaking. (7) Rinse the gel slice twice with 500 water for 1 min each time. (8) Centrifuge and remove water. (1) Incubate the gel slice in 200 uL CH3CN for 10 min. (2) Centrifuge and remove CH3CN. (3) SpeedVac Vacuum Centrifugal Evaporation Concentration Gel Slices for 15 min. (4) Incubate the gel pieces in 100 )nL 100 mmol/L NH4 HCO3, 10 mmol/L DTT at 56°C for 45 min. (5) Centrifuge and remove the D T T solution. (6) Add 100 100 mmol/L NH4 HCO3, 55 mpol/L iodoacetamide and incubate for 30 min at room temperature in the dark. (7) Centrifuge and remove the iodoacetamide solution. (8) Rinse the gel slice for lOmin in 100 100 mmol/L NH4 HCO3 with shaking. (9) Centrifuge and remove the NH4 HCCV solution. (10) Dehydrate gel pieces in 100 pL CH3C N for 5 min at room temperature. (11) Centrifuge and remove CH3CN (12) Rinse the gel slice in 100 uL of 100 mmol/L NH4 HCO3 for 5 min with shaking. (13) Centrifuge and remove the NH4 HCO3 solution. (14) Dehydrate the gel pieces in 100 uL CH3CN for 5 min at room temperature. (15) Centrifuge and remove CH3CN. (16) Concentrate gel pieces by SpeedVac vacuum centrifugal evaporation for 15 min. (17) The gel pieces were soaked with 20 50 mmol/L NH4 HCO3 containing 500 ng trypsin for 30 min on ice. (18) Remove the residual trypsin solution and cover the gel pieces with 50 mmol/L trypsin-free NH4 HCO3. (19) Digest the proteins in the gel overnight at 37°C. (20) Centrifuge and transfer the supernatant to a new club. (21) Add 50 uL of 50% C H 3 C N , 0 . 1 % trifluoroacetic acid to the gel sheet and sonicate in a cold water bath for 3 min. (22) Centrifuge and transfer the supernatant to the initial supernatant. (23) Repeat steps 2 1 and 22 twice more. (24) SpeedVac Vacuum Centrifugal Evaporation concentrates the supernatant until the volume is less than 10 uL. (25) Add 0 . 1 % trifluoroacetic acid to 10 uL. (1) Prior to MALDI-TOF analysis, trypsin-digested peptides were purified using a ZipTip C18 pipette tip according to the manufacturer's instructions. (2) Spot the purified peptides on M A L D I plates with 2 uL substrate. (3) Mass spectrometry was performed in delayed extraction and reflection mode using the following instrumental settings: 20 k V accelerating voltage, 150 ns delay time, 70 % gate voltage, 0.05 % guidewire voltage, 128 laser emission, laser intensity 2000, mass gate from 800 to 2800. m/z 842.5 1 and m/z 1045.5 6 of trypticase auto-degradation product were used for internal mass internal mass calibration (Figure 2). (4) The masses of the single isotope peaks were selected and the proteins were identified by searching the Swiss-Prot and TrEMBL databases using the search engine PeptIdent (http://www.expasy.ch/tools/peptident.html), matching the corresponding theoretical masses within 50 ppm®. (1) Repeat the preparation of the 2D gel as described in 3. 2. (2) Transfer the protein spots from the unstained gel to the nitrocellulose membrane in a Hoefer SE 600 bath containing Towbm buffer according to the manufacturer's user manual. (3) Seal the membrane with 5% skimmed milk solution. (4) Incubate the membrane in 5 ¼ skimmed milk containing anti-EphA2 -anti for lh at room temperature. (5) The membrane was rinsed four times in TBS "T for 5 min each time. (6) Incubate the membrane in skimmed milk solution containing HRP-conjugated donkey anti-rabbit IgG for lh. (7) Rinse the membrane four times in TBST for 5 min each time. (8) Immunoreactive spots were visualized with Western Lightning chemiluminescent reagent according to the manufacturer's protocol (Fig. 3). Caveat (1) Complete solubilization, depolymerization, denaturation, and reduction of sample proteins are critical to the success of protein separation on 2D gels. Therefore, protein precipitates need to be solubilized in R B by sonication for at least 3 min. Because urea can be degraded to isonitriles at higher temperatures, leading to protein aminomethylation, sonication is performed with a cuphorn sonicator under cooling in an ice-water bath.(2) The IPG adhesive strip tank is made of ceramic, so be careful when using it.(3) To avoid contamination, work with gloves. Do not allow air bubbles to remain under the IPG tape.(4) The water used here to prepare the solution must have a resistivity of above.(5) A 20 cm long glass tube with a lid is ideal for equilibration.(6) Avoid air bubbles between the IPG tape and the SDS glue.(7) Silver staining is the most sensitive visualization method. For high quality results, use high purity reagents and wear gloves to avoid contamination. For more product details, please visit Aladdin Scientific website.
Dithiothreitol (DTT).
Methylenedioxyacrylamide. Store at 4°C.
Transfer the supernatant (cytoplasmic component) to a new tube and retain the starch (microsomal component).
(1) Immerse the gel in fixative for 30 min.
