7. N-Terminal and Internal Amino Acid Sequence Analysis and Homology Search of Amino Acid Sequence

Transcription

1 Protocol 1. Sample Preparation 2. Two Dimensional Polyacrylamide Gel Electrophoresis 3. Image acquisition and Data Analysis 4. Cleveland Peptide Mapping 5. Western Blotting 6. Deblocking of Blotted Proteins 7. N-Terminal and Internal Amino Acid Sequence Analysis and Homology Search of Amino Acid Sequence 8. Mass Spectrometry 1

2 1. Sample Preparation 2.1. Chemical Lysis buffer (10ml) Urea (9.5 M) 4.8 g NP-40 (2%) 0.2 ml Ampholine 2% (ph ) 0.2 ml B-mercaptoethanol(5%) 0.5 ml PVP g Water 9.10 ml Note. This buffer may be stored frozen at 20 o C. Do not continuously freeze and thaw it. Use an aliquot once and discard the remainder 2.2. Method a. Collect sample and homogenize in lysis buffer. b. Centrifuged at 15,000 rpm for 10 min c. Take the supernatant and centrifuge again at 15,000 g for 10 min d. Take the supernatant and use for 2D-PAGE 2

3 2. Two Dimensional Polyacrylamide Gel Electrophoresis 2.1. First Dimensional Isoelectric Focussing (IEF) Materials. a. 30% acrylamide stock solution (28.38% acrylamide) g (1.62% BIS) 1.62 g Make the volume to 100 ml with water Acrylamide is a potent neurotoxin and should never be mouth pipeted. solution is light sensitive and should be stored in the dark at 4 o C. b. Anode electrode solution Acrylamide 0.02N H 3 PO 4 (1M stock: ml phosphoric acid to 100 ml water) c. Cathode electrode solution 0.02 N NaOH (1M stock: 4 g Sodium Hydroxide to 100ml water) d. SDS Sample buffer ( ph 6.8) 0.06 M Tris HCl g 2 % SDS 10 g 5% B-mercaptoethanol 25 ml 10% Glycerol 50 ml Make the volume up to 500 ml with water (MQ) Method a. Mark 2cm from the top of clean, dry 13 cm long and 3 mm diameter, 1d gel tubes to indicate the desired height of the gel. Place a rubber band around them so that they form a tight bundle. Hold the bundle vertically on a flat surface. Seal the bottom with two layers of parafilm b. Prepare the solution Urea 4.8 g 30% acrylamide stock 1.16 ml NP-40 2 ml Water 2.84 ml Ampholines (ph5-7) 0.25 ml Ampholines (ph ) 0.25 ml 10% APS 15 µl TEMED 10 µl 3

4 Note. Dissolve the urea by swirling in water bath whose temperature should not higher than 60 o C c. Using a long narrow guage hypodermic needle, fill the gel tubes with the solution of step no 3 to the mark. d. Overlay the gel mix with 20 µl of water and allow it to polymerize it for one hour e. Remove the para-film from the bottom of the tubes and water is removed from the top of the gel. f. Seat the tubes in the holes of the upper buffer reservoir of the tube cell. Plug any unused hole with rubber stopper g. The lower chamber is filled with 0.02 N Phosphoric acid. The samples are loaded in a volume µl and overlaid with 20 µl of 1/2 lysis buffer. The upper chamber is filled with 0.02 N Sodium Hydroxide. h. Run the current as follows 200 V for 30 min 400 V for h 600 V for 1 h i. Extrude the gel from the tubes using the water pressure from a syringe. Wash each gel in 5 ml SDS sample buffer for 15 minutes. Change the buffer and wash it again for 15 minutes. At this point the gel can be stored at 20 o C for several week or used immediately First Dimensional Immobilized ph Gradient (IPG) a. Seat the tubes in the holes of the upper buffer reservoir of the tube cell. Plug any unused hole with rubber stopper b. The lower chamber is filled with 0.02 N Sodium Hydroxide. c. The water is removed from the top of the gel. The samples are loaded in a volume µl with a syringe and overlaid with 20 µl of 1/2 lysis buffer. The upper chamber is filled with 0.02 N Phosphoric acid. Connect positive terminal with lower chamber (acidic) and negative terminal to upper chamber (basic). Connect positive terminal to lower chamber (basic) and negative terminal to upper chamber (acidic). Run the current as follow 400 V for 1 h 1,000 V for 16 h 2,000 V for 1 h d. Remove the tubes and force the gels out onto a parafilm. Wash each gel in 5ml SDS sample buffer for 15 minutes. Change the buffer and wash it again for 15 minutes. At this point the gel can be stored at 20 o C for several week or used immediately 2.3. Second Dimension with IEF and IPG 4

5 Materials a. Separating gel buffer (ph 8.8) Tris HCl (1M) g SDS (0.27 %) 0.27 g Make the volume to 100 ml with water (MQ) b. Stacking gel buffer (ph. 6.8) Tris HCl (0.25 M) 3.63 g SDS (0.2%) 0.2 g Make the volume to 100 ml with water (MQ) c. Acrylamide for separating gel Acrylamide 30 g BIS g Make the volume to 100 ml with water (MQ) d. Acrylamide for stacking gel Acrylamide 29.2g BIS 0.8g Make the volume to 100 ml with water (MQ) e. 1% agarose 1 g of agarose /100 ml of water f. Running buffer Tris-HCl 9.0 g Glycine 43.2 g SDS 3.0 g Make the volume to 3 L with water (MQ). g. Bromophenol blue solution BPB 0.01 g /100 ml of 10 % glycerol Method a. Assembled detergent cleaned gel plates. The separating gel is cast first, followed by the casting of the upper stacking gel. For a gel with dimension 0.75 mm x 14cm x 14cm, about 20 ml of separating gel mix and about 5ml of stacking gel mix is 5

6 needed. b. Prepare 15% polyacrylamide gel as shown below Acryalamide for separating gel 8.5 ml Separating gel buffer (ph.8.8) 6.3 ml Water 2.0 ml 10% APS 120 µl EMED 20 µl For one plate Swirl to mix and pour the gel immediately. Gently overlay with 1cm of water. Allow the gel to polymerize for 45 to 60 minutes c. Preparation of Stacking gel solution Stacking gel acrylamide 1.0 ml Stacking gel buffer 3.0 ml Water 2.0 ml APS 30 µl TEMED 20 µl Remove the overlaid water and pour the stacking gel solution immediately. Leave it to polymerize for minutes d. The first dimension is applied directly on the top of the stacking gel. The first dimension is overlaid by 1% agarose. e. The slab gel is assembled for running. A few drops of tracking dye (bromophenol blue) is added f. Run the sample at 35 ma (constant current) until the tracking dye reaches the bottom of the separating gel. g. Separate the gel plates and either stain the gel or process for western blot. The gel is stained either with silver staining or Coomassie Brilliant Blue 6

7 3. Image Acquisition and Data Analysis (1) CBB-stained gels were analyzed with Image Master 2D-Elite software. Specially, spot detection, spot measurement, background subtraction and spot matching were performed. Following automatic spot detection, gel images were carefully edited. Prior to performing spot matching between gel images, one gel image was selected as the reference gel. (2) After automatic matching, the unmatched spots of the member gels were added to the reference gel. The amount of a protein spot was expressed as the volume of the spot which was defined the sum of the intensities of all the pixels that make up the spot. In order to correct the variability due to CBB staining and reflect the quantitative variations in intensity of protein spots, the spot volumes were normalized as a percentage of the total volume in all of the spots present in gel. (3) The resulting data from image analysis were transferred to 2D Elite software for querying protein showing quantitative or qualitative variations. 7

8 4. Cleveland Peptide Mapping (1) Prepared samples are separated by 2D-PAGE. Then the gels are stained with Coomassie brilliant blue, and gel pieces containing protein spots are removed. (2) The protein is electroeluted from the gel pieces using an electrophoretic concentrator run at 2 W constant power for 2 h. After electroelution, the protein solution is dialyzed against deionized water for 2 d and lyophilized. a. Cut out stained protein spots from 2D gels and soak in deionized water. b. Fill 750 μl of electroelution buffer in the 2-mL Eppendorf tube containing protein spots (5 20 gel pieces). Shake for 30 min. c. Cut 12- to 15-cm-long seamless cellulose tubing (small size, no. 24, Wako, Osaka, Japan) as space is needed for clipping. Fill a 300-mL beaker with250 ml deionized water, boil it for 5 min, and keep the tubing membrane in it. Wet the small pieces of cellophane film in a small beaker with deionized water. d. Close the bottom of the small part of cup with a cellophane film, open the bottom of the large part of cup, connect and twist the tubing membrane, and close the distal end by clipping (Fig. 1A). e. Fix the cup in the electrophoretic concentrator (Fig. 1B; Nippon Eido, Tokyo, Japan). Deposit gel pieces containing proteins on the cellophane film (thesmall part of the cup), and add 750 μl of the electroelution buffer from the Eppendorf tube. Fill the small part of the cup with electroelution buffer, and then fill the large part of the cup with electroelution buffer in such a way thata layer of buffer joins both the cup parts, allowing movement of protein fromthe small part of the cup to the tubing membrane. Fill the apparatus with electroelution buffer. The small part of cup containing protein spots should be toward the positive side. f. Run at 2 W constant power for 2 h. g. Remove the tubing membrane and clip to close the end. Dialyze in a cold room (4 C). Change the deionized water three times the first day. The next day, change the deionized water two times. h. Transfer the protein solution to two to six 2-mL Eppendorf tubes. Freeze-dry overnight. i. Dissolve the protein in 30 μl of SDS sample buffer. (3) The protein is dissolved in 20 μl of SDS sample buffer (ph 6.8) and applied to a sample well of an SDS-PAGE gel. The sample solution is overlaid with 20 μl of a solution containing 10 μl of Staphylococcus aureus V8 protease (Pierce, Rockford, IL, USA) (0.1 μg/μl) in deionized water and 10 μl of SDS sample buffer (ph 6.8). Electrophoresis is performed until the sample and protease are stackedin the stacking gel. The power is switched off for 30 min to allow digestion of theprotein, 8

9 and then electrophoresis is continued. a. Fix glasses (100 x 140 x 1 mm) with clip, keeping a 1-mm space between the plates. b. Prepare separating gel solution in a 100-mL beaker. Mix the solutions, and fill the plates about 3 cm from top. (Caution: Pour the solutions into the plates immediately after adding 10% APS and TEMED.) c. Overlay the separating gel solution with 1 ml water (MQ). d. Leave the gel for 40 to 60 min at room temperature for polymerization. e. Remove the overlaid water and pour the following stacking gel solution. f. Prepare the stacking gel solution in a 100-mL beaker. Mix well, pour on the separating gel, and insert comb. g. Leave the gel for 20 min at room temperature for polymerization. h. Take out the comb, clips, and silicon tubes. i. Clean the wells with a syringe. j. Fix the gel plates with the apparatus. Pour SDS-PAGE running buffer. k. Dissolve the protein in 20 μl of SDS sample buffer (ph 6.8), and apply to asample well in SDS-PAGE. Overlay the sample with 20 μl of a solution containing10 μl of Staphylococcus aureus V8 protease with 1 μg/μl in deionized water and 10 μl of SDS sample buffer (ph 6.8). Add 30 μl BPB solution. l. Electrophoresis was performed until the sample and protease were stacked in the upper gel and interrupted for 30 min to digest the protein. m. Run the gel at 35 ma until the BPB line reaches about 5 mm near the bottom. n. Disconnect the electricity, and take out the plates. o. Separate two plates with a spatula. p. Separate the stacking gel, and take out the separating gel. 9

10 5. Western Blotting Following separation by 2D-PAGE or by the Cleveland method, the proteins are electroblotted onto a PVDF membrane (Pall, Port Washington, NY) (Fig. 2), using a semidry transfer blotter (Nippon-Eido, Tokyo Japan), and detected by CBB staining. a. Cut the PVDF membrane equal to the size of the gel. b. Cut the Whatman 3MM filter paper equal to the size of the gel. c. Wash the PVDF membrane in methanol for a few seconds, and transfer the membrane to 100 ml blotting solution C; shake for 5 min. d. Wet two blotting papers (3 MM) in A, B, and C blotting solutions. e. Place the separating gel in 100 ml blotting solution C, and shake for 5 min. f. Wet the semidry blotting apparatus with water (MQ). Place blotting paper A on the blotting plate and then blotting paper B. Remove air bubbles, if any. Place the PVDF membrane on the plate and then the gel and blotting paper C. g. Connect the power supply. Run the blotting at 1 ma/cm2 for 90 min. h. Wash the membrane in 100 ml water (MQ). i. Stain the gel for 2 to 3 min in CBB stain. j. Destain the gel in 60% methanol for 3 min twice. k. Wash with MQ and air-dry at room temperature. 10

11 6. Deblocking of Blotted Proteins 6.1. Acetylserine and Acetylthreonine Proteins with acetylserine and acetylthreonine at their N-termini separated by 2D-PAGE are electroblotted onto a PVDF membrane. The region of the PVDF membrane carrying the protein spot is excised and treated with trifluoroacetic acid (TFA) at 60 C for 30 min. They are then directly subjected to protein sequencing Formyl Group N-formylated protein separated by 2D-PAGE is electroblotted onto a PVDF membrane. The region of the PVDF membrane carrying the protein spot is excised and treated with 300 μl of 0.6 M HCl at 25 C for 24 h. The membrane is washed with deionized water, dried, and applied to the protein sequencer Pyroglutamic Acid (1) Proteins with pyroglutamic acid at their N-termini separated by 2D-PAGE are electroblotted onto a PVDF membrane. (2) The region of the PVDF membrane carrying the protein spot is excised and treated with 200 μl of 0.5% (w/v) polyvinylpyrrolidone (PVP)-40 in 100 mm acetic acid 37 C for 30 min (see Note 3). (3) The PVDF membrane is washed at least 10 times with 1 ml of deionized water. (4) The PVDF membrane is soaked in 100 μl of 0.1 M phosphate buffer (ph 8.0) containing 5 mm dithiothreitol and 10 mm EDTA acid. (5) Pyroglutamyl peptidase (5 μg) is added, and the reaction solution is incubated at 30 C for 24 h. (6) The PVDF membrane is washed with deionized water, dried, and applied to the protein sequencer. 11

12 7. N-Terminal and Internal Amino Acid Sequence Analysis and Homology Search of Amino Acid Sequence (1) The stained protein spots or bands are excised from the PVDF membrane and applied to the upper glass block of the reaction chamber in a gas-phase protein sequencer, Procise 494 or clc (Applied Biosystems, Foster City, CA, USA), and/or PPSQ (Shimazu, Kyoto, Japan). Edman degradation is performed according to the standard program supplied by Applied Biosystems and/or Shimazu. The released phenylthiohydantoin (PTH) amino acids are separated by an online high-performance liquid chromatography (HPLC) system and identified by retention time. (2) The amino acid sequences obtained are compared with those of known proteins in the Swiss-Prot, PIR, Genpept, and PDB databases with the web-accessible search program FastA. 12

13 8. Mass Spectrometry 8.1. Preparation of chemicals Chemicals Methanol (MeOH) for blotting sequence type Acetonitrile CH 3 CN), HPLC grade Trifluroacetic acid (TFA) HPLC grade Stock solutions (Preserve at 4 o C) 100 mm NH 4 HCO mm EDTA.2Na Washing solution (25% MeOH / 7% Acetic acid) 10 mm Tris-HCl (ph 8.0) 3% TFA 0.1% TFA / 100% CH 3 CN 0.1% TFA / 100% H 2 O (Freezer at -20 o C) 1 M Dithiothreitol (DTT) 10 pm Trypsin / 10 mm Tris-HCl Chemicals to be prepared during use Destaining solution (50 mm NH 4 HCO 3 / 50% MeOH) 100 mm NH 4 HCO ml 100% MeOH 2.5 ml a. Reduction solution ( 10 mm EDTA.2Na / 10 mm DDT / 100 mm NH 4 HCO mm EDTA.2Na 20 µl 1 M DDT 10 µl 100 mm NH 4 HCO µl b. Alkylation solution (10 mm EDTA. 2Na / 40 mm Iodoacetamide / 100 mm NH 4 HCO mm EDTA.2Na 20 µl Iodoacetamide 7.4 mg 100 mm NH 4 HCO µl c. Trypsin solution 10 pm Trypsin / 10 mm Tris-HCl 50 µl 10 mm Tris-HCl (ph 8.0) 450 µl 8.2. Methods a. Cut the gel (spot) Cut the selected protein spots from the 2D-gels. Place the gels on light box and cut the protein spots with a spatula. Collect the gel pieces in an eppendorf tube. Wash 13

14 the gel pieces with water (MQ), drain out water. This can be preserved in freezer at 20 o C. b. Washing and destaining Add 1 ml wash solution (25% MeOH / 7% Acetic acid) to the eppendorf tube, fix them in a rotor and rotate over night at room temperature. c. Destaining procedure depends on the method of staining: In case of CBB staining: Wash with water (MQ), add 0.5 ml destaining solution (50 mm NH 4 HCO 3 / 50% MeOH), incubate at 40 o C until the samples (gel pieces) are destained (may take 30 to 60 min). After destaining, dry out destaining solution then wash with water (MQ). In case of silver staining: Add 0.3 ml solution (20 mm EDTA. 2Na / 50 mm Tris-Hcl (ph8.0) / 30% CH 3 CN), wait for 5 min at room temperature. Drain out supernatant, repeat this step once more. Then wash with water (MQ) for two times. d. Reduction and Alkylation i) Dry the samples in speed Vac. concentrator (may skip this step) ii) Add 50 µl reduction solution (10 mm EDTA.2Na / 10 mm DTT / 100 mm NH 4 HCO 3 ) to the eppendorf tube to make the gels wet. Incubate for 1 h at 60 o C placing on a cool block. iii) Discard the solution and dry again using speed Vac. iv) Add 50 l alkylation solution (10 mm EDTA.2Na / 40 mm Iodoacetamide / 100 mm NH 4 HCO 3 ) to the gels in eppendorf tube, place in dark at room temterature for 30 min to carry out reaction. v) Add 1 ml water (MQ), wash the gels and drain the water. Repeat this step for 2 times. vi) Smear the gels with a Pelletier. vii) Dry the gels in a speed Vac. concentrator. e. Digestion i) Prepare 1 pmol trypsin solution (10 pm Trypsin / 10 mm Tris-HCl), add 50 µl to the gels in eppendorf tube. ii) Wrap the eppendorf tube with Para film, digest at 37 o C for 10 to 12 h. f. Elution i) Add 100 µl solution (0.1% TFA / 100% CH 3 CN) to the gels in eppendorf tube(a), sonicate for 10 min. 14

15 ii) Collect the supernatant in a new eppendorf tube (A1). iii) Add 100 µl solution (0.1% TFA / 100% MQ) to the gels in eppendorf tube (A), sonicate for 10 min. iv) Collect the supernatant in eppendorf tube (A1). v) Add 80 µl solution (0.1% TFA / 50% CH 3 CN) in the gels in eppendorf tube (A), sonicate for 10 min. vi) Collect the supernatant in eppendorf tube (A1). vii) Add µl solution (0.1% TFA / 100% CH 3 CN) to the gels in eppendorf tube (A), sonicate for 10 min. viii) Collect the supernatant in a new eppendorf tube (A1). ix) Concentrate the collected sample to µl in a speed Vac. (CH 3 CN will be evaporated). This sample is peptide solution. g. Desalt i) Using reverse phase column, desalt and concentrate the peptide solution. ii) Wash with ZipTip C18 (Millipore) filling and unfilling 10 µl 0.1% TFA / 50% CH 3 CN 3 times iii) Wash and stabilize ZipTip with 10 µl 0.1% TFA / 100% MQ, filling and unfilling 3 times iv) Take the whole peptide solution in ZipTip (10 µl each time) in repeated times, thus absorbing the whole peptides in ZipTip columniv) Wash and desalt the ZipTip with 10 µl 0.1% TFA / 100% MQ, repeat 3 times v) Dissolve the peptides absorved in column by 0.1% TFA / 50% CH 3 CN. The sample volume depends on the following operation. In case of analyzing using VOYAGER-RP: Use 2 µl peptide on the sample plate and 2 µl Matrix solution, mix them well, air dry and analyze. In case of analyzing using other Mass spectrometer The sample volume depends on requirement. In case of preservation for future analysis Take 5 in an eppendorf tube and keep in freezer at -20 o C. 15