研究方法進行步驟及執行進度：

1. Blood and tissue specimen for proteomics study
30 patients will be studied. Blood will be withdrawn from patient before the surgery or even before the pre-operative embolization. The collected blood will be kept cool for gene study, or centrifugated and stored in –80 degrees before peptide study.

During the procedure of surgical intervention for tumor excision, the soft tissue , nerve, and vessel within the tumor will be dissected out and put into liquid nitrogen for frozen and specimen for immunohistochemistry study.

2. 2D (two-dimensional) gel electrophoresis
Unstained frozen tissue sections are disrupted and grounded into powder, which are solubilized in 2D protein lysis buffer (0.01M Tris/HCl,pH 7.4, 8M urea, 5% NP40, 0.05M DTT, 6% pH3-10 ampholyte, 10% glycerol). The samples are stored at –70 o C until analyzed by 2D gel electrophoresis. For first dimension gel electrophoresis, the IPGphor until and pre-cast immobilized pH gradient (Amersham Pharmacia) using the protocol suggested by the manufacturer. The IPGphor unit is able to run 12 samples (6 pairs) simultaneously. The rehydration step was carried out with precast 18cm IPG strips for more than 10 h at low voltage of 30V IEF was run following a step-wise voltage increase procedure: 500V and 1000V for 1h each and 5000-8000V for about 10h with a total of 64KVh. After IEF, the strips were subjected to two-step equilibration in equilibration buffers for 10 min in 75mM Tris-HCl, pH 7.9, 3% SDS, 50 mM DTT, and 0.01 bromophenol blue. The second dimensional separation will be performed using 10% SDS-PAGE. The second dimension gel will apply the Hoefer DALT Vertical system (Amersham Pharmacia). The Hoefer DALT vertical system (25 x 18 cm) is allowed to run up to 12 gels simultaneously, thus providing high efficient of throughput time. After gel separation, the protein will be presented by using Coomassie-blue or silver staining method. Protein spots will be shown on an image scanner (Typhoon Variable Mode Imager) and analyzed using a program provided by the manufacturer. (ImageMaster 2D Elite, Amersham Bioscience)

3. Image Analysis
The protein spots that are differentially presented in control/muscle reduction, control/ muscle preservation, and muscle preservation/muscle reduction tissues will be isolated from the gel, followed by gel digestion by trypsin. Total of 50 clones of proteins will be selected, these 2D protein patterns produced can then be analyzed by gel-to-gel comparison using ImageMaster 2D Elite software (Amersham Bioscience). The trypsin digested products will be subjected to MALDI-TOF for sequencing analysis. Identification of the peptide will be obtained by immunoblots of cell lysates from ischemic limb or normal visceral organs using 2D electrophoresis.

4. In-gel tryptic digestion
Proteins corresponding to differential expressed spots will be cut from the gel and washed with 400 m l of 125mM ammonium carbonate:acetonitrile (CAN) 1:1 (v/v)

Solution. After briefly air-drying the gel, enzymatic cleavage will be initiated by first reswelling the gel in ammonium carbonate solution (125mM), then digestion by the addition with 50mM acetic acid containing 7 units of trypsin (Promega, Lyon, France). The gel slices will be placed in an eppendorf tube and a minimum volume of water will be added to totally immerse the gel pieces. The digestion will be carried for 12-16 hours at 30 o C. The liquid will be collected and the resulting peptides recovered after two extractions with a solution containing 45% CAN/10% formic acid. To recover very hydrophobic peptides, a third extraction with 95% CAN/5% formic acid will be performed. The extract will be finally dried using a Speed Vac-concentrator (Savant).

5. MALDI-TOF (mass spectrometry for peptide/protein identification)
MALDI-TOF analysis of tryptic digests will be performed in our core center, using a Vision 2000 (Finnigan, Bremen) instrument equipped with a 337-nm nitrogen laser. The instrument operated in reflector positive mode at an accelerating voltage of 6kV. Peptides will be resuspended in CH 3 CN/H 2 O (1:1) containing 0.5% formic acid, of which 0.5 m l will be mixed directly onto the target with 1 m l of 2,5-dihydroxybenzoic acid matrix solution(10mg/ml in CH 3 OH/H 2 O, 7:3). Mass measurements will be realized from spectra resulting from 30 to 50 laser spots after peak smoothing and internal calibration using the average mass of the two autolysis trypsin fragments. Protein sequence database searching will be performed using software provided by public domain ( www.matrixscience.com ).

預期完成之工作項目及成果：