Relativity of Liver Mitochondria Proteome and Energy Metabolism

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 21 (6) : , 3,,, ( ) 8 ( 4 ) Cy3 Cy5 8 Cy2 Cy3 Cy5 Cy2 DeCyder 112, 16, ATP NADH 2 A, Q49318,R22031 Relativity of Liver Mitochondria Proteome and Energy Metabolism : : (No ) 3 Tel : (0571) ,Fax : (0571) ,E2mail com Received :January 25,2005 ; Accepted : April 25,2005 in Shenyang Deficience Animals Induced by Hormone LU De2Zhao, WO Xing2De 3 Supported by National Natural Science Foundation of China (No ) 3 Corresponding author Tel : (0571) ,E2mail com, SHI Meng2Ru, LI Yi, TANGLi2Hua ( Department of Life Sciences, Zhejiang Traditional Chinese Medical College, Hangzhou , China) Abstract Two2dimensional differential gel electrophoresis (22D DIGE) was used to study the difference of liver mitochondria proteome between the shenyang deficience rats and normal rats The relation between shenyang dificience and energy metabolism were observed Eight samples (four samples from normal rats and four from shenyang dificience rats) were labeled with Cy3 or Cy5 Each Cy32labeled sample and Cy52labeled sample were mixed on the same 22D gel along with a Cy22labeled mixture of all eight samples as an internal standard The gels were imaged by Typhon 9400 using different emission filter respectively and spot2features were analyzed by DeCyder software in each paired normalπdisease comparison Using DIGE technology with internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference of gel2to2gel variation MALDI2TOF MS was used to provide sensitive and accurate mass spectral data for 16 unique proteins that were changed in abundance more than 1 2 folder up or down between normal and shenyang dificience rats, and 11 protein were successfully identified In these proteins, the expression of heat shock protein60π70,sarcosine dehydrogenase, carbamoylphosphate synthetase, ATP synthase, sulfite oxidase, mitochondrial aldehyde dehydrogenase precursor and NADH dehydrogenase increased, while the expression of ornithine amino2transferase, 22

2 oxoisovalerate dehydrogenase, acyl2coa dehydrogenase decreased in shenyang dificience rats These findings indicate that the change of the enzymes relation to energy metabolism in shenyang dificience animals involved in the deficiency2cold symptom in the clinic Key words proteome, shenyang deficience, liver mitochondria, energy metabolism 2,, 2, [1 ] 25 mgπkg 8,, 6 : [2,3 ] 0125 molπl 4 1Π10 ( WΠV) ph 714 MSHE (0122 molπl, 0107 molπl, 015 mmolπl EGTA, 011 %, 2 mmolπl HepesΠKOH ),600 g 5, min g 10 min 5 ml MSHE 20 ml 30 % Percoll ( difference gel (225 mmolπl, 1 mmolπl EGTA, 25 mmolπl electrophoresis,dige) Hepes, 011 % ), g 30 min gπml 40 ml MSHE g 10 min Percoll, 150 mmolπl KCl MSHE 2 1 ml D 10 [4 TRIS CHAPS SDS ph (24 gπ l, - 80 ] cm,ph 3 10),Ampholyte ph 3 10,22D clean up 115 Amersham CyDye (Cy2,Cy3,Cy5) - 20 (Na 2 S 2 O 3 ), 10 min, 25 l,, MilliQ g 30 s 2 l 200 pmolπ l 1 2 Ettan TM IPGphor TM, Ettan TM 400 pmol DALTSix,Typhoon min 1 l 10 mmolπl,decyder Image Quant TM V510, 10 min 2 Amersham P80MX (8 molπl, 130 mmolπl DTT, 2 % CHAPS, 2 %,UV8200 ph3210 Pharmalyte) 10 min HITACHI g SD 8, Cy3 Cy5 8 Cy2 2 4 (30 mmolπl Tris, 8 molπl, 4 % CHAPS, ph 815), 4 2 h, g 30 min 22D clean up kit, 50 g ph

3 6 : 809 Cy2,Cy3,Cy5 (8 molπl, 2 % CHAPS, 13 mmolπl DTT,015 % IPG H 2 OΠ5 %TFAΠ50 % 2,, % ) 450 l Amersham ( GE Healthcare Bio2Sciences), 8 MALDI2TOF2MS 4 ph 3 10 Mascot 22D :30 V 12 h ;500 V 1 h ;1000 V 1 h ;8 000 V 8 h,, 1 (1 % DTT, 50 mmolπl Tris2 Table 1 HCl, 6 molπl, 30 %, 2 % SDS, % Table 1 The search parameters of Mascot used for protein ) 2 (4 %, 50 mmolπl Tris2HCl, identification 6 molπl, 30 %, 2 %SDS, % ) 15 min, 1215 % SDS 015 %, :20 W,45 min ;60 W, 6 h 117 MilliQ Typhon9400, PTM : Cy2 : Blue2 NH 4 HCO 3 ( 55 mmolπl ) 1 h NH 4 HCO 3 ),35 50 l 45 % Parameter Database searched Taxonomy searched Enzyme used Parameter setting 100 mmolπl 3 NH 4 HCO 3,100 l, 100 mmolπl NH 4 HCO 3 Table ngπ l ( 25 mmolπl NCBInr Rattus Trypsin Missed cleavages Allow up to 1 Modifications Peptide mass tolerance Peptide masses are Carbamidomethyl (C) 100ppm Monoisotopic (488)Π600 ; Cy3 : Green (530)Π680 ; Cy5 : Red (633)Π590 2 DeCyder Image Quant TM V , 8 [5,6, Cy2 ] Cy3, 50 l Cy5 (50 mmolπl Na 2 S 2 O 3, 15 mmolπl K 3 Fe (CN) 6 ) 2 min, 100 l 25 mmolπl, NH 4 HCO 3 8 min, 100 l 8 min, 10 nmolπl DTT, 25 mmolπl NH 4 HCO l 57 1 h,,,, 100 l 25 mmolπl Table 2 2D2DIGE experimental design for biological variation analysis Gel No Cy2 labelled sample Cy3 labelled sample Cy5 labelled sample 1 Internal standard Normal rats Hormone deficient shenyang deficience rats 2 Internal standard Normal rats Hormone deficient shenyang deficience rats 3 Internal standard Normal rats Hormone deficient shenyang deficience rats 4 Internal standard Normal rats Hormone deficient shenyang deficience rats

4 SDS2PAGE 488 nm 530,, nm 633 nm 3 (Fig 1) Fig 1 22D protein map from gel 2(pH 3210NL, 24 cm) (A) Internal standard ; (B) Normal rat liver mitochondria ; (C) Shenyang deficience rat liver mitochondria % DeCyder510,Fig12 Fig 3 3, , Fig 2 spot map table and spot information Total of 1048 features were detected in the Master gel Average number of features were 962 and average number of spots matched were 856 Fig 3 Full image of gel 3 in shenyang deficience

5 6 : Fig 4 3,4, (Spot4) (Spot8 ), Fig 4 32D charts from Spot 4 and Spot 8 (A) : Spot 4 ; (B) : Spot [7 ] , 10 %, Table 4 112,4 112 Table 4 The identified protein spots by MALDI2TOF MS (Table 3) Fig 3, No Table 3 Targeted proteins for Mass spectrometry Spot No Master No T2test Ratio Spot Protein pi M r Ratio 1 Sarcosine dehydrogenase Carbamoyl2phosphate synthetase kd heat shock protein kd heat shock protein Mitochondrial aldehyde dehydrogenase precursor 22Oxoisovalerate dehydrogenase alpha subunit Ornithine aminotransferase Acyl2CoA dehydrogenase, short chain specific Sulfite oxidase ATP synthase alpha chain, mitochondrial precursor NADH dehydrogenase Table 4 : A,, ATP ATP NADH

6 (two2dimensional electrophoresis,22 3, 015 [10, ng, ],, 50 -, ( differential in2gel, electrophoresis,dige) [5,, ] Cy2, Cy3 Cy5 DIGE [3,8 ] camp cgmp [8 ] 100 %,, ( ), [9 ] [6 ], DE) 1975 O Farrell, 22D ,, % 4 12 SD, [2 ] : 10 % ,, 16, 11, 60 70, ATP NADH

7 6 : 813 A,,,,,, ATP ATP NADH, NADH A [12, ] 2 ATP, ATP 3 2 2, A, A NAD + NADH + H +,,, A 2,, 2 ATP A NAD + NADH + H + A A, A A, 2 A FAD, FADH 2 2, ATP 2, ATP 2 ATP ( References),,, A 2, 1,, (Dong Xin2Gang,Zhang Qing2Yi, Chen Yi2Ping Progress on the symptom of shenyangxu Chin J Interg Tradit West Nephrol), 2003,4(5) : ( Wu Qi2Duan, Xiong Dai2Shui, Liang Weng2Neng Reseach situation of shenyangxu animal model Form),2001,7(6) :54 56 Chin J Exp Tradit Med

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