蛋白質體學及應用1

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(1)蛋白質體學及應用. 中山大學生醫所. 黃弘文.

(2) 內容介紹 . 何謂蛋白質體學蛋白質體學研究方法蛋白質體學應用實驗技術.

(3) 何謂蛋白質體學 . 蛋白質體學與傳統生化學傳統研究“蛋白質”的科學 . 生物化學酵素學 …等.

(4) 蛋白質的構造 . 細胞的功能主要由蛋白質執行 . . . 代謝生化反應繁殖…. 蛋白質為一長鏈的胺基酸聚合物，鏈長由數十至數千。生物體約有20種胺基酸。.

(5) 胺基酸構造 Functional group: 1.-NH3 2.-COOH 3.-R (羧基) 4.鏡像結構.

(6) 20種胺基酸: 依R group分類: 1.非極性 2.極性 3.正電 4.負電.

(7) 蛋白質鏈方向性(N → C ).

(8) 蛋白質四元結構.

(9) 傳統研究蛋白質的方法 • • • •. 蛋白質濃度測定蛋白質(酵素)反應測定蛋白質分離純化蛋白質定序.

(10) ■各種蛋白質定量法：. Coomassie Arg Blue G. NH+ 2 C NH HN 2 C C C Fe C C O H C H C R R H R O O N CC C N C N C C C O C O R N C C C R C H H + (metal) Active N H R O H H H N Site N O C NH2 H2N R C 206 nm H N C C O C O C C (carbonyl) N C 尿素 O R O H urea 2+ R Cu. Special Binding Lys + Groups (heme) NH3. SO-3. N=N. OH. NH SO-. 3. SO-3 SO3. N=N. OH. NH SO-. 3. SO-3. 280 nm. Tyr. (aromatic) OH .... UV Absorbance. Biuret Method. phsophomolybdicphosphotungstate. (carbonyl). Lowry Method.

(11) ■ Bradford Method：. ■ Coomassie Brilliant Blue G-250 470 nm. CBG 是一種指示劑. 595 nm. 加入蛋白質酸性環境下呈茶色. 與蛋白質結合變藍色.

(12) 可能有回饋抑制. 反應物. 酵素. -. 生成物. +. NAD 移除生成物並轉成可測量者. 直接測量. NADH. 耦合反應. ■ 酵素反應及偵測方法：. 使用大量反應物 10 x Km. 能否直接測量？. 把生成物再轉換成可被測量的生成物.

(13) 蛋白質的分離 • 電泳(electrophoresis) • 色層分析法(column chromatography).

(14) ■ 電泳形式的演進：. -. +. 濾紙電泳 Cellulose Protein Denatured. 薄層電泳 (TLE) Cellulose acetate Partial Denatured. 膠體電泳 Starch → Gel.

(15) ■ 兩種主要蛋白質染色法：. Ammoniacal silver. Coomassie Blue R + H3N...Ag...NH3 Ag Arg + + Lys NH2 NH2 + + NH3 H2N...Ag C Glu NH2 還 HN C Glutaraldehyde 原 C NH C C C Lys O CC H C H C R R C C H R O O (Cys) C C N C N C C N C C O C O R C N C C R H H R C + O H H N H N HN O C C R N C C H C O O C C N O R H R 金屬銀沉澱. SO3. N=N. OH. NH SO-. 3. SO3 SO3. N=N. OH. SO3. NH SO-. 3.

(16) ISCO: Little Blue Tank Concentrator. ■ 電泳膠體蛋白質的溶離： ● 直接挖出膠體進行溶離或定序. Little Blue Tank.

(17) ■ 電泳槽及相關設備：轉印三明治. 電泳槽. 轉印槽. 鑄膠器. 供電器.

(18) ■ 轉印及免疫染色流程： A. B. 轉印三明治. 免疫染色流程及結果. 塑膠夾板. 轉印. Filter Paper. -. 抗體 HRP. + 電泳膠體 Gel Nitrocellulose 海綿墊. Coomassie Blue Staining. 轉印紙 Ponceau Staining. 抗體偵測呈色.

(19) ■ 澱粉磷解染色方法比較：. 硝酸銀. Disc-PAGE. 免疫轉印. 活性染色. SDS-PAGE. 膠體過濾法各分劃.

(20) 蛋白質體(Proteomics) . 二維電泳(2-Dimensional protein gel electrophoresis) and HPLC were long used in protein separation.. . 近年生物質譜技術快速發展(development of mass spectrometry in recent years, ~2000 AD). . 2002 Nobel prize:. • John B. Fenn (Virginia Commonwealth Univ.) • Koichi Tanaka (Shimadzu Corporation, Japen) • Soft desorption ionization(軟式脫附離子化) methods for mass spectrometric analysis.. . proteins annotation(蛋白質註解)..

(21) Functional Genomics (or Phenomics) The –OMICS – based sciences !. 基因體學轉譯體學蛋白體學代謝體學. 系統生物學.

(22) Summary of the systems . . -ome--- Compete set. -ics --- the utility for analyzing these domains. 功能基因體學 (Functional genomics) : hierachical approaches for studying the functional analysis of novel genes.

(23) Defining proteomics . Functional proteomics: 利用2DE及MS研究蛋白表現 • 二維電泳 (2DE): two dimensional gel analysis, 通常指: sample first run a 等電位電泳IEF (isoelectrofocusing) electrophoresis. 再以 SDS PAGE分離蛋白的方法. • 質譜(MS): mass spectrometry, basically, a method to “accurate” determine molecule weight..

(24) 比較蛋白質體學與蛋白質化學 Differences between protein chemistry and proteomics ---------------------------------------------------------------------------------------Protein chemistry Proteomics --------------------------------------------------------------------------------------Individual proteins complex mixtures Complete sequence analysis. partial sequence analysis. Emphasis on structure and function. emphasis on identification by database matching Structural biology systems biology ----------------------------------------------------------------------------------------. Conventional ideas: Using physical biochemistry or mechanistic enzymology, study one protein or multisubunit protein complex at a time. Proteomic ideas: Study multiprotein systems, in which the focus is on the interplay of multiple, distinct proteins in their roles as part of a larger system or network. Proteomics: to characterize the behavior of the system.

(25) ►蛋白質體學因應基因體學而來: ► Proteomics and Genomics ► Genomics Genome sequencing ►Complete. DNA sequences from various organisms. Bioinformatics ►Tools. for sequence comparison. Identification of homologues ►Tools. for sequence interpretation. Elucidation of coding sequences / protein functions. Gene expression (protein) profiling ►Transcriptomics. Differential expression of mRNAs (MicroArray)基因晶片. 已知mRNA表現,為何還須知道蛋白質表現?.

(26) 轉錄. DNA 基因. mRNA 功能性基因. t-RNA. t-RNA. 轉譯. 核糖體. 蛋白質. Microarray. 基因體學 (Genomics). (....). 轉譯後修釋. X (....) X. 蛋白質體學 (Proteomics). 功能性蛋白質 (生化反應). X. CHO 醣質化 PO4 磷酸化. Proteomic. 1.蛋白質體學與基因體學兩者相互互補(如表現量)但不一定相同。 2.蛋白質體學能發現在基因體學無法完成得到的結果(如醣質化及磷酸化)。 2001 Proteomics Group, Institute of Biological Chemistry, Academia Sinica.

(27) 蛋白質體的四個關鍵技術:. 1. 2. 3. 4.. Sample preparation, 樣本處理 Protein information,蛋白質資訊 Protein Identification and quantification,蛋白質註解及定量 Cell mapping: targeted proteomics (study of protein-protein interactions, posttranslational modifications (PTMs), cellular localization, etc.)..

(28) 蛋白質體目前瓶頸: Protein determination range . . 不同蛋白質表現量差異很大:~ 106. e.g., 10 copies/cell for transcription factor, up to 106 copies/cell for abundant molecules. 2D gels, which usually separate ~1,500 spots, present most abundant proteins if a crude protein mixture is used..

(29) Protein Copy number and cell quantity: 無法測到低表現蛋白質 Detection in Silver stain range and coomassie stain range..

(30) 蛋白質二維電泳2-D gel electrophoresis. . . 1st D - Isoelectric focusing (聚膠電泳) - separation on basis of pI 2nd D - SDS gel electrophoresis (SDS電泳) - separation on basis of mass Isoelectric focusing - separation on basis of pI - pH gradient - migrate to their pI and stabilize pH gradient.

(31) 1st -Dimensional gel Electrophoresis . 1st PAGE precast gels: • 提供高解析及高範圍生物材料分離 (“denatured” proteins, polypeptides or nucleotides) from 2,000,000 to 5,000 Da..

(32) . Polyacrylamide gel electrophoresis Acrylamide :. . CH2 = CH - CO – NH2. N,N' methylene bis acrylamide: CH2 = CH – CO – NH – CH2 – NH – CO – CH = CH2. . . Polymerization induced by free radicals produced from ammonium persulfate in presence of TEMED (N, N, N', N' – tetra methylethylendiamine) TEMED causes free radicals to form from persulfate.

(33) 利用起始劑自由基反應聚合: •Chemical: TEMED and APS. •Photochemical: riboflavin-5’phosphate, or methylene blue. 電泳膠孔徑(由單體濃度及比例決定): • %, and ratio: acrylamide and Bis-acrylamide • Polymerization temp. • Gel additives: •Urea (smaller pore size) •Polyethylene glycol: macroporous..

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(35) Isoelectric point (pI) • Separation by charge: 4. Stable pH gradient. 5. Low pH: Protein is positively charged. 6 7 8 9 10. High pH: protein is negatively charged. At the isolectric point the protein has no net charge and therefore no longer migrates in the electric field..

(36) pH值的選擇 pH梯度差越小,解析度越高. 根據不同的樣品選擇不同pH梯度, e.g., •The known extreme pI : acidic glycoprotein of the chimpanzee: pI=1.8, Lysozyme from human placenta: pI=11.7. . The most used pI range is pH=3 ~ 10..

(37) IEF Ideal Lysis Buffer (標準配方) 8M urea (or 7M urea + 2M thiourea in the case of fractions containing hydrophobic proteins) 2-4% CHAPS 1% IPG buffer (appropriate buffer for pH range to be analysed) 2 mg/ml DTT.

(38) Factors which may affect pI - amino acid composition - phosphorylation - glycosylation - other post-translational modifications.

(39) 2nd -Dimensional gel Electrophoresis . 第二維電泳通常以SDS PAGE進行. • 傳統SDS PAGE須使用聚膠電泳(stacking gel)及分離電泳(separating gel)合併使用 • 通常二維電泳中,因為第一維已聚膠，不使用stacking gel..

(40) ■ SDS (sodium dodecyl sulfate). 極性頭部. 非極性尾巴. 切勿吸入 SDS 塵埃以免引起呼吸困難.

(41) ■ SDS 在蛋白質表面均勻附上一層負電：. SDS. 原態蛋白質. boiling. 變性蛋白質成一線狀分子.

(42) Fig 2.3. Kohlrausch boundary, a limited high-voltage gradient. Stacking gel: pH6.8, resolving gel: pH8.8, electrode buffer, pH8.3 (Tris-glycine) Sampel(P1-P5) running between two solution: Cl-(leading) , and trailing (Gly-). Laemmli system.

(43) ■ 單元體分子量的測定： SDS-PAGE Mol mass. kDa 330 220. kDa 300 200. kDa. 94 67 60. 100 80 60 50 40 30. 67 43. 36. 30. 20. 20.1. 10. 0.5 Migration (Rf). 1.0. 18.5. 14.4. Pharmacia: Molecular Markers for electrophoresis.

(44) Multiphor™ II IEF System . . . Perform first- and second-dimension separations with the same system. Run up to 12 IPG strips (7-24 cm) simultaneously. With DryStrip Reswelling Tray, samples can be loaded during IPG strip rehydration by including them in the rehydration buffer..

(45) Step 1. Rehydration Use DryStrip Reswelling Tray.. Step 2. IEF Use the parallel grooves. Cap loading.

(46) Ettan IPGphor IEF System . . Accommodates all lengths of Immobline DryStrip gels (7, 11, 13, 18, and 24 cm) and can run 12 gels simultaneously. 8,000 V Max.

(47) BioRad system . Isoelectric focusing (IEF) . Pre-cast IPG strips • Variable pH range (e.g. 3-10, 4-7, 36, 6-12, 4-5) • Variable strip length (7, 11, 17, 24 cm) • Optimised protein loading • 10,000V Max.

(48) . IPGphor ceramic strip holders are available in five lengths: 7, 11, 13, 18, and 24 cm..

(49) . IPG strips are rehydrated with IPG Buffer in regular strip holders. The sample can be added to the buffer for efficient loading during rehydration..

(50) . With movable electrodes and sample cup, the Cup Loading Strip Holder accommodates IPG strips up to 24 cm long..

(51) 1. Apply rehydration solution.. 2. Remove protective film from. 3.Drystrip in strip holder. DryStrip gel.. 4. Apply Cover oil. 5. Cover on strip holder.. 6. Place assembled strip holder on Ettan™ IPGphor™ platform..

(52) TECHNICAL SPECIFICATIONS Instrument. Integrated Peltier cooling and highhigh-voltage power supply. Voltage. 0−8000 VDC. Current. 0−1.5 mA. Platform temperature. 18− 18−25 °C. IPG strip lengths. 7, 11, 13, 18, or 24 cm Immobiline™ Immobiline™ DryStrip Gels. Number of strips. 12 maximum. Reswelling. In strip holder or reswelling tray when using Cup Loading Strip Holder. Strip holder material. Base − aluminum oxide Cover − clear acrylic. Sample application. During rehydration or after rehydration when using Cup Loading Strip Holder. Software features. 10 protocols, 9 voltage ramps or steps each; set rehydration delay; set maximum current limit; set temperature. Ports. Serial port (RS232). Operating temperature. 15− 15−35 °C. Relative humidity. 0−90% nonnon-condensing. Line voltage. 90− 90−260 VAC. Maximum power. 100 W. Dimensions (H × W × D). 14 × 25 × 46 cm. Weight. 6.8 kg. Safety features. Automatic voltage shutshut-off when safety lid is opened. Safety certifications. CE 73/23/EEC (LV directive); UL3101UL3101-1; CSA22.2 10101010-1.

(53) Multiphor™ II and ExcelGel™ System . . . . Perform second-dimension separations using convenient precast gels and 7 11 18 or 24 cm IPG strips. Choose from ExcelGel precast SDS-polyacrylamide gels available as 12.5% homogeneous and 12-14% gradient gels. Prepare multiple 2-D maps on a single gel using three 7 cm or two 11 cm IPG strips side-by-side. Configure Multiphor II with MultiTemp III Thermostatic Circulator and the programmable EPS 1001 Power Supply for reproducible running conditions..

(54) Hoefer™ SE 600 Ruby™ Vertical System . . . . Perform second-dimension separations using 7 or 13 cm IPG strips. Prepare multiple 2-D maps on a single gel using two 7 cm IPG strips side-by-side. Run up to four gels simultaneously and perform eight second-dimension separations at once. Configure SE 600 with MultiTemp III Thermostatic Circulator and the programmable EPS 601 Power Supply for reproducible running conditions..

(55) 影像分析 . Complex protein patterns in 2D electrophoresis gels are captured in digital format to allow accurate, reproducible and quantifiable comparisons. • Image Master (Melanie) (Pharmacia) • PDQuest (BioRad).

(56) Image Scanner ImageScanner has an extremely broad optical density range to give the high resolution required for reliable quantification of Coomassie and silver stained gels. Fast scanning and image acquisition rates ensure short scan times..

(57) Typhoon • Typhoon unites four-colour fluorescence, filmless autoradiography and chemiluminescence into a single system. • Its broad dynamic range ensures accurate results at the first attempt. • Highly sensitive optics facilitate extremely low limits of fluorescence detection and enable direct chemiluminescence imaging without intermediate exposures to film or screens..

(58) 2-DE Sample prep.. 蛋白質體分析流程 Protein Mass. Rehydration. IEF. SDS-PAGE. Protein identify from database. MALDI-TOF In-Gel Digestion. Gel -Image Analysis. Reduction Alkylation. Extraction Peptide.

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