蛋白質(zhì)組學(xué)proteomicswxj課件

1、Proteomics 蛋白質(zhì)組學(xué)Proteome & Proteomics 定義Proteome：1994年，由澳大利亞Macguarie大學(xué)的Wilkins等首先提出：“蛋白質(zhì)組指由一個(gè)細(xì)胞或一個(gè)組織的基因組所表達(dá)的全部蛋白質(zhì)” “proteome”是由蛋白質(zhì)一詞的前幾個(gè)字母“prote”和基因組一詞的后幾個(gè)字母“ome”拼接而成Proteome & Proteomics 定義Proteomics：蛋白質(zhì)組學(xué)是從整體水平細(xì)胞內(nèi)蛋白質(zhì)的組成、結(jié)構(gòu)、功能及其動(dòng)態(tài)變化規(guī)律的科學(xué)。研究內(nèi)容包括分析蛋白質(zhì)組所有組分及它們的表達(dá)水平，確定各種組分的空間定位、修飾方法、互作機(jī)制、生物活性及相應(yīng)特定功能等。

2、由此獲得蛋白質(zhì)水平上的關(guān)于疾病發(fā)生，細(xì)胞代謝等過程的整體而全面的認(rèn)識(shí) Genome & ProteomeGenomics Transcriptomics & ProteomicsProteomics & Structural GenomicsProteomics & Functional Genomics特點(diǎn)之一整體性特點(diǎn)之二系統(tǒng)性特點(diǎn)之三動(dòng)態(tài)性Structural Proteomics 結(jié)構(gòu)蛋白質(zhì)組學(xué)Functional Proteomics 功能蛋白質(zhì)組學(xué) 分類Structural ProteomicsSeparationIdentificationPTM (post-translatio

3、nal modification) IdentificationComparison & Subtraction Analysis Functional Proteomics LocalizationProtein Complex DeterminationProtein-Protein Interaction/Interaction NetworkFunction of Protein (Metabolic and Regulatory Pathway; Signal Transduction Pathway) 蛋白質(zhì)鑒定：可以利用一維電泳和二維電泳并結(jié)合生物質(zhì)譜、Western印跡、蛋白質(zhì)

4、芯片等技術(shù)，對(duì)蛋白質(zhì)進(jìn)行全面的鑒定研究。 翻譯后修飾的鑒定：如磷酸化、糖基化、酶原激活等過程。 蛋白質(zhì)功能確定：包括蛋白質(zhì)定位研究，蛋白質(zhì)活性，蛋白質(zhì)相互作用，酶活性和確定酶底物，細(xì)胞因子的生物分析，配基-受體結(jié)合分析等。蛋白質(zhì)組學(xué)的主要任務(wù)蛋白質(zhì)組學(xué)研究思路與方法策略、技術(shù)、工具主要專業(yè)術(shù)語及其英文對(duì)照和縮寫IPG-IEF：固相pH梯度等電聚焦（immobilized pH gradients isoelectric focusing）SDS- PAGE：十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳（Sodium dodecyl sulfate- polyacrylamide gel electrop

5、horesis）2-DE：雙向電泳(Two Dimensional Electrophoresis)HPLC：高效液相色譜（High performance Liquid Chromatography）MALDI-TOF MS：基質(zhì)輔助激光解吸電離飛行時(shí)間質(zhì)譜（Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry)蛋白序列數(shù)據(jù)庫（SWISS-PROT/TrEMBL；http:/www.expasy.ch）基因序列數(shù)據(jù)庫（Genbank，http:/ EMBL； http:/www.ebi.ac.

6、uk/）蛋白模式數(shù)據(jù)庫（Prosite；http:/www.expasy.ch/sprot/prosite.html）蛋白質(zhì)二維凝膠電泳數(shù)據(jù)庫、蛋白三維結(jié)構(gòu)數(shù)據(jù)庫（PDB，http:/；FSSP，http:/www.embl-ebi.ac.uk）蛋白翻譯后修飾數(shù)據(jù)庫（O-GLYCBASE，http:/www.cbs.dtu.dk/databases/OGLYCBASE）蛋白質(zhì)組學(xué)研究相關(guān)數(shù)據(jù)庫研究策略兩條互補(bǔ)的實(shí)驗(yàn)流程基于凝膠的工作流程（Gel-based workflow）基于液相色譜的工作流程（LC-based workflow）Classical GE & LC to Protein I

7、D Gapelo (2010) J ProteomicsHPLC-MS蛋白質(zhì)組研究的主要手段雙向電泳（two dimensional gel electrophoresis, 2D-GE）差示電泳（differential gel electrophoresis, DIGE ）毛細(xì)管電泳(capillary electrophoresis, CE) 高效液相色譜(high performance liquid chromatography,HPLC) 分子篩柱層析 反向柱層析質(zhì)譜分析 ( mass spectrometry MS) 基質(zhì)輔助激光解吸離子化-飛行時(shí)間串聯(lián)質(zhì)譜(matrix-ass

8、isted laser disorption ionization-time of flight /tandem MS , MALDI-TOF/MS/MS) 電噴霧離子化串聯(lián)質(zhì)譜（electrospray ionization ESI-MS）生物信息學(xué)蛋白質(zhì)組學(xué)實(shí)驗(yàn)室所需的條件蛋白質(zhì)組學(xué)研究的基本技術(shù)路線蛋白質(zhì)樣品的制備雙向電泳或HPLC圖像分析凝膠中的蛋白溶液中的蛋白混合肽蛋白質(zhì)質(zhì)量N端測序肽序列質(zhì)譜數(shù)據(jù)肽指紋圖數(shù)據(jù)搜索新的或已知蛋白翻譯后修飾的鑒定酶解Sample Protein PreparationUsually the complexity of the protein and /o

9、r peptide mixture lies beyond the theoretical separation space of any separation method. Proteome Complexity. Genomic transcription in diversity; post-transcription processing; post-translational modification; constitution of combinatorial complex.It requires quite a long time for analysis of protei

10、n complex.Sample recovery low through separation: more steps, less overall recovery.The conc. of the proteins with a range of 6-order magnitude : in tissues, protein concentrations span a dynamic range of six orders of magnitudes (for regulatory protein or TFs a few molecules per cell, for housekeep

11、ing proteins million copies per cell). So, it is difficult to detect very low concentrated proteins in the presence of highly abundant proteins .The complex protein samples with limited stability due to existence of enzymes and proteases, enzyme inhibitors can only partly stabilize the sample.Many p

12、arameters influence the composition of proteome between induced and inherent biological variations, such as genetic differences, gender and age of patients and cell growth conditions. This requires sample replicates. (statisticians would demand at least five replicates, in many cases three replicate

13、s can deliver highly confident results. In clinical the number of required proteins are much high) . Membrane proteins are very difficult to solubilize, and easy to loss during sample preparation and separation by sticking to a surface or aggregation. Post-translational modifications (PTMs) like pho

14、sphorylation and glycosylation require sophisticated analysis tools like MSn, where the peptide ions generated several times fragmented. Sample Protein Preparation蛋白質(zhì)組研究的基本技術(shù) 樣品預(yù)分離樣品的制備（預(yù)處理）：組織細(xì)胞細(xì)胞器（線粒體、葉綠體、細(xì)胞核）蛋白質(zhì)組研究的基本技術(shù) 蛋白提取重要性：在制備時(shí)丟失的蛋白永遠(yuǎn)不能在后面實(shí)驗(yàn)中彌補(bǔ)原則：使所有待分析的蛋白樣品全部處于溶解狀態(tài) 防止樣品在聚焦時(shí)發(fā)生蛋白的聚集和沉淀 防止在樣品制

15、備過程中發(fā)生樣品的抽提后化學(xué)修飾（如酶性或化學(xué)性降解等）完全去除樣品中的核酸和某些干擾蛋白 蛋白質(zhì)組研究的基本技術(shù) 蛋白提取步驟：破碎沉淀蛋白去除雜質(zhì)蛋白質(zhì)組研究的基本技術(shù) 蛋白提取樣品制備流程 破碎盡可能減少蛋白水解/其它形式蛋白降解原則機(jī)械法（超聲波法、高壓法 、機(jī)械勻漿法 ）化學(xué)法（去污劑法、酶裂解法 ）物理法（液氮研磨法、反復(fù)凍融法、滲透法 、玻璃珠破碎法 ）蛋白質(zhì)組研究的基本技術(shù) 蛋白提取樣品制備流程 沉淀蛋白 去雜濃縮后蛋白可溶性是關(guān)鍵 三氯醋酸（TCA）-丙酮沉淀法 TCA沉淀法 引起降解/修飾 丙酮沉淀法 硫酸銨沉淀法 影響IEF醋酸銨沉淀法 步驟繁瑣 2D電泳結(jié)果影響因素分析

16、蛋白質(zhì)組研究的基本技術(shù) 蛋白提取樣品制備流程 去除雜質(zhì) 關(guān)鍵是盡量不丟失蛋白和減少蛋白修飾 核酸的清除 （DNase/RNase）多糖的清除 （超離心 、TCA沉淀等）去污劑的清除 （丙酮沉淀法等）鹽離子和外源帶電小分子的清除（透析、TCA-丙酮沉淀法） 2D電泳結(jié)果影響因素分析可能原因：樣品含高豐度Pr可能原因：TCA殘留致使Pr丟失蛋白質(zhì)組研究的基本技術(shù) 蛋白提取樣品制備注意事項(xiàng)：蛋白質(zhì)水解 蛋白酶抑制劑(PMSF等)特殊樣品的制備 (低豐度 、強(qiáng)堿性蛋白質(zhì) （核糖體）、極端分子量 )樣品定量 重復(fù)性Two Dimensional Gel Electrophoresis (2D-GE)Di

17、fferential Gel Electrophoresis (DIGE) Gel ElectrophoresisWorkflow for Two-Dimensional Electrophoresis (2-DE)GE (2004) 2-D Electrophoresis two dimensional gel electrophoresis (2D-GE)Isoelectric Focusing Electrophoresis (IEF)IEF mainly applied for the following purposes: 1st dimensional fractionation

18、of protein mixtures in high-resolution 2-D electrophoresis Pre-fractionation of protein mixtures according to chargeFor the separation of very heterogeneous mixtures of tryptic peptides instead of strong cation exchange chromatography in MDLC-MSIPG immobilized pH gradientIPG Strip for IEFpH10.0 Cath

19、ode (-)pH 3.0Anode (+)pH103IEF is performed in a pH gradient gel.Proteins Charged with Anion(-) or Cation(+) depend on (i) their molecular traits due to their acidic and basic groups, and (ii) the environmental pH. Environmental pH : in basic buffer, the acidic groups of proteins with negative charg

20、e; in acidic buffer, the basic groups with positive charge. Protein Isoelectric Point (pI) : At a pH value, the net charge of a protein is zero.39IEF Theoretical Background 40The Principle of Isoelectric Focusing ElectrophoresisIEF PrincipleAmpholytes to Create A pH Gradient: A Heterogeneous Mixture

21、 of Isomers of aliphatic oligoamino-oligocarboxylic acids under electricity field could align themselves due to individual molecule trait & create a pH gradient along cathode with high-pH to anode with low-pH. Ampholyte Properties : (i) high buffering and solubility in its pI (ii) good and regular e

22、lectric conductivity at its pI (iii) absence of biological effect (iv) low molecular weightThe Ampholyte-Gel : the ampholyte 2% (w/v), the gel 4% (T) & 3% (C)Immobile pH Gradient Strip for Proteins Separation (IPG) : (i) proteins applied to a position on the IPG strip could be charged with anion, ca

23、tion and zero (ii) protein-cation moves forward cathode and stop the site where the pH value is equal to the pI of protein-cation; similarity for protein-anion to move forward anode and stop; protein-zero to be naive to move.4142Immobilized pH Gradient PolyacrylamideThe General Structure of Immobili

24、ne (Ampholyte)雙向凝膠電泳（2-DE）是等電聚焦電泳和SDS的組合即先進(jìn)行等電聚焦電泳（按照pI分離）然后再進(jìn)行SDS（按照分子大小）凝膠經(jīng)染色得到二維分布的蛋白質(zhì)圖 蛋白質(zhì)組研究的基本技術(shù)2D-SDS：樣品制備第一向IPG-IEF電泳IPG平衡第二向SDS電泳染色（銀染）及 圖譜分析目標(biāo)蛋白獲取及其鑒定（MC分析）2-DE第一向IPG-IEF電泳IEF是一種根據(jù)樣品的等電點(diǎn)不同而使它們在pH梯度中相互分離的一種電泳技術(shù)將等電點(diǎn)不同的蛋白質(zhì)混合物加入有pH梯度的凝膠介質(zhì)中，在電場內(nèi)經(jīng)過一定時(shí)間后，各組分將分別聚焦在各自等電點(diǎn)相應(yīng)的pH位置上，形成分離的蛋白質(zhì)區(qū)帶從而得知其等電點(diǎn)信

25、息IPG-IEF電泳2-DE第二向垂直SDS電泳聚丙烯酰胺凝膠形成網(wǎng)狀結(jié)構(gòu)，具有濃縮效應(yīng)、電荷效應(yīng)、分子篩效應(yīng) 。SDS與蛋白質(zhì)形成雪茄狀帶負(fù)電荷復(fù)合物，從而消除了蛋白間的電荷及形狀差異，電泳速度僅與分子量有關(guān) 從而得知其分子量信息第二向垂直SDS電泳凝膠濃度與其對(duì)應(yīng)的分離范圍膠濃度 分離范圍(KD) 5% 36-200 7.5% 24-200 10% 14-200 12.5% 14-100 15% 14-60第二向垂直SDS電泳Ettan Dalt twelve電泳系統(tǒng)第二向垂直SDS電泳Ettan Dalt six電泳系統(tǒng)2-DE 凝膠蛋白質(zhì)斑點(diǎn)的檢測染色： 1）考馬斯亮蘭染色法； 2）銀

26、染法； 3）負(fù)染法； 4）熒光染色法； 5）放射性同位素標(biāo)記法等安全（safety）靈敏（sensitivity）簡單（simplicity）特異性（specificity）快速（speed）穩(wěn)定（stability）兼容性（synergy）理想顯色劑的7S有機(jī)染料和銀染考馬斯亮藍(lán)染色靈敏度為30100ng，線性范圍是20倍；硝酸銀染色的線性范圍是40倍，靈敏度是考染的100倍。膠體考馬斯亮藍(lán)染色技術(shù)可實(shí)現(xiàn)PAGE的無背景染色，其極限靈敏度為810ng，但這種染液會(huì)對(duì)蛋白質(zhì)進(jìn)行修飾而影響質(zhì)譜分析的結(jié)果。氨基黑常用于轉(zhuǎn)印至聚偏二氟乙烯（PVDF）和/或硝酸纖維素膜上的蛋白質(zhì)的染色。銀染的缺點(diǎn)是：

27、對(duì)某些種類的蛋白質(zhì)染色效果差，對(duì)其后的蛋白質(zhì)測序和質(zhì)譜分析造成影響。這兩類染色技術(shù)都可減少膠內(nèi)蛋白質(zhì)產(chǎn)量。 Coomassie Brilliant Blue &Silver Stain 負(fù) 染能專門提高PAGE膠上蛋白質(zhì)的回收率，但不能用于膜上染色。結(jié)果表現(xiàn)為膠面著色而蛋白質(zhì)點(diǎn)透明。速度快（515min），蛋白質(zhì)的生物活性能保持：一旦用絡(luò)合劑如EDTA或Tris/甘氨酸轉(zhuǎn)移緩沖液來絡(luò)合金屬離子就可進(jìn)行提取來轉(zhuǎn)移蛋白質(zhì)。它主要適用于蛋白質(zhì)顯色、完整蛋白質(zhì)的膠上被動(dòng)提取以及質(zhì)譜分析。該技術(shù)主要包括金屬鹽染料、鋅咪唑染料等的使用。膠體擴(kuò)散染料主要用于高靈敏度檢出電轉(zhuǎn)印至硝酸纖維素和PVDF膜上的蛋白

28、質(zhì)，不用于膠內(nèi)染色。最好的膠體金染色的靈敏度與PAGE膠內(nèi)的銀染類似。這種技術(shù)主要包括麗春紅、印度墨水染料、膠體金染料等。 有機(jī)熒光團(tuán)染料包括共價(jià)結(jié)合和非共價(jià)結(jié)合的熒光團(tuán)染料兩類。后者最為常用，其典型代表是已經(jīng)商品化的SYPRO Red、 Orange、 Ruby等熒光染料。這三種染料可對(duì)SDS膠內(nèi)蛋白質(zhì)進(jìn)行一步染色，約3060min完成，靈敏度為210ng。染色后的凝膠用標(biāo)準(zhǔn)的實(shí)驗(yàn)室300nm紫外透射儀進(jìn)行照像保存，其線性范圍為3個(gè)數(shù)量級(jí)。這三種染料的電泳染色結(jié)果與在酵母中通過SAGE所獲得的基因表達(dá)水平的動(dòng)態(tài)范圍相匹配。在Tris/甘氨酸轉(zhuǎn)印緩沖液中染色后，蛋白質(zhì)可被轉(zhuǎn)印至膜上并進(jìn)行免疫染

29、色或Edman測序來鑒定蛋白質(zhì)。熒光染色金屬螯合染料這是一類與現(xiàn)代蛋白質(zhì)組學(xué)研究相兼容的、相對(duì)較新的蛋白質(zhì)顯色試劑，其設(shè)計(jì)專門與常用微量化學(xué)表征過程兼容。它們不包含戊二醛、甲醛或Tween-20等，很容易和集成化蛋白質(zhì)組學(xué)平臺(tái)（包括自動(dòng)化凝膠染色儀、圖像分析工作站、機(jī)器人剪切儀器、蛋白質(zhì)酶解工作站和質(zhì)譜儀等）相結(jié)合。其中SYPRO Ruby也是一種基于釕的金屬發(fā)光染料。同位素標(biāo)記，放射自顯影 靈敏度 20pg2DE重復(fù)性The same protocol and sample, however not the same resultsRec: similar; cir: different D

30、IGE & Proteins Labeled with CyDyesProteins labeled with CyDyes : with dyes cyanine (Cy2 blue, Cy3 red, Cy5 green), the dyes cannot influence protein property as well as its molecular weight much.The Mixed Run on 2-DE : the mixed ratio at 1:1, run on the same 2-DE, the same kind of proteins with diff

31、erent dyes from different samples can co-migrate. The Mixed Gel Scanned : with laser scanner to scan the mixed ran gel at different wavelengths according to CyDyeThe Result Read out : the position and the color of the protein spots on DIGE image, the position to represent the protein ID, the color,

32、according to standard color calibrator to infer each dye the proportion to the dye mix, to represent the relative amount of the same kind of protein among different samples.64650C x 30 minGE (2004) 2-D ElectrophoresisLysine Labeling (minimal labeling)In practice 400 pmol of dye is added to 50ug of p

33、rotein.In this way only 3-5% of the proteins will receive a tag (95-97% remain unlabeled)Labeling reaction: no IPG buffer, no reductant, pH 8.5, 37, 30 min with dye, the epsilon-amino side group of lysine is labeled with dye. 66Cysteine Labeling (saturation labeling)The high sensitivity, down to 100

34、0 human cells (around 2.5 ug protein) could provide good 2-DE Labeling reaction : no IPG buffer; TCEP reductant pH 8.0, 37 , 1h; dye added pH 8.0, 37 30 min; the thio group of cysteine labeled with dyes (Cy3 or Cy5).67Workflow for 2-DE of Proteins Labeled with CyDyes68Workflow for DIGE of Proteins L

35、abeled with CyDyesGE (2004) 2-D Electrophoresis69Proteins Labeled with CyDyesMix Labeled Proteins 2-DELaser Scanner with Different WavelengthsIndividual & Overlap Image Analysis (Protein Spot Location & Color)Identification of Protein ID & Abundance1st IEF (pI)2nd SDS(Mw)Example for DIGE Images Scan

36、ned with Difference WavelengthProteomics in Practice p69702-DE 凝膠蛋白質(zhì)斑點(diǎn)的檢測圖像掃描和分析Image Scanner II2-DE 凝膠蛋白質(zhì)斑點(diǎn)的檢測全自動(dòng)斑點(diǎn)切取系統(tǒng)（Ettan Spot Picker） 質(zhì)譜分析 ( MS)質(zhì)譜原理：樣本分子離子化后,根據(jù)不同離子間質(zhì)荷比(m/e )差異,分離樣本,確定分子量2-DE 凝膠蛋白質(zhì)斑點(diǎn)的檢測Principle for Mass Spectrometry74Schematic of Quadrupole TOF Hybrid AnalyzerWestermeier (200

37、8) Proteomics in Practice電噴霧質(zhì)譜樣品溶于固定的底物中形成晶體，用激光脈沖使其離子化，離子被加速后通過飛行管時(shí)分離，所有離子均可被檢測，常用來測蛋白質(zhì)、多肽、核酸和多糖等生物大分子MALDI-TOF MS通過質(zhì)譜分析，可以獲得分析樣品的分子量、分子式、分子中同位素構(gòu)成和分子結(jié)構(gòu)等多方面的信息SARS病毒N蛋白整體分子量 蛋白質(zhì)經(jīng)過酶解成肽段后，獲得所有肽段的分子質(zhì)量，形成一個(gè)特異的肽質(zhì)量指紋圖譜（peptide mass fingerprinting，PMF），通過數(shù)據(jù)庫搜索與比對(duì)，便可確定待分析蛋白質(zhì)分子的性質(zhì)。Peptide Mass Fingerprinting

38、 (PMF)Peptide Mass Fingerprinting (PMF)Peptide Mass Fingerprinting (PMF)用PMF方法未能鑒定的蛋白質(zhì)可通過質(zhì)譜技術(shù)獲得該蛋白質(zhì)一段或數(shù)段多肽的串連質(zhì)譜信息（氨基酸序列）并通過數(shù)據(jù)庫檢索來鑒定該蛋白質(zhì)?；旌系鞍踪|(zhì)酶解后的多肽混合物直接通過（多維）液相色譜分離，然后進(jìn)入質(zhì)譜進(jìn)行分析。用串聯(lián)質(zhì)譜（MS/MS）鑒定蛋白質(zhì)用串聯(lián)質(zhì)譜（MS/MS）鑒定蛋白質(zhì)多肽氨基酸序列分析串聯(lián)質(zhì)譜（Tandem-MS）,第一級(jí)質(zhì)譜得到肽的分子離子,選取目標(biāo)肽的離子作為母離子，與惰性氣體碰撞，使肽鏈中的肽鍵斷裂，形成一系列離子，即N端碎片離子系列（B

39、系列）和C端碎片離子系列（Y系列），將這些碎片離子系列綜合分析，可得出肽段的氨基酸序列?！皃rotein ladder sequencing”的方法，通過對(duì)Edman降解法的修改，產(chǎn)生一系列截去N端殘基的肽段，用MALDI-MS測得這些肽段的質(zhì)量，從而推測N端序列。 84The protonated total peptide mass is 1410.6.The peptide to be ionized into N-terminal ions (b-ions) and C-terminal ions (y-ions).The alignment of these ions of the

40、peptide from small to large according to their m/z, a unique mass spectrum for the peptide.The signal intensity (the height) of each ion represents the abundance of the fragment ion in system.Contrast of b-ions to y-ions could infer the peptide sequence.多肽氨基酸序列分析多肽氨基酸序列分析Isotope Labeling Protein Sam

41、ples via Metabolism Campbell (2002) Discovering p189 86Different Isotopes to label different samples via metabolism.Combine the samples at a ratio of one to one from diff sources and to subject to separation, and digestion.Analysis by MS to produce characteristic mass spectrum (1) at every m/z point

42、, two peaks always occurred in couple, representing the same molecule with diff mass isotopes : the front with light isotope and the following with heavy one.(2) at every m/z point, the ratio of two peaks to represent the same molecule relative abundance from diff sources.Isotope Tag ICAT for Labeli

43、ng of Protein Samples in vitro 87ICAT consists of three parts (i) biotin affinity tag, bind reversibly to avidin (ii) a linker, contain 8 stable isotopes (iii) thiol group, bind to cysteine of protein In ICAT molecule, if X groups present with H (=1), the ICAT is the light isotope tag.If X groups present with D (deuterium=2), the ICAT is the heavy isotope tag.The mass difference between H and D in ICAT could be discriminated by MS.88Workflow of Protein Tag