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1、Proteomics,蛋白质组学,Proteome&Proteomics 定义,Proteome：1994年，由澳大利亚Macguarie大学的Wilkins等首先提出：“蛋白质组指由一个细胞或一个组织的基因组所表达的全部蛋白质”“proteome”是由蛋白质一词的前几个字母“prote”和基因组一词的后几个字母“ome”拼接而成,Proteome&Proteomics 定义,Proteomics：蛋白质组学是从整体水平细胞内蛋白质的组成、结构、功能及其动态变化规律的科学。研究内容包括分析蛋白质组所有组分及它们的表达水平，确定各种组分的空间定位、修饰方法、互作机制、生物活性及相应特定功能等。由
2、此获得蛋白质水平上的关于疾病发生，细胞代谢等过程的整体而全面的认识,Genome&Proteome,Genomics Transcriptomics&Proteomics,Proteomics&Structural Genomics,Proteomics&Functional Genomics,特点之一整体性,特点之二系统性,特点之三动态性,Structural Proteomics 结构蛋白质组学Functional Proteomics 功能蛋白质组学,分类,Structural Proteomics,SeparationIdentificationPTM(post-translation
3、al modification)IdentificationComparison&Subtraction Analysis,Functional Proteomics,LocalizationProtein Complex DeterminationProtein-Protein Interaction/Interaction NetworkFunction of Protein(Metabolic and Regulatory Pathway;Signal Transduction Pathway),蛋白质鉴定：可以利用一维电泳和二维电泳并结合生物质谱、Western印迹、蛋白质芯片等技术，
4、对蛋白质进行全面的鉴定研究。翻译后修饰的鉴定：如磷酸化、糖基化、酶原激活等过程。蛋白质功能确定：包括蛋白质定位研究，蛋白质活性，蛋白质相互作用，酶活性和确定酶底物，细胞因子的生物分析，配基-受体结合分析等。,蛋白质组学的主要任务,蛋白质组学研究思路与方法,策略、技术、工具,主要专业术语及其英文对照和缩写,IPG-IEF：固相pH梯度等电聚焦（immobilized pH gradients isoelectric focusing）SDS-PAGE：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（Sodium dodecyl sulfate-polyacrylamide gel electrophores
5、is）2-DE：双向电泳(Two Dimensional Electrophoresis)HPLC：高效液相色谱（High performance Liquid Chromatography）MALDI-TOF MS：基质辅助激光解吸电离飞行时间质谱（Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry),蛋白序列数据库（SWISS-PROT/TrEMBL；http:/）基因序列数据库（Genbank，http:/EMBL；http:/）蛋白模式数据库（Prosite；http:/）蛋白质二维凝
6、胶电泳数据库、蛋白三维结构数据库（PDB，http:/；FSSP，）蛋白翻译后修饰数据库（O-GLYCBASE，http:/databases/OGLYCBASE）,蛋白质组学研究相关数据库,研究策略,两条互补的实验流程基于凝胶的工作流程（Gel-based workflow）基于液相色谱的工作流程（LC-based workflow）,Classical GE&LC to Protein ID Gapelo(2010)J Proteomics,HPLC-MS,蛋白质组研究的主要手段,双向电泳（two dimensional gel electrophoresis,2D-GE）差示电泳（dif
7、ferential gel electrophoresis,DIGE）毛细管电泳(capillary electrophoresis,CE)高效液相色谱(high performance liquid chromatography,HPLC)分子筛柱层析 反向柱层析质谱分析(mass spectrometry MS)基质辅助激光解吸离子化-飞行时间串联质谱(matrix-assisted laser disorption ionization-time of flight/tandem MS,MALDI-TOF/MS/MS)电喷雾离子化串联质谱（electrospray ionization
8、ESI-MS）生物信息学,蛋白质组学实验室所需的条件,蛋白质组学研究的基本技术路线,蛋白质样品的制备,双向电泳或HPLC,图像分析,凝胶中的蛋白,溶液中的蛋白,混合肽,蛋白质质量,N端测序,肽序列质谱数据,肽指纹图,数据搜索,新的或已知蛋白,翻译后修饰的鉴定,酶解,Sample Protein Preparation,Usually the complexity of the protein and/or peptide mixture lies beyond the theoretical separation space of any separation method.Proteome
9、Complexity.Genomic transcription in diversity;post-transcription processing;post-translational modification;constitution of combinatorial complex.It requires quite a long time for analysis of protein complex.Sample recovery low through separation:more steps,less overall recovery.The conc.of the prot
10、eins 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 housekeeping proteins million copies per cell).So,it is difficult to detect very low concentrated proteins in the prese
11、nce 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 parameters influence the composition of proteome between induced and inherent biological variations,such as geneti
12、c 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 replicates can deliver highly confident results.In clinical the number of required proteins are much high).Membrane proteins ar
13、e 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 phosphorylation and glycosylation require sophisticated analysis tools like MSn,where the peptide ions generated several times f
14、ragmented.,Sample Protein Preparation,蛋白质组研究的基本技术-样品预分离,样品的制备（预处理）：组织细胞细胞器（线粒体、叶绿体、细胞核）,蛋白质组研究的基本技术-蛋白提取,重要性：在制备时丢失的蛋白永远不能在后面实验中弥补原则：使所有待分析的蛋白样品全部处于溶解状态 防止样品在聚焦时发生蛋白的聚集和沉淀 防止在样品制备过程中发生样品的抽提后化学修饰（如酶性或化学性降解等）完全去除样品中的核酸和某些干扰蛋白,蛋白质组研究的基本技术-蛋白提取,步骤：破碎沉淀蛋白去除杂质,蛋白质组研究的基本技术-蛋白提取,样品制备流程-破碎尽可能减少蛋白水解/其它形式蛋白降解原
15、则机械法（超声波法、高压法、机械匀浆法）化学法（去污剂法、酶裂解法）物理法（液氮研磨法、反复冻融法、渗透法、玻璃珠破碎法）,蛋白质组研究的基本技术-蛋白提取,样品制备流程-沉淀蛋白 去杂浓缩后蛋白可溶性是关键 三氯醋酸（TCA）-丙酮沉淀法 TCA沉淀法 引起降解/修饰 丙酮沉淀法 硫酸铵沉淀法 影响IEF醋酸铵沉淀法 步骤繁琐,2D电泳结果影响因素分析,蛋白质组研究的基本技术-蛋白提取,样品制备流程-去除杂质 关键是尽量不丢失蛋白和减少蛋白修饰 核酸的清除（DNase/RNase）多糖的清除（超离心、TCA沉淀等）去污剂的清除（丙酮沉淀法等）盐离子和外源带电小分子的清除（透析、TCA-丙酮沉
16、淀法）,2D电泳结果影响因素分析,可能原因：样品含高丰度Pr,可能原因：TCA残留致使Pr丢失,蛋白质组研究的基本技术-蛋白提取,样品制备注意事项：蛋白质水解-蛋白酶抑制剂(PMSF等)特殊样品的制备(低丰度、强碱性蛋白质（核糖体）、极端分子量)样品定量 重复性,Two Dimensional Gel Electrophoresis(2D-GE)Differential Gel Electrophoresis(DIGE),Gel Electrophoresis,Workflow for Two-Dimensional Electrophoresis(2-DE)GE(2004)2-D Elect
17、rophoresis,two dimensional gel electrophoresis(2D-GE),Isoelectric Focusing Electrophoresis(IEF),IEF mainly applied for the following purposes:1st dimensional fractionation of protein mixtures in high-resolution 2-D electrophoresis Pre-fractionation of protein mixtures according to chargeFor the sepa
18、ration of very heterogeneous mixtures of tryptic peptides instead of strong cation exchange chromatography in MDLC-MS,IEF 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.En
19、vironmental pH:in basic buffer,the acidic groups of proteins with negative charge;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.,39,IEF Theoretical Background,40,The Principle of Isoelectric Focusing Electrophor
20、esis,IEF Principle,Ampholytes to Create A pH Gradient:A Heterogeneous Mixture of Isomers of aliphatic oligoamino-oligocarboxylic acids under electricity field could align themselves due to individual molecule trait protein-zero to be naive to move.,41,42,Immobilized pH Gradient Polyacrylamide,The Ge
21、neral Structure of Immobiline(Ampholyte),双向凝胶电泳（2-DE）,是等电聚焦电泳和SDS-PAGE的组合即先进行等电聚焦电泳（按照pI分离）然后再进行SDS-PAGE（按照分子大小）凝胶经染色得到二维分布的蛋白质图,蛋白质组研究的基本技术,2D-SDS-PAGE：样品制备第一向IPG-IEF电泳IPG平衡第二向SDS-PAGE电泳染色（银染）及 图谱分析目标蛋白获取及其鉴定（MC分析）,2-DE,第一向IPG-IEF电泳IEF是一种根据样品的等电点不同而使它们在pH梯度中相互分离的一种电泳技术将等电点不同的蛋白质混合物加入有pH梯度的凝胶介质中，在电场
22、内经过一定时间后，各组分将分别聚焦在各自等电点相应的pH位置上，形成分离的蛋白质区带从而得知其等电点信息,IPG-IEF电泳,2-DE,第二向垂直SDS-PAGE电泳聚丙烯酰胺凝胶形成网状结构，具有浓缩效应、电荷效应、分子筛效应。SDS与蛋白质形成雪茄状带负电荷复合物，从而消除了蛋白间的电荷及形状差异，电泳速度仅与分子量有关 从而得知其分子量信息,第二向垂直SDS-PAGE电泳,凝胶浓度与其对应的分离范围,胶浓度 分离范围(KD)5%36-200 7.5%24-200 10%14-200 12.5%14-100 15%14-60,第二向垂直SDS-PAGE电泳,Ettan Dalt twelv
23、e电泳系统,第二向垂直SDS-PAGE电泳,Ettan Dalt six电泳系统,2-DE 凝胶蛋白质斑点的检测,染色：1）考马斯亮兰染色法；2）银染法；3）负染法；4）荧光染色法；5）放射性同位素标记法等,安全（safety）灵敏（sensitivity）简单（simplicity）特异性（specificity）快速（speed）稳定（stability）兼容性（synergy）,理想显色剂的7S,有机染料和银染,考马斯亮蓝染色灵敏度为30100ng，线性范围是20倍；硝酸银染色的线性范围是40倍，灵敏度是考染的100倍。胶体考马斯亮蓝染色技术可实现PAGE的无背景染色，其极限灵敏度为81
24、0ng，但这种染液会对蛋白质进行修饰而影响质谱分析的结果。氨基黑常用于转印至聚偏二氟乙烯（PVDF）和/或硝酸纤维素膜上的蛋白质的染色。银染的缺点是：对某些种类的蛋白质染色效果差，对其后的蛋白质测序和质谱分析造成影响。这两类染色技术都可减少胶内蛋白质产量。,Coomassie Brilliant Blue&Silver Stain,负 染,能专门提高PAGE胶上蛋白质的回收率，但不能用于膜上染色。结果表现为胶面着色而蛋白质点透明。速度快（515min），蛋白质的生物活性能保持：一旦用络合剂如EDTA或Tris/甘氨酸转移缓冲液来络合金属离子就可进行提取来转移蛋白质。它主要适用于蛋白质显色、完整
25、蛋白质的胶上被动提取以及质谱分析。该技术主要包括金属盐染料、锌咪唑染料等的使用。,胶体扩散染料,主要用于高灵敏度检出电转印至硝酸纤维素和PVDF膜上的蛋白质，不用于胶内染色。最好的胶体金染色的灵敏度与PAGE胶内的银染类似。这种技术主要包括丽春红、印度墨水染料、胶体金染料等。,有机荧光团染料,包括共价结合和非共价结合的荧光团染料两类。后者最为常用，其典型代表是已经商品化的SYPRO Red、Orange、Ruby等荧光染料。这三种染料可对SDS-PAGE胶内蛋白质进行一步染色，约3060min完成，灵敏度为210ng。染色后的凝胶用标准的实验室300nm紫外透射仪进行照像保存，其线性范围为3个
26、数量级。这三种染料的电泳染色结果与在酵母中通过SAGE所获得的基因表达水平的动态范围相匹配。在Tris/甘氨酸转印缓冲液中染色后，蛋白质可被转印至膜上并进行免疫染色或Edman测序来鉴定蛋白质。,荧光染色,金属螯合染料,这是一类与现代蛋白质组学研究相兼容的、相对较新的蛋白质显色试剂，其设计专门与常用微量化学表征过程兼容。它们不包含戊二醛、甲醛或Tween-20等，很容易和集成化蛋白质组学平台（包括自动化凝胶染色仪、图像分析工作站、机器人剪切仪器、蛋白质酶解工作站和质谱仪等）相结合。其中SYPRO Ruby也是一种基于钌的金属发光染料。,同位素标记，放射自显影 灵敏度 20pg,2DE重复性,T
27、he same protocol and sample,however not the same resultsRec:similar;cir:different,DIGE&Proteins Labeled with CyDyes,Proteins 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 mix
28、ed ratio at 1:1,run on the same 2-DE,the same kind of proteins with different 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 o
29、n DIGE image,the position to represent the protein ID,the color,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.,64,65,Lysine Labeling(minimal labeling)In practice 400 pmol of
30、dye is added to 50ug of protein.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.,66,Cysteine Labeling(saturation labeling)The high sensitiv
31、ity,down to 1000 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).,67,Workflow for 2-DE of Proteins Labeled with CyDyes,68,Workflow for DIGE of Pro
32、teins Labeled with CyDyesGE(2004)2-D Electrophoresis,69,Example for DIGE Images Scanned with Difference WavelengthProteomics in Practice p69,70,2-DE 凝胶蛋白质斑点的检测,图像扫描和分析Image Scanner II,2-DE 凝胶蛋白质斑点的检测,全自动斑点切取系统（Ettan Spot Picker）,质谱分析(MS),质谱原理：样本分子离子化后,根据不同离子间质荷比(m/e)差异,分离样本,确定分子量,2-DE 凝胶蛋白质斑点的检测,Pri
33、nciple for Mass Spectrometry,74,Schematic of Quadrupole TOF Hybrid AnalyzerWestermeier(2008)Proteomics in Practice,电喷雾质谱,样品溶于固定的底物中形成晶体，用激光脉冲使其离子化，离子被加速后通过飞行管时分离，所有离子均可被检测，常用来测蛋白质、多肽、核酸和多糖等生物大分子,MALDI-TOF MS,通过质谱分析，可以获得分析样品的分子量、分子式、分子中同位素构成和分子结构等多方面的信息,SARS病毒N蛋白整体分子量,蛋白质经过酶解成肽段后，获得所有肽段的分子质量，形成一个特异的肽
34、质量指纹图谱（peptide mass fingerprinting，PMF），通过数据库搜索与比对，便可确定待分析蛋白质分子的性质。,Peptide Mass Fingerprinting(PMF),Peptide Mass Fingerprinting(PMF),Peptide Mass Fingerprinting(PMF),用PMF方法未能鉴定的蛋白质可通过质谱技术获得该蛋白质一段或数段多肽的串连质谱信息（氨基酸序列）并通过数据库检索来鉴定该蛋白质。混合蛋白质酶解后的多肽混合物直接通过（多维）液相色谱分离，然后进入质谱进行分析。,用串联质谱（MS/MS）鉴定蛋白质,用串联质谱（MS/M
35、S）鉴定蛋白质,多肽氨基酸序列分析,串联质谱（Tandem-MS）,第一级质谱得到肽的分子离子,选取目标肽的离子作为母离子，与惰性气体碰撞，使肽链中的肽键断裂，形成一系列离子，即N端碎片离子系列（B系列）和C端碎片离子系列（Y系列），将这些碎片离子系列综合分析，可得出肽段的氨基酸序列。“protein ladder sequencing”的方法，通过对Edman降解法的修改，产生一系列截去N端残基的肽段，用MALDI-MS测得这些肽段的质量，从而推测N端序列。,84,The protonated total peptide mass is 1410.6.The peptide to be io
36、nized into N-terminal ions(b-ions)and C-terminal ions(y-ions).The alignment of these ions of the 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-
37、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 Samples via Metabolism Campbell(2002)Discovering p189,86,Different Isotopes to label different samples via metabolism.Combine the samples at a ratio of one to one from diff sources and to subject to separat
38、ion,and digestion.Analysis by MS to produce characteristic mass spectrum(1)at every m/z point,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 repre
39、sent the same molecule relative abundance from diff sources.,Isotope Tag ICAT for Labeling of Protein Samples in vitro,87,ICAT 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
40、 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.,88,Workflow of Protein Tagged with ICAT for LC-MS Campbell(2002)Discovering,Proteins Labe
41、led with ICAT in vitro,Analyzed by MS Campbell(2002)Discovering p191,89,In vitro proteins from diff sources to be labeled with different ICAT,light and heavy mass.Combine the samples(the light with the heavy)and digest it.With affinity purification against ICAT to isolate protein fragments labeled w
42、ith ICAT.The ICAT-label fragments to be analyzed by MS,similar to that of labeled with N14 and N15.At a m/z point,there are two mass peaks,the same molecule,from different sources,labeled with the light and the heavy ICAT.The peak ratio represents the same molecule with relative abundance according
43、to diff sources.,90,Quantification and Identification Proteins by ICATCampbell(2002)Discovering,蛋白质功能研究技术,功能蛋白质组学,Techniques for Study of Protein-Protein Interactions,Protein MicroarrayPull down Immunoprecipitation（Co-IP）&Western BlottingY2H System(yeast two-hybrid system)Fluorescence Resonance Ener
44、gy Transfer through the protein complex(FRET),Pull down,Co-IP,Yeast two-hybrid system,蛋白质组在医学研究中的现状和前景,蛋白质组在医学研究中的现状和前景,人类疾病的蛋白质组研究直肠癌：Sanchez等对15例结肠癌和13例正常人的结肠上皮进行2-DE。建立了包括882和861个斑点的结肠癌及正常人结肠粘膜的标准胶图。结果发现在分子量为13kD和pI值为5.6处的蛋白质仅出现在结肠癌的组织中。经鉴定为：钙粒蛋白B（calgranulin B）及钙卫蛋白（calprotectin）,蛋白质组在医学研究中的现状和前景,致病微生物的蛋白质组研究 检测博氏疏螺旋体与免疫有关的蛋白质 弓形体抗原的检测白色念珠菌-对真菌细胞壁蛋白分析筛选抗药真菌药物,蛋白质组学研究趋势,蛋白质组学研究趋势,在应用研究方面蛋白质组学将成为寻找疾病分子标记和药物靶标最有效的方法之一 在技术发展方面研究方法将更强调各种方法间的整合和互补，以适应不同蛋白质的不同特征蛋白质组学与其它学科的交叉互动,
