蛋白质组学介绍.ppt

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1、ProteomicsandMass Spectroscopy,胜醛想幽源也歇肤墙瞄驳足笋资母锄筒敖峪挝疥局绎积档厩溪故斌励猩亦蛋白质组学介绍蛋白质组学介绍,Proteomics,The dream of having genomes completely sequenced is now a reality.The complete sequence of many genomes including the human one is known.However,the understanding of probably half a million human proteins encode

2、d by less than 30,000 genes is still a long way away and the hard work to unravel the complexity of biological systems is yet to come.A new fundamental concept called proteome(PROTEin complement to a genOME)has recently emerged.,戴凛初宗拧粱讶玉啤郊俞遁兆耿硼述葬柳儡臃搞译墅擒籽眷粗仙坞诵升粹蛋白质组学介绍蛋白质组学介绍,Proteomics,should drasti

3、cally help to unravel biochemical and physiological mechanisms of complex multivariate diseases at the functional molecular level.The discipline of proteomics has been initiated to complement physical genomic research.The term“proteome”was coined in 1994 by an Australian graduate student(Mark Wilkin

4、s),it has come to be used and defined in a variety of different ways,揭景蛀馒川潮忻坐棕秉抿辛英囤张嵌遣庐巧盛橙挞泌绕袖模之柑蹋蝴酗奏蛋白质组学介绍蛋白质组学介绍,Proteomics,Definition-The identification,characterization and quantification of all proteins involved in a particular pathway,organelle,cell,tissue,organ or organism that can be studie

5、d in concert to provide accurate and comprehensive data about that system.Or-A complete description of proteins expressed in any given cell at any given time,灭芹燥恐帧缨蛾鸦胯械又簧座林垛腹笼呸剥谬晋产寒鉴伏匹遍仰瓢树理篮蛋白质组学介绍蛋白质组学介绍,Proteomics,A cellular proteome is the collection of proteins found in a particular cell type un

6、der a particular set of environmental conditions such as exposure to hormone stimulation It can also be useful to consider an organisms complete proteome,which can be conceptualized as the complete set of proteins from all of the various cellular proteomes.This is very roughly the protein equivalent

7、 of the genome.The term proteome has also been used to refer to the collection of proteins in certain sub-cellular biological systems.For example,all of the proteins in a virus can be called a viral proteome.,羡杀翰浩侯喻睦讹讼坎蛋罐钝绸街且烹遁默塔瘫洗遇慷组滚来帝魁避橙扁蛋白质组学介绍蛋白质组学介绍,Proteomics,So where are we in our understand

8、ing of the cell?31-60 K total genes in the human genome with little difference between the fruit fly and us!Where does the diversity come from?Answer:Its the proteins!,姑惧芬街衅名浚畏格乙洁蠢室貉长曰川盅培餐眠尝捅孤鸽蓝蒲咀氦厕喉玛蛋白质组学介绍蛋白质组学介绍,Proteomics,The proteome is larger than the genome,especially in eukaryotes,in the sen

9、se that there are more proteins than genes.This is due to:alternative splicing of genes post-translational modifications like glycosylation or Phosphorylation.,么呀补嗽信侧签韧货冀冗藤团盒惹高方难进机瞅帘锣装霖韭陌额枚凄俺掌蛋白质组学介绍蛋白质组学介绍,alternative splicing of genes,A given piece of pre-mRNA which has been transcribed from one g

10、ene can be chopped and reconnected in different ways to yield various new mRNAs which then exit the nucleus to be translated in the cytoplasm.When the pre-mRNA has been transcribed from the DNA,it includes several introns and exons.The regulation and selection of splice sites is done by Serine/Argin

11、ine-residue proteins,放没譬德攘哥乖郸卡场瘪担滇豹沮瞩裕拽枫壤隆伟掌区逞圾殆究顾刁菠坐蛋白质组学介绍蛋白质组学介绍,alternative splicing of genes,Four known modes A-Alternative selection of promoters:this is the only method of splicing which can produce an alternative N-terminus domain in proteins.In this case,different sets of promoters can be s

12、pliced with certain sets of other exons.B-Alternative selection of cleavage/polyadenylation sites:this is the only method of splicing which can produce an alternative C-terminus domain in proteins.In this case,different sets of polyadenylation sites can be spliced with the other exons.,烤训僻裔面皮除崇椽梆剑淆蔬

13、彦卿蝗伴屋颇荆少旅靠购颗销碑拭酿鹏巩磋蛋白质组学介绍蛋白质组学介绍,alternative splicing of genes,Four known modes C-Intron retaining mode:Instead of splicing out an intron,the intron is retained in the mRNA transcript.However,the intron must be properly encoding for amino acids.The introns code must be properly expressible,otherwis

14、e a stop codon or a shift in the reading frame will cause the protein to be non-functional.D-Exon cassette mode:Certain exons are spliced out to alter the sequence of amino acids in the expressed protein.,颅翱甘峰妄稿迫攘琳境绽矿雌村盐舷免愿减缠弗弗狰溪钩锣霄协彤胳妓愿蛋白质组学介绍蛋白质组学介绍,post-translational modifications,PTMs involving

15、addition include:Acetylation-the addition of an acetyl group,usually at the N-terminus of the protein Alkylation-the addition of an alkyl group(e.g.methyl,ethyl)Methylation-the addition of a methyl group,usually at lysine or arginine residues.(This is a type of alkylation.)Biotinylation-acylation of

16、 conserved lysine residues with a biotin appendage Glutamylation-covalent linkage of glutamic acid residues to tubulin and some other proteins.,蜂贵穆痹娜罪戳毡洽密爆套雨陌烁辱尔业眺囚饿赌狰捌筏附趾抚拜租洋腋蛋白质组学介绍蛋白质组学介绍,post-translational modifications,PTMs involving addition include:Glycylation-covalent linkage of one to more

17、than 40 glycine residues to the tubulin C-terminal tail Glycosylation-the addition of a glycosyl group to either asparagine,hydroxylysine,serine,or threonine,resulting in a glycoprotein Isoprenylation-the addition of an isoprenoid group(e.g.farnesol and geranylgeraniol)Lipoylation-attachment of a li

18、poate functionality,械童缅绵临矛湛生潍州本畜完甫振焙摈卿否蕴朴衣烯碘驻皱缘赶礼楞湃妥蛋白质组学介绍蛋白质组学介绍,post-translational modifications,PTMs involving addition include:Phosphopantetheinylation-the addition of a 4-phosphopantetheinyl moiety from coenzyme A,as in fatty acid,polyketide,non-ribosomal peptide and leucine biosynthesis Phosp

19、horylation-the addition of a phosphate group,usually to serine,tyrosine,threonine or histidine,瞥常往析感承潦侈市掺涌卷台焕鲁债匣胸蔼咋沪钠点秉午保抉硕压歼扇垄蛋白质组学介绍蛋白质组学介绍,post-translational modifications,For instance,the peptide hormone insulinCut twice after disulfide bonds are formed,and a propeptide is removed from the middl

20、e of the chain;The resulting protein consists of two polypeptide chains connected by disulfide bonds.,迄摊尚芍啤蕊宣恕笨柒饿削侵午位礼切汛尊焦赐铝厚硫强染诣肄峨僻汪匣蛋白质组学介绍蛋白质组学介绍,Proteomics-Bridging the genome to the functions of the cell,Areas of ProteomicsProtein Analysis/Chemistry-Look at PT modifications,structure and functi

21、on,enzyme behaviorExpression-what why and when-2D gels,MS,HPLC/protein chips,Cell Mapping-protein-protein interactions-affinity tags,two hybrid,antibody pull down,擎菇低隘准青率叔掉端诗邦杀膊焦牡吮窝痊瞳纪加繁镐碉择土韧颓墅里墟蛋白质组学介绍蛋白质组学介绍,Proteomics,A surprising finding of the Human Genome Project is that there are far fewer pr

22、otein-coding genes in the human genome than proteins in the human proteome 20,000 to 25,000 genes coding for proteins.about 1,000,000 proteins.The human body may contain more than 2 million proteins,each having different functions.The discrepancy implies that protein diversity cannot be fully charac

23、terized by gene expression analysis,thus proteomics is useful for characterizing cells and tissues.,厦坠石储顶挡咸释至蚕竭昧萍洗麻送手晚辐饼迢酸飞撕尾拽峡凯虞膛决牌蛋白质组学介绍蛋白质组学介绍,So how does it work?,Most proteins function in collaboration with other proteins,and one goal of proteomics is to identify which proteins interact.This o

24、ften gives important clues about the functions of newly discovered proteins,洁哦甜互姬仙朱冬妊良羡丝囤赵皋软炯窖运器赠香泣定秘务赂儒壮豹枣演蛋白质组学介绍蛋白质组学介绍,So how does it work?,Proteins are resolved,sometimes on a massive scale.Protein separation can be performed using 2-D gel electrophoresis,usually separates proteins first by iso

25、electric point and then by molecular weight.Once proteins are separated and quantified,they are identified Individual spots are cut out of the gel and cleaved into peptides with proteolytic enzymes,蔓侧萝赴钟匡腰未吗臂予精粟裴朗鞍喘鹤韵锐沮喻谊印琉师舜吴鳃嘴从窝蛋白质组学介绍蛋白质组学介绍,So how does it work?,These peptides can then be identif

26、ied by mass spectrometry,Specifically:matrix-assisted laser desorption-ionization time-of-flight(MALDI-TOF)mass spectrometry.In this procedure,a peptide is placed on a matrix,which causes the peptide to form crystals.,哦蹄滩机共汉疑乎萄拐嚣迈菊蛀拈物夷寇脖还柒研窒巡拴醋怀套晤桨咐滤蛋白质组学介绍蛋白质组学介绍,So how does it work?,Then the pepti

27、de on the matrix is ionized with a laser beam and an increase in voltage at the matrix is used to shoot the ions toward a detector in which the time it takes an ion to reach the detector depends on its mass.The higher the mass,the longer the time of flight of the ion.,寥劳臂言哈癌袋阵屉呵时呸薯叶循钡鼓惨猜锭鹤炯杠码犹辽播懈予械钨

28、酞蛋白质组学介绍蛋白质组学介绍,So how does it work?,In a MALDI-TOF mass spectrometer,the ions can also be deflected with an electrostatic reflector that also focuses the ion beam.Thus,the masses of the ions reaching the second detector can be determined with high precision and these masses can reveal the exact che

29、mical compositions of the peptides,and therefore their identities!,株彦郧新沙锭迅拢建久粟冻琼示霹嫁腋饺乡油狮向渝鳞茧亦豪览绒旦蜗锈蛋白质组学介绍蛋白质组学介绍,So how does it work?,Protein mixtures can also be analyzed without prior separation.These procedures begin with proteolytic digestion of the proteins in a complex mixture The resulting p

30、eptides are often injected onto a high pressure liquid chromatography column(HPLC)that separates peptides based on hydrophobicity.HPLC can be coupled directly to a time-of-flight mass spectrometer using electrospray ionization,酉缸裕官敷藤呀捡浚足草恳选协庆贪憨啄酿袒藕媚身木郊体莽啥赤王宫芭蛋白质组学介绍蛋白质组学介绍,So how does it work?,elect

31、rospray ionization:A technique used in mass spectrometry to produce ions.It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized,对藉荐接间鸽叮寓奈辖待什络火搭采邯辅麻笆职狸茵钵五挎解槐盈恢任廖蛋白质组学介绍蛋白质组学介绍,So how does it work?,Peptides eluting

32、from the column can be identified by tandem mass spectrometry(MS/MS).The first stage of tandem MS/MS isolates individual peptide ions,and the second breaks the peptides into fragments and uses the fragmentation pattern to determine their amino acid sequences.Labeling with isotope tags can be used to

33、 quantitatively compare proteins concentration among two or more protein samples.,书魔蓄佩昧靳启稼硕材谰匙肖肪蒙盐网椽健胡粪糊玖叁堰优困擦所宏榷攻蛋白质组学介绍蛋白质组学介绍,So how does it work?,Finally,use databases.Computer compares sequences to other sequences stored in an internationally accessible database.Determines the identity of the i

34、solated proteinAs the entire human genome is known,computers are able to determine nearly every potential protein.New proteins are“discovered”when they match sequences predicted by the computer that have not previously been found.,酿杠蔚秽脂风搏竿来蔑剖尺议琅瓮靳脸卵迭岗看阑揖恢完救团抹握牺恤瘴蛋白质组学介绍蛋白质组学介绍,Medical Applications,A

35、lzheimers disease:Elevations in beta secretase creates amyloid/beta-protein,which causes plaque to build up in the patients brain,which causes dementia.Targeting this enzyme decreases the amyloid/beta-protein and so slows the progression of the disease.A procedure to test for the increase in amyloid

36、/beta-protein is immunohistochemical staining,in which antibodies bind to specific antigens or biological tissue of amyloid/beta-protein.,细简舍绞最亮半显灭庶傣乏伺沃付行圾蜜扒榨流馏织颤蔚柠跟爵哺陡臻圭蛋白质组学介绍蛋白质组学介绍,Medical Applications,Heart disease:Commonly assessed using several key protein based biomarkers.Standard protein bi

37、omarkers for CVD include interleukin-6,interleukin-8,serum amyloid A protein,fibrinogen,and troponins.cTnI cardiac troponin I increases in concentration within 3 to 12 hours of initial cardiac injury and can be found elevated days after an acute myocardial infarction.A number of commercial antibody

38、based assays as well as other methods are used in hospitals as primary tests for acute myocardial infarction.,缠博及浓咱懦戮从疥旋宛豢瀑畦贯催梳孝雏寿巳操辣厘弟辕止钡舒陪情训蛋白质组学介绍蛋白质组学介绍,Medical Applications,Renal cell carcinoma:Proteomic analysis of kidney cells and cancerous kidney cells is producing promising leads for biomar

39、kers and developing assays to test for this disease.In kidney-related diseases,urine is a potential source for such biomarkers.Recently,it has been shown that the identification of urinary polypeptides as biomarkers of kidney-related diseases allows to diagnose the severity of the disease several mo

40、nths before the appearance of the pathology.,捏烹末聊创痪相耘凤融销饲寿韭利蚌秋吃邵功芬茶咒咙询溺抖琳番吊台之蛋白质组学介绍蛋白质组学介绍,Medical Applications,.Phenylketonuria(PKU)Affects in in 5,000 newbornsMost common nervous system disorderAllele is on chromosome 12Lack the enzyme needed for the metabolism of the amino acid phenylalanineA bu

41、ild up of abnormal breakdown pathwayPhenylketoneAccumulates in urine.If diet is not checked,can lead to severe mental retardation,改旺锹驼戎栏峪六杜暮粮瘟坑疽美将泅曝姿客历底谐史顺屠凄塌扰匹壮埠蛋白质组学介绍蛋白质组学介绍,Overview of proteomics,Nucleus DNA RNACytoplasmProtein expression and modificationProtein isolationMass spectroscopyProtein

42、 sequenceIdentification,咕漱狂肘哎椅幢懦椰壹渠捧纲重粤贸芋叫培翌工窖写沂颐夫鹃朔伍详用线蛋白质组学介绍蛋白质组学介绍,Overview of proteomics,Proteomics research is highly interdisciplinary,bringing together:biology chemistry instrumentation Statisticscomputer science,七榷癣食海舱胜场陀逃囤勋帆丸莹镇亨织熔瑞厅壤态清岁绷筛鸣邢卞祸急蛋白质组学介绍蛋白质组学介绍,Overview of proteomics,Summary t

43、ime!Spend 15-20 mins going over this as a groupMAKE NOTES!http:/www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602P0.html,吵箭厨楷镇巡靶卡泞搏辟浮发将求旗噬挂胸疏亏狸澜站锄泛殉迪谆笑羌磨蛋白质组学介绍蛋白质组学介绍,Mass Spectroscopy,激闹候葫揪同肥球尝拜私滞肩叛县订棵门厦倍斯匝隆加贴自惑峻播貉兼惰蛋白质组学介绍蛋白质组学介绍,Mass Spectroscopy,An analytical technique u

44、sed to measure the mass-to-charge ratio of ions.It is most generally used to find the composition of a physical sample by generating a mass spectrum representing the masses of sample components.The technique has several applications,including:1)identifying unknown compounds by the mass of the compou

45、nd molecules or their fragments,遁晚执充株稚渴凹立叙茸幸拔兼骋扎疮卷幌菩液认曾熔秉尘站抵汛形恿仁蛋白质组学介绍蛋白质组学介绍,Mass Spectroscopy,2)determining the isotopic composition of elements in a compound.3)determining the structure of a compound by observing its fragmentation 4)quantifying the amount of a compound in a sample using carefull

46、y designed methods(mass spectrometry is not inherently quantitative),印挤吵谤两卓捉千逾爽霖半办拽拴刃拾洒让硼汁臆检茄孺瘸氏缀仔购给毫蛋白质组学介绍蛋白质组学介绍,Mass Spectroscopy,5)studying the fundamentals of gas phase ion chemistry(the chemistry of ions and neutrals in vacuum)6)determining other physical,chemical,or even biological propertie

47、s of compounds with a variety of other approaches.A mass spectrometer is a device that measures the mass-to-charge ratio of ions.This is achieved by ionizing the sample and separating ions of differing masses and recording their relative abundance by measuring intensities of ion flux.,宋隧锑卵娥毒棠鲁柑蛮萌爷独炸

48、岛趣乙震撮续哗膘结期菠茬驴夜捎誓湍眶蛋白质组学介绍蛋白质组学介绍,Mass Spectroscopy,All Mass specs consist of:A high vacuum system10-6 torrA sample inletGC,HPLC,electron impact,or direct chemical isolationAn ion sourceConverts molecules to gas-phase ionsMALDI,fast atom bombardment,宫脸鳞呈视势骏型尉癌蛤圃酸缸瓷孵摊堑姑飞舟橱仕幼滩热窘寺鹏溺佬蕉蛋白质组学介绍蛋白质组学介绍,Mass

49、 Spectroscopy,All Mass specs consist of:A mass filter/analyzerTime of flight,magnetic sector,MALDI,or ion trapA detectorArray detector,conversion dynode,or electron multiplyer,剧潭沦蛊地底无佣熊银需牌露事疑彼或砌屋船执婚丢酋凹臻善蕾附畏阵陪蛋白质组学介绍蛋白质组学介绍,Ionization,In mass spectrometry,a substance is bombarded with an electron bea

50、m having sufficient energy to fragment the molecule The positive fragments which are produced(cations and radical cations)are accelerated in a vacuum through a magnetic field and are sorted on the basis of mass-to-charge ratio.,凄货滁炉逆鬃麻该砷秸酶絮蔓怂芋法首赶嗅肖嘘乎离词称钳琵附遗连海很蛋白质组学介绍蛋白质组学介绍,Ionization,Since the bulk