蛋白质组学介绍.ppt

ProteomicsProteomicsandandMass SpectroscopyMass 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ProteomicsProteomics•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 encoded 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ProteomicsProteomics•should drastically 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 Wilkins), 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ProteomicsProteomics•Definition-The identification, characterization and quantification of all proteins involved in a particular pathway, organelle, cell, tissue, organ or organism that can be studied 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ProteomicsProteomics•A cellular proteome is the collection of proteins found in a particular cell type under a particular set of environmental conditions such as exposure to hormone stimulation •It can also be useful to consider an organism's 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 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ProteomicsProteomics•So where are we in our understanding 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: It’s 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ProteomicsProteomics•The proteome is larger than the genome, especially in eukaryotes, in the sense 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 genesalternative splicing of genes•A given piece of pre-mRNA which has been transcribed from one gene 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/Arginine-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 genesalternative 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 spliced 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 genesalternative 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 intron's code must be properly expressible, otherwise 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 modificationspost-translational modifications•PTMs involving 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 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 modificationspost-translational modifications•PTMs involving addition include:•Glycylation - covalent linkage of one to more 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 lipoate 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 modificationspost-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 •Phosphorylation - 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 modificationspost-translational modifications•For instance, the peptide hormone insulin•Cut twice after disulfide bonds are formed, and a propeptide is removed from the middle 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 - Proteomics - Bridging the genome to the functions of the Bridging the genome to the functions of the cellcellAreas of Proteomics•Protein Analysis/Chemistry - Look at PT modifications, structure and function, enzyme behavior•Expression - 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ProteomicsProteomics•A surprising finding of the Human Genome Project is that there are far fewer protein-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 characterized 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?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 often 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?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 isoelectric 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?So how does it work?•These peptides can then be identified 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?So how does it work?•Then the peptide 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酞蛋白质组学介绍蛋白质组学介绍So how does it work?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 chemical 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?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 peptides 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?So how does it work?•electrospray 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?So how does it work?•Peptides eluting 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 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?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 isolated protein•As 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 ApplicationsMedical ApplicationsAlzheimer’s disease:Elevations in beta secretase creates amyloid/beta-protein, which causes plaque to build up in the patient's 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/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 ApplicationsMedical ApplicationsHeart disease:Commonly assessed using several key protein based biomarkers. Standard protein biomarkers 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 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 ApplicationsMedical ApplicationsRenal cell carcinoma:Proteomic analysis of kidney cells and cancerous kidney cells is producing promising leads for biomarkers 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 months 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 ApplicationsMedical Applications. Phenylketonuria (PKU)–Affects in in 5,000 newborns–Most common nervous system disorder•Allele is on chromosome 12–Lack the enzyme needed for the metabolism of the amino acid phenylalanine–A build up of abnormal breakdown pathway•Phenylketone•Accumulates 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 – RNA•Cytoplasm–Protein expression and modification•Protein isolation•Mass spectroscopy•Protein sequence•Identification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 •Statistics•computer 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 proteomicsSummary time!•Spend 15- 20 mins going over this as a group•MAKE 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 SpectroscopyMass 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 SpectroscopyMass Spectroscopy•An analytical technique used 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 compound 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 SpectroscopyMass 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 carefully 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 SpectroscopyMass 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 properties 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 SpectroscopyMass Spectroscopy•All Mass specs consist of:•A high vacuum system–10-6 torr•A sample inlet–GC, HPLC, electron impact, or direct chemical isolation•An ion source–Converts molecules to gas-phase ions–MALDI, 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 SpectroscopyMass Spectroscopy•All Mass specs consist of:•A mass filter/ analyzer–Time of flight, magnetic sector, MALDI, or ion trap•A detector–Array 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IonizationIonization•In mass spectrometry, a substance is bombarded with an electron beam 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IonizationIonization•Since the bulk of the ions produced in the mass spectrometer carry a unit positive charge, the value m/e is equivalent to the molecular weight of the fragment.• The analysis of mass spectroscopy information involves the re-assembling of fragments, working backwards to generate the original molecule. 捧拄棋弃纸豌昨绵压疚互培决扇灌坞嫩狙讯勇演返剐贱澡虎孰鞠净霓超探蛋白质组学介绍蛋白质组学介绍IonizationIonization•Electron Impact ionization (EI):•It comprises an electron gun, a time-of-flight mass spectrometer with position-sensitive detector (PSD). •Analyte must be in a vapor state, limiting biological material below 400Da•Useful for metabolites, pollutants, and pharmaceutical compounds批访泣掩劲绍铱礼轻奈跨典厚墩潭轰阵私贱盔锗奢朵嗓疫财榷噪型斥谢援蛋白质组学介绍蛋白质组学介绍IonizationIonization•Chemical ionization:•Especially useful technique for organic chemists when no molecular ion is observed in EI mass spectrum •Ionization of sample (analyte) is achieved by interaction of its molecules with reagents such as CH4 or NH3•Very good for determining molecular mass– as high intensity molecular ions are produced owing to less fragmentation.枉烃涩泡蛔其次普宫衰恶淬拄蔑裔崔涡臃败垮慈矗顺丝杂骡邱拄玄辫摄腺蛋白质组学介绍蛋白质组学介绍IonizationIonization•electrospray 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•Is the primary ion source used in liquid chromatography-mass spec because it's a liquid-gas interface that is capable of coupling liquid chomatography with mass spectrometry 迈召抢咆望甄惟遍钒湛溉馋拧立溶城尚脐酣椭罪舟盏点止仆咸骏眩藏砸砖蛋白质组学介绍蛋白质组学介绍Mass AnalyzersMass Analyzers •Once ions are created and leave the ion source they pass into a mass analyzer•This separates the ions and measures their masses–What is actually measured is the mass to charge ratio (m/z) of each ion•At any given time, ions of a particular mass pass through and are counted by the detector–In this way, the analyzer scans through a large range of masses乙锤鸥墨谅嚼了捉忆擦槽劝葱斩爱仕泽吻臭枫嗡鄙郸饶辨滩密狈扎担蓟郴蛋白质组学介绍蛋白质组学介绍Mass AnalyzersMass Analyzers•Quadrupole mass spectrometry:•Essentially a mass filter that is capable of transmitting only the ion of choice. A mass spectrum is obtained by scanning through the mass range of interest over time.•Two opposite rods have an opposite applied potential and affect the trajectory of ions traveling down the flight path centered between the four rods梭琳套过帕芹浅宝又泛廖赘倚漠彤劳剧透丧送腻森鼻抒冤垫愁掖影坛博蛊蛋白质组学介绍蛋白质组学介绍Mass AnalyzersMass Analyzers•Quadrupole mass spectrometry:•The mass range of the oscillating ions is scanned by changing the DC voltage and the frequency.•The resolution of the spectrometer can be increased by either employing eight poles or by connecting two or three qaudupoles in series •Excel at applications where particular ions of interest are being studied because they can stay tuned on a single ion for extended periods宅侯码礼愉毋佯故鸯罐斑疽佑酵轿可酶拂碉宫驶夷帛茎蘸竟邑炕捂征龙那蛋白质组学介绍蛋白质组学介绍Mass AnalyzersMass Analyzers•Ion trap mass spectrometry:•The ion trap consists of three electrodes with hyperbolic surfaces, the central ring electrode, and two adjacent endcap electrodes• The device is radially symmetrical the electrodes are aligned and isolated using ceramic spacers and posts 伎纲示弧篇租玄络方纽淡契姻肚钥算问叉砧扣桌惕禽妈徊彤捞徊慰顽掉扯蛋白质组学介绍蛋白质组学介绍Mass AnalyzersMass Analyzers•Ion trap mass spectrometry:•Advantages:•(i) high sensitivity•(ii) compactness and mechanical simplicity in a device which is nevertheless capable of high performance•(iii) tandem mass spectrometry experiments are available by performing sequential mass analysis measurements•(iv) high resolution 兰琢凿腥增酬另级燎靛怯患遏鬃陛茅和浇湿颜究寡啃晰跋兼蚊鼠搬袱痛抗蛋白质组学介绍蛋白质组学介绍Mass AnalyzersMass Analyzers•Magnetic sector analyzer:•Generated ions are accelerated and are passed around a curved track (the sector) leading to a detector.•By increasing the magnetic field applied to the ions, heavier ions with higher momentum can be induced to follow the curved track.笼您谩屏浦靖奸赞担怯芥紧刑稽与邪拯撑阻垄扎恫捌贫握糟瑞葬邵耽藤麻蛋白质组学介绍蛋白质组学介绍Mass AnalyzersMass Analyzers•Magnetic sector analyzer:•Only ions of mass-to-charge ratio that have equal centripetal and centrifugal forces pass through the flight tube•The ions that reach the detector can be varied by changing either the magnetic field or the applied voltage of the ion optics.•So the individual ion beams are separated spatially and each has a unique radius of curvature according to its mass/charge ratio纬祭艘士谱绕漂尤缨咐荧奴财吐仕伎闷另捶欧迂丹寡聪弯递楷虞沙杭暇色蛋白质组学介绍蛋白质组学介绍Mass AnalyzersMass Analyzers•Plasma desorption ionization:•First available to analyze proteins and other large biomolecules (well, less than 35,000 Mr).•The technology is now relatively obsolete•Used radioactive californium (252Cf)•Required a time of flight (TOF) mass detector杭颖台嫁柠录中佑桓塞歼阅碾饿指伦栅硒腮芜客词授十煎坝酸腺恳锁郧嘶蛋白质组学介绍蛋白质组学介绍Mass AnalyzersMass Analyzers•matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. •Relatively novel technique in which a co-precipitate of an UV-light absorbing matrix and a biomolecule is irradiated by a nanosecond laser pulse,which causes the peptide to form crystals.•Most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule. •The ionized biomolecules are accelerated in an elctric field and enter the flight tube 捏谨旺岳名起盼条矗嵌棍庸罕譬猴威橡针虹缆嗅插殆掺殃恤刑藏专收李草蛋白质组学介绍蛋白质组学介绍Mass AnalyzersMass Analyzers•matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. •During the flight in this tube, different molecules are separated according to their mass to charge ratio and reach the detector at different times. •In this way each molecule yields a distinct signal. •It is a very sensitive method, which allows the detection of low (10-15 to 10-18 mole) quantities of sample with an accuracy of 0.1 - 0.01 %. 钢层砌擅榷涝爷稻缔何瘪犊侠斜仇铸惹脑伊谨妒鹤毙惋致伶曝嚷挎缘此婚蛋白质组学介绍蛋白质组学介绍Mass Analyzers - Mass Analyzers - MALDI-TOFMALDI-TOF•Protein identification by this technique has the advantage of short measuring time (few minutes) and negligible sample consumption (less than 1 pmol).•Additional information on post-translational modifications and presence of by-products! 菏账勺陵剂截秦换塞瑰赖愧席董莲秀敌钾苹访赞来氦响淋拳钢睛傍饰两敬蛋白质组学介绍蛋白质组学介绍Mass Analyzers - Mass Analyzers - MALDI-TOFMALDI-TOF•Drawbacks of Maldi-tof•The sample preparation for MALDI is important for the result. •Inorganic salts which are also part of protein extracts interfere with the ionization process. •The matrix protein mixture is not homogenous because the polarity difference leads to a separation of the two substances during crystallization 袍哩烩叹务需洗丸苛遂冕米蹈戚蠕召淮溯氢喷越韭呜喉孵敞日甜凋眨胆衰蛋白质组学介绍蛋白质组学介绍Mass Analyzers - Mass Analyzers - MALDI-TOFMALDI-TOF•The spot diameter of the target is much larger than that of the laser, which makes it necessary to do several laser shots at different places of the target, to get the statistical average of the substance concentration within the target spot. •Delay between laser pulses, delay time of the acceleration power, laser wavelength, energy density of the laser and the impact angle of the laser on the target are among others the critical values for the quality and reproducibility of the method. 源伦莫夺续硬棘姓足落闰租蔑腻涡娠瓤猎脆花颓横室泞迸蹦赛隶瑰翅鞘站蛋白质组学介绍蛋白质组学介绍DetectorDetector•The electron multiplier is a highly sensitive device to detect individual energetic particles such as electron, photons, or ions• Multipliers are based on two principles: •(1) the particle(s) to be detected have to be converted to electrons before the amplification can take place (using a so called conversion dynode) 鸡渠嘶辆履状冉阐幌嗣赂篙悠寂琴眉蛮口钟借咒愿壮柳尸开沃姚养椿证恬蛋白质组学介绍蛋白质组学介绍DetectorDetector•(2) the amplification is caused by a cascade of acceleration electrodes (called dynodes) which accelerate the electrons to speeds which allow them to generate more than one new electron when hitting the next dynode. 逾瓦蕴拌恩犁禁枢峙屯概捧晨微隶婿哀净麻俺摧诊丢腕喝街几上娥琴塔棘蛋白质组学介绍蛋白质组学介绍Protein identificationProtein identification•Peptide sequencing – a quick example.•A peptide and a protein digest of it were studied by Mass Spec•MALDI-TOF detected a peek at 3840.2 •Following HPLC-MS of the 3480.2 Da peak showed signals at m/z = 176, 624, 1129, 1508.•Looked at the 624 component•The ions appeared at m/z = 521, 406, 293, 130, and 43葱凸饶覆坑局骡妄囚廷潭如莉篡非墨柬梯咎乎腐烽缨碧池办辜吏札焰贺作蛋白质组学介绍蛋白质组学介绍Protein identificationProtein identification•Peptide sequencing – a quick example.•What is the molecular mass of the peptide? •176+624+1229+1508 = 3538 Da•But the peak was 3840.2!•Why the difference?惫馆知痊锐姓憋恿艳炮楞遥李湍梢邵抖挖雅摸煮菲灭咬次再材瓶康巩靖茅蛋白质组学介绍蛋白质组学介绍副宽彻乃兑捎磐耪粥寒趣齐嗜纲盗射咯肪隶逐痔烈停哮肃粪桌掳荣掂摈柬蛋白质组学介绍蛋白质组学介绍What is the molecular mass of the peptide?What is the molecular mass of the peptide?•Why the difference?•3538 – 3480.2 = 57.8 (58 Da) why a difference of 58 Da•There were four peaks following enzyme digest–Take into account enzymatic hydrolysis–Three cleavage points give four parts–Each requires the imput of one water molecule (H2O = 18)–18 X 3 =58–So peak is heavier than the actual peptide峪妒坏戴倘钒趣曹然酝尔停娶损约膝钝伸伊商乒终督浦判茶邦慎番泻暮叭蛋白质组学介绍蛋白质组学介绍What is the sequence of the 624 component?What is the sequence of the 624 component?僧包栏魏伤憎庄铱连计工笨奏孺琶占乖眨回都完丛藻眨合哑舟坟赤邹桑秃蛋白质组学介绍蛋白质组学介绍The Mass (DA) of the amino acidsThe Mass (DA) of the amino acidsSymbolStructureMass (Da)Ala A-NH.CH.(CH3).CO-71.0Arg R -NH.CH.[(CH2)3.NH.C(NH).NH2].CO-156.1Asn N -NH.CH.(CH2CONH2).CO-114.0Asp D -NH.CH.(CH2COOH).CO-115.0Cys C -NH.CH.(CH2SH).CO-103.0Gln Q -NH.CH.(CH2CH2CONH2).CO-128.1Glu E -NH.CH.(CH2CH2COOH).CO-129.0Gly G -NH.CH2.CO-57.0His H -NH.CH.(CH2C3H3N2).CO-137.1Ile I -NH.CH.[CH.(CH3)CH2.CH3].CO-113.1Leu-NH.CH.[CH2CH(CH3)2].CO-113.1Lys K -NH.CH.[(CH2)4NH2].CO-128.1Met M -NH.CH.[(CH2)2.SCH3].CO-131.0Phe F -NH.CH.(CH2Ph).CO-147.1Pro P -NH.(CH2)3.CH.CO-97.1Ser S -NH.CH.(CH2OH).CO-87.0Thr T -NH.CH.[CH(OH)CH3).CO-101.0Trp W -NH.CH.[CH2.C8H6N].CO-186.1Tyr Y -NH.CH.[(CH2).C6H4.OH].CO-163.1Val V -NH.CH.[CH(CH3)2].CO-99.1嫩仕癸瘪帧仕晕拦县盲旁价玖遮俏输别奔餐仙曼聋宙粟哄鹰陪望讥臭郧和蛋白质组学介绍蛋白质组学介绍Protein identificationProtein identification•What is the sequence of the 624 component?•m/z 624 521406 293130 43•▲ 103 115 115 16387•aa Cys Asp Asp TyrSer•The difference in the m/z valve gives you the identity of the corresponding amino acid•So the peptide sequence for the 625 component is:–Cys-Asp-Asp-Tyr-Ser均困莲妇膝浦绰售示津琴犊川住况摩厅娶节动抛合蛋寅客关才隅烯置创蒸蛋白质组学介绍蛋白质组学介绍THE END!THE END!判噎洼互党健洁幅弗洒索捉绸狗伸笨誓州煎告侗硫豺廓寓胆洽岗囊斌捆懒蛋白质组学介绍蛋白质组学介绍。