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1、proteomics and mass 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 understandin

2、g 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

3、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 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

4、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 proteomics Definition-The identific

5、ation, 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

6、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 under a particular set of environmental conditions such as exposure to hormone stimulation It can al

7、so 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 of the genome. The term “proteome“ has also been used to refer to the collection of proteins in

8、 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 understanding of the cell? 31-60 K total genes in the hu

9、man 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 th

10、e 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 genes A given pie

11、ce 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 regulatio

12、n 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 genes Four known modes A - Alternative selection of promoters: this is the only method of splicing which can

13、 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 pro

14、teins. 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 genes Four known modes C - Intron retaining mode: Instead of splicing out an intron, t

15、he intron is retained in the mRNA transcript. However, the intron must be properly encoding for amino acids. The introns 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 spl

16、iced 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 addition include: Acetylation - the addition of an acetyl group, usually at the N-termin

17、us 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 - coval

18、ent 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 than 40 glycine residu

19、es 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 fun

20、ctionality 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-ribos

21、omal 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 modifications For instance, the peptide hormon

22、e 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 proteomic

23、s - Bridging the genome to the functions of the cell Areas 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 h

24、ybrid, 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 protein-coding genes in the human genome than proteins in the human proteome 20,000 to 25,000

25、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

26、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 often gives important clues about the fun

27、ctions 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 firs

28、t 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

29、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

30、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 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 depen

31、ds 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? In a MALDI-TOF mass spectrometer, the ions can also be deflected with an electrostatic reflector that also f

32、ocuses 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

33、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 peptides are often injected onto a high pressure liquid chromatography column (HPLC) that separa

34、tes 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? electrospray ionization: A technique used in mass sp

35、ectrometry 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

36、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 use

37、d 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? Finally, use databases. Computer compares sequences to other sequences stored in an internationally a

38、ccessible 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

39、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 Alzheimers 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 amyloi

40、d/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

41、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 biomarkers for CVD include interleukin-6, interleukin-8, serum amyloid A protein, fibrinogen, and troponins. cTnI cardiac troponin I increases in

42、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

43、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 biomarkers and developing assays to test for this disease. In kidney-related diseases, urine is a

44、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

45、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 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

46、 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 sp

47、ectroscopy 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

48、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 time! Spend 15- 20 mins going over this as a group MAKE NOTES! http:/www.childrenshospital.org/cfapps/researc h/data_admin/Site602/mainpageS602P0.html 吵箭厨楷镇巡靶卡泞搏辟浮发将求旗噬挂胸

49、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 used to measure the mass-to-charge ratio of ions. It is most generally used to find the composition of a physical sample by genera

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