1、第四講 疾病蛋白質(zhì)組學(xué)（一） disease proteomics,一、基本概念和總體研究概況,疾病蛋白質(zhì)組學(xué)disease proteomics,運(yùn)用蛋白質(zhì)組學(xué)研究手段，通過(guò)比較正常和病理情況下細(xì)胞、組織或體液中蛋白質(zhì)在組成成分、表達(dá)水平、表達(dá)位置和修飾狀態(tài)上的差異，尋找疾病診斷和預(yù)后的特異性蛋白質(zhì)(群)，包括特異性抗原及相關(guān)抗原、受體、酶等，以及藥物治療的靶標(biāo)等。通過(guò)深入了解這些疾病特異性蛋白質(zhì)的結(jié)構(gòu)和功能，揭示疾病過(guò)程中細(xì)胞內(nèi)全部蛋白質(zhì)的活動(dòng)規(guī)律，為多種疾病發(fā)生、發(fā)展機(jī)制的闡明和早期診斷及治療提供理論根據(jù)和解決途徑。,研究進(jìn)展,腫瘤蛋白質(zhì)組： 研究細(xì)胞的增殖、分化、異常轉(zhuǎn)化、腫瘤形成 白

2、血病、乳腺癌、結(jié)腸癌、膀胱癌、前列腺癌、肺癌、腎癌、肝細(xì)胞癌和神經(jīng)母細(xì)胞瘤等 聯(lián)合激光捕獲微切割技術(shù)(Laser capture mierodisseetion，LCM)，直接從腫瘤組織中提取純腫瘤細(xì)胞， 以克服組織內(nèi)異質(zhì)性的問(wèn)題 ，為腫瘤蛋白質(zhì)組研究提供了技術(shù)上的保障。 鑒定了一批腫瘤相關(guān)蛋白，為腫瘤的早期診斷、藥靶的發(fā)現(xiàn)、療效和預(yù)后的判斷提供了重要依據(jù)。 在心臟、肺部 、內(nèi)分泌系統(tǒng)、神經(jīng)系統(tǒng)疾病、藥物成癮性 、環(huán)境毒理學(xué) 、傳染病、內(nèi)耳相關(guān)疾病等方面，蛋白質(zhì)組研究成果也為其提供了新的診療方向。 國(guó)內(nèi)：重點(diǎn)在肝病、惡性腫瘤、心血管、神經(jīng)系統(tǒng)疾病和新發(fā)傳染病等方面,存在問(wèn)題和發(fā)展趨勢(shì),利用蛋白

3、質(zhì)組研究的人類疾病的范圍雖然日趨擴(kuò)大，但仍停留在初級(jí)比較階段。 進(jìn)一步鑒定、驗(yàn)證，發(fā)展成應(yīng)用于臨床的生物標(biāo)志物 開展全方位的蛋白質(zhì)組相互作用網(wǎng)絡(luò)的分析 進(jìn)一步提高蛋白分離和鑒定的通量、靈敏度和規(guī)模； 提高生物信息學(xué)應(yīng)用范圍與準(zhǔn)確率，進(jìn)行信息綜合，準(zhǔn)確地分析蛋白質(zhì)的相互作用，界定相互作用連鎖群；,二、心血管疾病蛋白質(zhì)組學(xué) Cardiovascular Proteomics,the cardiovascular (CV) system is composed of a number of specialized cell types including cardiac myocytes, fibr

4、oblast, neurons, endothelial and smooth muscle cells and newly discovered stem and progenitor cells. To date, the proteome of these cells are not well characterized nor has the interplay between the cell types been established in health or disease. This remains a significant challenge as CV disease

5、is the number one killer world wide.,Research Focus,The myofilament proteome. Redox modifications in the cardiac proteome. Cardiac biomarkers. Secretory microvesicles Proteomics of the secretome,The myofilament proteome,The myofilament （肌絲）proteins are responsible for the contractile nature of the c

6、ardiac myocytes. the myofilament subproteome allows the heart to act as a pump. The myofilament proteins are highly regulated by a number of specific post-translational modifications (PTMs) some of which have been discovered through proteomic studies. PTMs of myofilament proteins can directly impact

7、 on the contractility of the heart.,A simplified illustration of the cardiac myofilament proteins. The thick filament proteins consist of myosin heavy chain (MHC), myosin-binding protein C (MyBP-C), and two myosin light chains (MLC1 and MLC2). The thin filament proteins consist of actin, tropomyosin

8、 (Tm), and the three components of troponin; troponin I (TnI), troponin C (TnC) and troponin T (TnT). Phosphorylation sites on the myofilament proteins are indicated with a small diamond. The large scaffolding protein, titin, which spans the sarcomere, is not included in this illustration.,肌球蛋白重鏈(MH

9、C): myosin heavy chain 肌球蛋白輕鏈-1,2(MLC1,2): myosin light chain-1,2 肌動(dòng)蛋白：Actin 肌球蛋白結(jié)合蛋白C(MyBP-c): myosin binding protein C） 肌鈣蛋白(TnT, TnI, TnC): troponin T, I ，C -原肌球蛋白(Tm)： -tropomyosin 肌聯(lián)蛋白: titin,Structure of a region of the overlap region of a cardiac sarcomere in diastole on the left and during s

10、ystole on the right with indications of major and functionally significant protein phosphorylation sites.,Post-translational modifications of myofilament proteins,Sample preparation,There are two commonly used myofilament protein-enrichment strategies. Both methods are compatible with 1-DE and 2-DE

11、analysis： TFA (trifluoroacetic acid, 三氟醋酸) extraction ：cells are lysed with low ionic buffer, and myofilament proteins are extracted from the resulting pellet with 1% TFA v/v. applied to extract myofilament proteins from minute amounts (20,50 mg) of biopsy samples.（ref: Proteomics 2002, 2, 978987.)

12、Myofibril isolation：intact myofibrils can be isolated form detergent-skinned (detergent extraction) heart muscle and stored in 50% glycerol at -20 C. (ref: FASEB J. 2005, 19, 11371139.),Detection Methods for Protein modification,phosphorylation changes: 1-D-IEF (phosphorylation significantly decreas

13、es protein pI values) Western blots with phosphorylation-site-specific antibodies MS analysis: MALDI-TOF coupled with phosphatase treatment or Post source decay (PSD) immobilized metal affinity column (IMAC) enrichment and LC separation followed by MS/MS analysis,Immobilized metal affinity column (I

14、MAC),Schematic of affinity binding of phosphopeptides to immobilized metal ion affinity columns.,Detection Methods for Protein modification,Protein degradation: 1-D-gel separation followed by Western blot 2-DE, 2-D DIGE direct sequencing from the N terminus or MS (exact site of degradation) oxidatio

15、n and nitrosylation: gel electrophoresis(change apparent MWand pI values ) nano-ESI LC/MS/MS (identify nitrotyrosine residues) “top-down” MS (傅里葉轉(zhuǎn)換離子回旋共振質(zhì)譜）,文獻(xiàn)閱讀,Proteomics Clin. Appl. (2008) Chao Yuan, R. John Solaro. Myofilament proteins: From cardiac disorders to proteomic changes (p 788-799) Wen

16、hai Jin, Anna T. Brown, Anne M. Murphy. Cardiac myofilaments: from proteome to pathophysiology(p 800-810),2. Redox modifications in the cardiac proteome,Myocardial ischemia results in oxidative stress, which involves the mitochondria and many/all aspects of myocyte function. Due to the susceptibilit

17、y of cardiac protein to oxidative damage, proteomics can help to discover, quantify, and characterize the redox signaling and oxidative PTMs. Nitric oxide is a key mediator of CV cellular response in acute and chronic disease settings. New approaches in the proteomics can help identify and define im

18、portant pathway of nitric oxide-induced PTMs.,Outline of potential consequences of oxidative stress in cell system,Oxidants can react with proteins to cause one of two broad consequences. They can oxidise cellular components such as proteins, rendering them dysfunctional, which negatively affects ce

19、ll function and promotes disease. In this scenario, antioxidants can prevent the cellular proteins from being oxidised and so provide protection. In contrast, oxidants can induce regulatory post-translational oxidative protein modifications, which are important for stress adaptation. Thus, antioxida

20、nts can interfere with homeostatic control and might explain why antioxidant therapies can be detrimental in some cases.,Mechanisms of ROS generation. Sequential reduction of molecular oxygen to generate superoxide, hydrogen peroxide and then hydroxyl radical. List of amino acids particularly suscep

21、tible to modification.,Diagram showing the production of NO and RNS, with their effects on biological targets. At high concentrations, NO reacts mainly with oxygen superoxide forming peroxynitrite (ONOO) and peroxynitrous acid (ONOOH). In this way, NO is intimately linked with ROS. Moreover, the rea

22、ction of NO with O2 leads to the formation of the highly poisonous nitrogen dioxide (NO2), dinitrogen tetroxide (N2O4), or both. At low concentrations, the direct effects of NO predominate (dashed arrow) and haems and redox metals at ironsulphur centres in proteins are the main targets. Ni-NOR, nitr

23、ite:nitric oxide reductase; Ni, nitrite reductase; NOS, nitric oxide synthase; NR, nitrate reductase; RSNOs, S-nitrosothiols.,Structure of common redox modifications of amino acid side chains. ROS and RNS can chemically modify amino acids, particularly the side chains of those outlined here. Clearly

24、, cysteine thiols are subject to a diverse range of alterations.,亞磺酸,磺酸,次磺酸,亞砜,亞硝基硫醇,羰基化,硝基化酪氨酸,Commonly observed oxidative modifications of protein amino acids (A) cysteine; (B) methionine; (C) tyrosine; (D) tryptophan. All the amino acids are schematically represented as part of a polypeptide chai

25、n. However, the names shown are those of free amino acids for convenience.,List of the most utilized methods in redox proteomics,Biotin switch method A hypothetical protein is indicated with cysteines in either the free thiol, disulphide, or nitrosothiol conformations. In the first step, free thiols

26、 are blocked using MMTS. Next, nitrosylated cysteine residues are selectively reduced with ascorbate and the newly generated free thiols are finally S-biotinylated with biotin-HPDP.,The biotinylated proteins can be detected directly by Western blotting with antibodies specific for biotin or using av

27、idin or streptavidin. Antibodies can be radiolabelled, fluorescently or enzymatically labelled, as is known in the art. Additionally, tagged proteins can also be isolated from affinity columns or beads. PSH, protein sulphhydryl groups; PSNO, Snitrosated proteins.,Isotope Coded Affinity Tagging (ICAT

28、) (a). The reagent consists of three moieties: an affinity tag biotin, a linker that can incorporates stable isotopes, and a maleimide (順丁烯二酰亞胺) group which reacts specifically with the thiol group of cysteine. Two labelled forms of the reagent are used, the heavy containing eight deuteriums (氘)and

29、the light with none.,(b) Proteins from two different cell states are labelled with the light or heavy ICAT reagents. The samples are then combined and digested. The ICAT-labelled peptides are isolated by affinity chromatography using an avidin column and then analysed HPLC-MS (/MS) directly or by MA

30、LDI of the collected HPLC fractions. The ratio of the peaks areas for specific ICAT-labelled pairs defines the relative abundance of its parent proteins between the two cell states,quantification of protein cysteine oxidation,List of cardiac proteins demonstrated to undergo oxidative modification,Re

31、f: Proteomics Clin. Appl. 2008, 2, 823836,3. Cardiac biomarkers,Diagnosis of MI relies on the detection in serum of a cardiac specific isoform of the myofilament protein, troponin I which is released into the blood when the cardiac myocyte dies due severe ischemia Earlier detection of MI or diagnosi

32、s of myocardial ischemia prior to cell death will help to allow even earlier intervention to save “potentially viable” heart muscle. proteomic discovery pipeline for analysis of human plasma samples for patients with induced and control MI helped to set the stage for earlier detection of patents at

33、high risk.,Current gold standard markers of CV distress,(i) electrophysiological and functional changes as monitored by electrocardiography and echocardiography respectively (ii) elevated serum levels of cardiac specific proteins ： myofilament proteins and cardiac troponin-I and -T （myocardial infar

34、ction） brain natriuretic peptide and inflammation-related proteins, including C-reactive protein (CRP), （heart failure）. cardiac enzymes lactate dehydrogenase and creatine kinase (CK),Several approaches currently used to quantitativelyprofile global proteomic expression patterns,fluorescence 2-D DIG

35、E coupled to MS analysis Protein arrays in vitro and in vivo stable isotope label LC-MS techniques Significant cost of using labeled reagents in large-scale studies. the apparent bias of these techniques towards labeling the relatively most abundant species in a complex mixture, More recently, “l(fā)abe

36、l-free” differential (d)MS (無(wú)標(biāo)記的質(zhì)譜定量方法),Workflow for label-free dMS analysis of plasma samples. (A) Workflow chart. The six stages of the process are represented within this figure including sample preparation, addition of internal standards and MS analysis. Each stage plays an important role in lea

37、ding to a successful of determination of meaningful differentials.,4. Secretory microvesicles,Vascular secretory protein and membrane vesicles can affect homeostasis and communication within entire CV system in response to injury.,Schematic figure of the use of proteomics for the characterization of

38、 the non-cellular protein fractions relevant in atherosclerosis. The figure represents an atherosclerotic plaque and its cellular components. The cells involved in atheroma formation release soluble proteins and membrane bodies that modify the vascular microenvironment. Proteomics can be applied to

39、the characterization of these non-cellular components of the atherosclerotic microenvironment.,The limitations of plasma proteomics,plasma and serum are routinely used for biomarker discovery in proteomics. the high-abundance proteins, notably albumin and immunoglobulins, which together with haptogl

40、obulin, antitrypsin and transferrin, typically constitute more than 90% of the total protein mass in human plasma. prospective biomarkers: pgng/ml; albumin: 3550 mg/ml the limited ability of proteomics to detect low-abundance plasma proteins,Proteomics of extracellular secretoryvesicles,(3) Matrix v

41、esicles are extracellular membrane particles observed in the initial stages of arterial calcification and contain high levels of calcium-binding acidic phospholipids. (4) Apoptotic bodies are large particles released from cells at the later stages of programmed cell death and characterized by large

42、diameter, nuclear content, and surface ligands for phagocytic cell receptors. (5) Heterogeneous population or secretory microvesicles.,(1) Microparticles are released from the plasma membrane of stimulated or apoptotic cells. Their protein composition may vary in response to different stimuli (high

43、shear stress, apoptosis, etc.). (2) Exosomes are the smallest of the secretory membrane particles and are secreted as a consequence of the fusion of the plasma membrane with the multivesicular bodies (MVB). MVB are late components of the endocytic pathway.,The critical patho-physiological role of mi

44、croparticles,In the vascular context, microparticles are released by endothelial cells, smooth muscle cells, lymphocytes, monocytes, erythrocytes and platelets. Plasma levels of microparticles are markedly elevated in patients with vein thrombosis, acute coronary disease, ischemic stroke, diabetes,

45、myocardial infarction, and hypertension. Microparticles show pro-coagulant activity, pro-inflammatory, and pro-atherosclerotic activities. modulating the endothelial secretion of prostacyclin and nitric oxide; promote monocyte-endothelium interaction by direct transfer of arachidonic acid to the pla

46、sma membrane; physically mediate leukocyte-leukocyte and leukocyte-endothelium interactions via direct binding of cell surface receptors,Proteomics of microparticles,Proteomic analysis of protein expression in human plasma microparticles. Microparticles derived from the peripheral blood by centrifug

47、ation were lysed and labelled with Cydyes (green and red colour in A and B, respectively). Using DIGE, microparticle and microparticle-depleted plasma proteins were co-separated in large format 2-D gels. Images were acquired on a fluorescence scanner and proteins identified by LC-MS/MS. Actin and ha

48、emoglobin are enriched in microparticles, compared to microparticle-depleted plasma.,characterisation of microparticles released by a particular cell type in vitro by proteomics,Besides the investigation of the mixture of microparticles contained in human plasma, proteomics can be applied to the cha

49、racterisation of microparticles released by a particular cell type in vitro. platelet microparticles (J. Proteome Res. 2005; 4: 15161521) surface proteins typical of platelets, such as integrin aIIb, integrin b3 and P-selectin, and chemokines, such as CXCL4, CXCL7 and CCL5, 380 proteins not previous

50、ly identified in platelets Endothelial cells in response to stimulation with (TNFa). (Proteomics 2005; 5:44434455) cytoskeleton and cytoskeleton-binding proteins (tubulin, actin, cofilin, vimentin, etc.) membrane-associated proteins that control transport and signalling (caveolin, annexins, dynein,

51、etc.) foldingchaperones (calnexin, calreticulin, etc.) Adhesion molecules, such as ICAM-1 and integrins b1, a5 and a2,The role of Exosomes,modulate immune response regulate haemostatic balance support thrombin generation and induce expression and secretion of plasminogen activator inhibitor-1 by end

52、othelial cells attenuating fibrinolysis and promoting pro-thrombotic conditions ability to be absorbed to the cell surface and mediate cell-cell interactions in the cardiovascular system,Proteomics of exosomes,dendritic cell-derived exosomes (J. Immunol. 2001, 166, 73097318.) endocytic proteins were

53、 abundant components of the proteome of exosomes. 21 new exosomal proteins were identified, including cytoskeleton-related proteins, such as cofilin, profilin I or elongation factor 1a, and intracellular membrane transport proteins, such as annexins, rab7, 11, rap 1B, and syntenin. a series of apoptosis-related proteins, including thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. mast-cell derived exosomes (Arterioscler. Thromb.Vasc. Biol. 2005, 25, 17441749) regulate the secretion of plasminogen a