1、第四講 疾病蛋白質組學（一）一、基本概念和總體研究概況疾病蛋白質組學disease proteomics運用蛋白質組學研究手段，通過比較正常和病理情況下細胞、組織或體液中蛋白質在組成成分、表達水平、表達位置和修飾狀態上的差異，尋找疾病診斷和預后的特異性蛋白質(群)，包括特異性抗原及相關抗原、受體、酶等，以及藥物治療的靶標等。通過深入了解這些疾病特異性蛋白質的結構和功能，揭示疾病過程中細胞內全部蛋白質的活動規律，為多種疾病發生、發展機制的闡明和早期診斷及治療提供理論根據和解決途徑。研究進展腫瘤蛋白質組：研究細胞的增殖、分化、異常轉化、腫瘤形成白血病、乳腺癌、結腸癌、膀胱癌、前列腺癌、肺癌、腎癌、

2、肝細胞癌和神經母細胞瘤等聯合激光捕獲微切割技術(Laser capture mierodisseetion，LCM)，直接從腫瘤組織中提取純腫瘤細胞， 以克服組織內異質性的問題 ，為腫瘤蛋白質組研究提供了技術上的保障。鑒定了一批腫瘤相關蛋白，為腫瘤的早期診斷、藥靶的發現、療效和預后的判斷提供了重要依據。在心臟、肺部 、內分泌系統、神經系統疾病、藥物成癮性 、環境毒理學 、傳染病、內耳相關疾病等方面，蛋白質組研究成果也為其提供了新的診療方向。國內：重點在肝病、惡性腫瘤、心血管、神經系統疾病和新發傳染病等方面存在問題和發展趨勢利用蛋白質組研究的人類疾病的范圍雖然日趨擴大，但仍停留在初級比較階段。進

3、一步鑒定、驗證，發展成應用于臨床的生物標志物開展全方位的蛋白質組相互作用網絡的分析進一步提高蛋白分離和鑒定的通量、靈敏度和規模；提高生物信息學應用范圍與準確率，進行信息綜合，準確地分析蛋白質的相互作用，界定相互作用連鎖群；二、心血管疾病蛋白質組學 Cardiovascular Proteomicsthe cardiovascular (CV) system is composed of a number of specialized cell types including cardiac myocytes, fibroblast, neurons, endothelial and smoot

4、h 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 is the number one killer world wide.Re

5、search FocusThe myofilament proteome.Redox modifications in the cardiac proteome.Cardiac biomarkers.Secretory microvesiclesProteomics of the secretomeThe myofilament proteomeThe myofilament （肌絲）proteins are responsible for the contractile nature of the cardiac myocytes. the myofilament subproteome a

6、llows 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 on the contractility of the heart.A simplifie

7、d 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 (Tm), and the three components of troponin; tr

8、oponin 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(MHC):myosin heavy chain肌球蛋白輕鏈-1,2(MLC1,2): myosin

9、light chain-1,2肌動蛋白：Actin肌球蛋白結合蛋白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肌聯蛋白: titinStructure of a region of the overlap region of a cardiac sarcomere in diastole on the left and during systole on the right with indications of major and function

10、ally significant protein phosphorylation sites.Post-translational modifications of myofilament proteinsSample preparationThere are two commonly used myofilament protein-enrichment strategies. Both methods are compatible with 1-DE and 2-DE analysis：TFA (trifluoroacetic acid, 三氟醋酸) extraction ：cells a

11、re 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.)Myofibril isolation：intact myofibrils can be isolated form dete

12、rgent-skinned (detergent extraction) heart muscle and stored in 50% glycerol at -20 C. (ref: FASEB J. 2005, 19, 11371139.)Detection Methods for Protein modificationphosphorylation changes:1-D-IEF (phosphorylation significantly decreases protein pI values) Western blots with phosphorylation-site-spec

13、ific 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 analysisImmobilized metal affinity column (IMAC) Schematic of affinity binding of phosphopeptides to immobilized

14、metal ion affinity columns. Detection Methods for Protein modificationProtein degradation:1-D-gel separation followed by Western blot2-DE, 2-D DIGEdirect sequencing from the N terminus or MS (exact site of degradation)oxidation and nitrosylation:gel electrophoresis(change apparent MWand pI values )n

15、ano-ESI LC/MS/MS (identify nitrotyrosine residues)“top-down” MS (傅里葉轉換離子回旋共振質譜）文獻閱讀Proteomics Clin. Appl. (2008)Chao Yuan, R. John Solaro. Myofilament proteins: From cardiac disorders to proteomic changes (p 788-799)Wenhai Jin, Anna T. Brown, Anne M. Murphy. Cardiac myofilaments: from proteome to pa

16、thophysiology(p 800-810)2. Redox modifications in the cardiac proteomeMyocardial ischemia results in oxidative stress, which involves the mitochondria and many/all aspects of myocyte function. Due to the susceptibility of cardiac protein to oxidative damage, proteomics can help to discover, quantify

17、, 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 important pathway of nitric oxide-induced PTMs.Outline of potential consequences of ox

18、idative stress in cell systemOxidants 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 cell function and promotes disease. In this scenario, antioxidants can prevent the cellu

19、lar 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, antioxidants can interfere with homeostatic control and might explain why antioxidant therapies

20、 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 susceptible to modification.Diagram showing the production of NO and RNS, with their effects

21、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 reaction of NO with O2 leads to the formation of the highly poisonous nitrogen dioxide (NO2

22、), 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, nitrite:nitric oxide reductase; Ni, nitrite reductase; NOS, nitric oxide synthase; NR, nitra

23、te 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, 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

24、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 chain. However, the names shown are those of free amino acids for convenience.List of the most uti

25、lized methods in redox proteomicsFrom: Journal of Experimental Botany, Vol. 59, No. 14, pp. 37813801, 2008Biotin switch method A hypothetical protein is indicated with cysteines in either the free thiol, disulphide, or nitrosothiol conformations. In the first step, free thiols are blocked using MMTS

26、. 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 avidin or streptavidin. A

27、ntibodies 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) (a). The reagent consi

28、sts 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 the light with none.(b)

29、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 MALDI of the collected HPLC

30、 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 cysteineoxidationList of cardiac proteins demonstrated to undergo oxidative modificationRef: Proteomics Clin. Appl. 20

31、08, 2, 8238363. Cardiac biomarkersDiagnosis 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 ischemiaEarlier detection of MI or diagnosis of myocardial ischemia prior

32、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 high risk.Current gold standard

33、 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 infarction）brain natriuretic peptide a

34、nd 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 patternsfluorescence 2-D DIGE coupled to MS analysisProtein array

35、sin 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, “label-free” differential (d)MS (無標記的質譜定量方法)Wo

36、rkflow 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 leading to a successful of determination of m

37、eaningful differentials.4. Secretory microvesiclesVascular 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 the non-cellular protein fractions relevant

38、 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 the characterization of these non-cellular c

39、omponents of the atherosclerotic microenvironment.The limitations of plasma proteomicsplasma and serum are routinely used for biomarker discovery in proteomics.the high-abundance proteins, notably albumin and immunoglobulins, which together with haptoglobulin, antitrypsin and transferrin, typically

40、constitute more than 90% of the total protein mass in human spective biomarkers: pgng/ml; albumin: 3550 mg/mlthe limited ability of proteomics to detect low-abundance plasma proteinsProteomics of extracellular secretoryvesicles(3) Matrix vesicles are extracellular membrane particles observ

41、ed 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 diameter, nuclear content, and surface ligands for

42、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 shear stress, apoptosis, etc.). (2) Exosomes are th

43、e 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 microparticlesIn the vascular context, microparticles

44、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, myocardial infarction, and hypertension.Microparticles

45、 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 plasma membrane; physically mediate leukocyte-leukocyte and

46、 leukocyte-endothelium interactions via direct binding of cell surface receptorsProteomics of microparticlesProteomic analysis of protein expression in human plasma microparticles. Microparticles derived from the peripheral blood by centrifugation were lysed and labelled with Cydyes (green and red c

47、olour 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 haemoglobin are enriched in microparticles, compared to micr

48、oparticle-depleted plasma.characterisation of microparticles released by a particular cell type in vitro by proteomicsBesides the investigation of the mixture of microparticles contained in human plasma, proteomics can be applied to the characterisation of microparticles released by a particular cel

49、l 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 previously identified in plateletsEndothelial cells in response to stim

50、ulation 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, etc.) foldingchaperones (calnexin, calreticulin, etc.)Adhesion mol

51、ecules, such as ICAM-1 and integrins b1, a5 and a2The role of Exosomesmodulate immune responseregulate haemostatic balancesupport thrombin generation and induce expression and secretion of plasminogen activator inhibitor-1 by endothelial cellsattenuating fibrinolysis and promoting pro-thrombotic con

52、ditionsability to be absorbed to the cell surface and mediate cell-cell interactions in the cardiovascular systemProteomics of exosomesdendritic cell-derived exosomes (J. Immunol. 2001, 166, 73097318.)endocytic proteins were abundant components of the proteome of exosomes. 21 new exosomal proteins w

53、ere 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 p