疾病蛋白质组学.ppt

　　疾病蛋白质组学

　　疾病蛋白质组学PPT课件疾病蛋白质组学PPT课件

　　一、基本概念和总体研究概况

　　疾病蛋白质组学disease proteomics

　　运用蛋白质组学研究手段，通过比较正常和病理情况下细胞、组织或体液中蛋白质在组成成分、表达水平、表达位置和修饰状态上的差异，寻找疾病诊断和预后的特异性蛋白质(群)，包括特异性抗原及相关抗原、受体、酶等，以及药物治疗的靶标等。通过深入了解这些疾病特异性蛋白质的结构和功能，揭示疾病过程中细胞内全部蛋白质的活动规律，为多种疾病发生、发展机制的阐明和早期诊断及治疗提供理论根据和解决途径。

　　研究进展

　　肿瘤蛋白质组： 研究细胞的增殖、分化、异常转化、肿瘤形成 白血病、乳腺癌、结肠癌、膀胱癌、前列腺癌、肺癌、肾癌、肝细胞癌和神经母细胞瘤等 联合激光捕获微切割技术(Laser capture mierodisseetion，LCM)，直接从肿瘤组织中提取纯肿瘤细胞， 以克服组织内异质性的问题 ，为肿瘤蛋白质组研究提供了技术上的保障。 鉴定了一批肿瘤相关蛋白，为肿瘤的早期诊断、药靶的发现、疗效和预后的判断提供了重要依据。 在心脏、肺部 、内分泌系统、神经系统疾病、药物成瘾性 、环境毒理学 、传染病、内耳相关疾病等方面，蛋白质组研究成果也为其提供了新的诊疗方向。 国内：重点在肝病、恶性肿瘤、心血管、神经系统疾病和新发传染病等方面

　　存在问题和发展趋势

　　利用蛋白质组研究的人类疾病的范围虽然日趋扩大，但仍停留在初级比较阶段。 进一步鉴定、验证，发展成应用于临床的生物标志物 开展全方位的蛋白质组相互作用网络的分析 进一步提高蛋白分离和鉴定的通量、灵敏度和规模; 提高生物信息学应用范围与准确率，进行信息综合，准确地分析蛋白质的相互作用，界定相互作用连锁群;

　　二、心血管疾病蛋白质组学 Cardiovascular Proteomics

　　the cardiovascular (CV) system is composed of a number of specialized cell types including cardiac myocytes, fibroblast, 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 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 (肌丝)proteins are responsible for the contractile nature of the cardiac 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 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 (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.

　　肌球蛋白重链(MHC): myosin heavy chain 肌球蛋白轻链-1,2(MLC1,2): myosin light chain-1,2 肌动蛋白：Actin 肌球蛋白结合蛋白C(MyBP-c): myosin binding protein C) 肌钙蛋白(TnT, TnI, TnC): troponin T, I ，C ?-原肌球蛋白(Tm)： ?-tropomyosin 肌联蛋白: titin

　　Structure 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 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 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, 978–987.) 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, 1137–1139.)

　　Detection Methods for Protein modification

　　phosphorylation changes: 1-D-IEF (phosphorylation significantly decreases 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 (IMAC)

　　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) oxidation and nitrosylation: gel electrophoresis(change apparent MWand pI values ) nano-ESI LC/MS/MS (identify nitrotyrosine residues) “top-down” MS (傅里叶转换离子回旋共振质谱)

　　文献阅读

　　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 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 susceptibility 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 important 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 cell 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, antioxidants 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 susceptible 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 reaction 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 iron–sulphur centres in proteins are the main targets. Ni-NOR, nitrite: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, cysteine thiols are subject to a diverse range of alterations.

　　亚磺酸

　　磺酸

　　次磺酸

　　亚砜

　　亚硝基硫醇

　　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 chain. However, the names shown are those of free amino acids for convenience.

　　List of the most utilized methods in redox proteomics

　　(Gp:) From: Journal of Experimental Botany, Vol. 59, No. 14, pp. 3781–3801, 2008

　　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 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 avidin 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) (a). The reagent consists of three moieties: an affinity tag biotin, a linker that can incorporates stable isotopes, and a maleimide (顺丁烯二酰亚胺) 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) 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 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

　　Ref: Proteomics Clin. Appl. 2008, 2, 823–836

　　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 diagnosis 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 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 infarction) 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 quantitativelypro proteomic expression patterns

　　fluorescence 2-D DIGE 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, “label-free” differential (d)MS (无标记的质谱定量方法)

　　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 leading 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 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 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 haptoglobulin, antitrypsin and transferrin, typically constitute more than 90% of the total protein mass in human plasma. prospective biomarkers: pg~ng/ml; albumin: 35–50 mg/ml the limited ability of proteomics to detect low-abundance plasma proteins

　　Proteomics of extracellular secretoryvesicles

　　(3) Matrix vesicles 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 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 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 microparticles

　　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, 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 plasma 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 centrifugation 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 haemoglobin 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 characterisation of microparticles released by a particular cell type in vitro. platelet microparticles (J. Proteome Res. 2005; 4: 1516–1521) 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 platelets Endothelial cells in response to stimulation with (TNFa). (Proteomics 2005; 5:4443–4455) 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 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 endothelial 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, 7309–7318.) endocytic proteins were 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, 1744–1749) regulate the secretion of plasminogen activator inhibitor-1 by endothelial cells possibly by prothrombinase complex TNFa angiotensinogen precursors.

　　5. Proteomics of the secretome

　　“secretome” referred to the complex collection of proteins secreted by a particular type of cell - often maintained in vitro. the analysis of the secretome of different blood and vascular cell types could be of critical importance in the clarification of heterogeneous cell-cell interactions and their regulation by autocrine and paracrine factors. The limited complexity of the secretome makes it suitable for the application of a proteomic approach

　　Challenges to Proteomics of the secretome

　　It is difficult to completely avoid cross-contamination with proteins of the serum supplement commonly used in cell cultures, although the conditioned medium is usually sampled after extensive washing and during incubation in serum-free medium. Any variation in the carry-over of serum proteins has a profound impact on quantitative comparisons Cell death and cytoplasmic protein release in the culture medium is a source of false positives, which further impairs the reliability of proteomic analysis of the secretome.

　　Technical innovations to improve the capability of secretome analysis

　　protein-enrichment by precipitation (e.g. carrier-assisted TCA precipitation) [Proteomics 2007, 7: 1757–1770] high-abundance serum protein depletion (e.g. sodium chloride/ethanol precipitation) [Proteomics 2005, 5: 2656–2664]), LC fraction (e.g. RP tC2 Sorbent) [J. Proteome Res. 2006, 5: 899–906]) dialysis/ultrafiltration methods [J. Microbiol. Methods 2007, 68: 396–402].

　　SUMMARY

　　一、疾病蛋白质组学(disease proteomics)概念和总体研究概况 二、心血管疾病蛋白质组学 The myofilament proteome. Redox modifications in the cardiac proteome. Cardiac biomarkers. Secretory microvesicles Proteomics of the secretome

　　(Gp:) 谢谢!