疾病蛋白质组学ppt课件

1、第四讲第四讲 疾病蛋白质组学一疾病蛋白质组学一 disease proteomics一、根本概念和总体研讨概略一、根本概念和总体研讨概略疾病蛋白质组学疾病蛋白质组学disease proteomics 运用蛋白质组学研讨手段，经过比较正常和病理情况下细运用蛋白质组学研讨手段，经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、表达程度、表达位胞、组织或体液中蛋白质在组成成分、表达程度、表达位置和修饰形状上的差别，寻觅疾病诊断和预后的特异性蛋置和修饰形状上的差别，寻觅疾病诊断和预后的特异性蛋白质白质( (群群) )，包括特异性抗原及相关抗原、受体、酶等，以，包括特异性抗原及相关抗原、受体

2、、酶等，以及药物治疗的靶标等。经过深化了解这些疾病特异性蛋白及药物治疗的靶标等。经过深化了解这些疾病特异性蛋白质的构造和功能，提示疾病过程中细胞内全部蛋白质的活质的构造和功能，提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、开展机制的阐明和早期诊断及动规律，为多种疾病发生、开展机制的阐明和早期诊断及治疗提供实际根据和处理途径。治疗提供实际根据和处理途径。研讨进展研讨进展肿瘤蛋白质组：肿瘤蛋白质组：研讨细胞的增殖、分化、异常转化、肿瘤构成研讨细胞的增殖、分化、异常转化、肿瘤构成白血病、乳腺癌、结肠癌、膀胱癌、前列腺癌、肺癌、肾癌、肝细胞白血病、乳腺癌、结肠癌、膀胱癌、前列腺癌、肺癌、肾

3、癌、肝细胞癌和神经母细胞瘤等癌和神经母细胞瘤等结合激光捕获微切割技术结合激光捕获微切割技术(Laser capture mierodisseetion，LCM)，直，直接从肿瘤组织中提取纯肿瘤细胞，接从肿瘤组织中提取纯肿瘤细胞， 以抑制组织内异质性的问题以抑制组织内异质性的问题 ，为，为肿瘤蛋白质组研讨提供了技术上的保证。肿瘤蛋白质组研讨提供了技术上的保证。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、疗效和预后的判别提供了重要根据。预后的判别提供了重要根据。在心脏、肺部在心脏、肺部 、内分泌系统、神经系统疾病、药物成瘾性、内

4、分泌系统、神经系统疾病、药物成瘾性 、环境毒、环境毒理学理学 、传染病、内耳相关疾病等方面，蛋白质组研讨成果也为其提、传染病、内耳相关疾病等方面，蛋白质组研讨成果也为其提供了新的诊疗方向。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等方面存在问题和开展趋势存在问题和开展趋势 利用蛋白质组研讨的人类疾病的范围虽然日趋扩展，但利用蛋白质组研讨的人类疾病的范围虽然日趋扩展，但仍停留在初级比较阶段。仍停留在初级比较阶段。 进一步鉴定、验证，开展成运用于临床的生物标志物进一步鉴定、验证，开展成运用于临床的

5、生物标志物 开展全方位的蛋白质组相互作用网络的分析开展全方位的蛋白质组相互作用网络的分析 进一步提高蛋白分别和鉴定的通量、灵敏度和规模；进一步提高蛋白分别和鉴定的通量、灵敏度和规模； 提高生物信息学运用范围与准确率，进展信息综合，准提高生物信息学运用范围与准确率，进展信息综合，准确地分析蛋白质的相互作用，界定相互作用连锁群；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二、心血管疾病蛋白质组学 Cardiovascular Proteomics the cardiovascular (CV) system is composed of a number of speci

6、alized 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. T

7、his remains a significant challenge as CV disease is the number one killer world wide.Research FocusThe myofilament proteome.Redox modifications in the cardiac proteome.Cardiac biomarkers.Secretory microvesiclesProteomics of the secretomeThe myofilament proteome The myofilament 肌丝肌丝proteins are resp

8、onsible 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

9、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 filam

10、ent 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 no

11、t included in this illustration.肌球蛋白重链肌球蛋白重链(MHC):myosin heavy chain肌球蛋白轻链肌球蛋白轻链-1,2(MLC1,2): myosin light chain-1,2肌动蛋白：肌动蛋白：Actin肌球蛋白结合蛋白肌球蛋白结合蛋白C(MyBP-c): myosin binding protein C肌钙蛋白肌钙蛋白(TnT, TnI, TnC):troponin T, I ，C-原肌球蛋白原肌球蛋白(Tm)：-tropomyosin肌联蛋白肌联蛋白: titinStructure of a region of the overla

12、p 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 proteinsSample preparation There are two commonly used myofilament protein-enri

13、chment strategies. Both methods are compatible with 1-DE and 2-DE analysis： TFA (trifluoroacetic acid, 三氟醋酸三氟醋酸) 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 am

14、ounts (20,50 mg) of biopsy samples.ref: Proteomics 2019, 2, 978987.) 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. 2019, 19, 11.)Detection Methods for Protein modificationphosphoryl

15、ation 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 follo

16、wed by MS/MS analysisImmobilized metal affinity column (IMAC) Schematic of affinity binding of phosphopeptides to immobilized 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

17、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 (傅里叶转换离子盘旋共振质谱傅里叶转换离子盘旋共振质谱文献阅读文献阅读 Proteomics Clin. Appl. (2019) Chao Yuan, R. John Solaro. Myofilament protei

18、ns: 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 proteomeMyocardial ischemia results in oxidative stress, which involves the mitochondria and

19、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

20、 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 systemOxidants can react with proteins to cause one of two broad consequences. They can oxidise cellular components such as proteins

21、, 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,

22、 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

23、 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

24、 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 ironsulph

25、ur 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, part

26、icularly the side chains of those outlined here. Clearly, cysteine thiols are subject to a diverse range of alterations.亚磺酸亚磺酸磺酸磺酸次磺酸次磺酸亚砜亚砜 亚硝基硫醇亚硝基硫醇 羰基化羰基化 硝基化酪氨酸硝基化酪氨酸Commonly observed oxidative modifications of protein amino acids (A) cysteine; (B) methionine; (C) tyrosine; (D) tryptophan. All

27、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 proteomicsFrom: Journal of Experimental Botany, Vol. 59, No. 14, pp. 37813801, 2019Biotin switch method A hy

28、pothetical 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-bio

29、tinylated 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

30、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 (顺丁烯二酰亚胺顺丁烯二酰亚胺) gro

31、up which reacts specifically with the thiol group of cysteine. Two labelled forms of the reagent are used, the heavy containing eight deuteriums (氘氘)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

32、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 pro

33、teins between the two cell states quantification of protein cysteineoxidationList of cardiac proteins demonstrated to undergo oxidative modificationRef: Proteomics Clin. Appl. 2019, 2, 8238363. Cardiac biomarkers Diagnosis of MI relies on the detection in serum of a cardiac specific isoform of the m

34、yofilament 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

35、 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 echocardi

36、ography respectively (ii) elevated serum levels of cardiac specific proteins ：myofilament proteins and cardiac troponin-I and -T myocardial infarctionbrain natriuretic peptide and inflammation-related proteins, including C-reactive protein (CRP), heart failure.cardiac enzymes lactate dehydrogenase a

37、nd creatine kinase (CK)Several approaches currently used to quantitativelyprofile global 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 stud

38、ies. the apparent bias of these techniques towards labeling the relatively most abundant species in a complex mixture, More recently, “label-free differential (d)MS (无标志的无标志的质谱定量方法质谱定量方法)Workflow for label-free dMS analysis of plasma samples. (A) Workflow chart. The six stages of the process are rep

39、resented 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 microvesiclesVascular secretory protein and membrane vesicles can affect h

40、omeostasis 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 inv

41、olved 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 rou

42、tinely 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: pgng/ml; albumin:

43、 3550 mg/ml the limited ability of proteomics to detect low-abundance plasma proteinsProteomics 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 phosph

44、olipids. (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

45、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 plasm

46、a 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 are released by endothelial cells, smooth muscle cells, lymphocytes, monocytes, erythrocytes and platelets.

47、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 endotheli

48、al 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 receptorsProteomics of microparticle

49、sProteomic 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

50、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

51、 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 cell type in vitro.platelet microparticles (J. Proteome Res. 2019; 4: 15161521)surface proteins typical of pla

52、telets, 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 stimulation with (TNFa). (Proteomics 2019; 5:44434455)cytoskeleton and cytoskeleton-binding proteins (tubulin,

53、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 a2The role of Exosomes modulate immune response regulate ha

54、emostatic 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 cardiovasc

55、ular systemProteomics of exosomesdendritic cell-derived exosomes (J. Immunol. 2019, 166, 73097318.)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. B