疾病蛋白质组学医学课件.ppt

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1、第四讲 疾病蛋白质组学（一） disease proteomics,第四讲 疾病蛋白质组学（一） disease pr,一、基本概念和总体研究概况,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

2、决途径。,疾病蛋白质组学disease proteomics运用蛋白,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

3、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,研究进展肿瘤蛋白质组：,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；,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,二、心血管疾病蛋白质组学 Cardiovascular Proteomics,the cardio

4、vascular (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 th

5、e cell types been established in health or disease. This remains a significant challenge as CV disease is the number one killer world wide.,二、心血管疾病蛋白质组学 Cardiovascular,Research Focus,The myofilament proteome.Redox modifications in the cardiac proteome.Cardiac biomarkers.Secretory microvesiclesProteo

6、mics of the secretome,Research FocusThe myofilament,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 sp

7、ecific 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.,The myofilament proteomeThe my,A simplified illustration of the cardiac myofilament proteins. The thick fil

8、ament 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). Phosphoryl

9、ation 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 prote

10、in C）肌钙蛋白(TnT, TnI, TnC):troponin T, I ，C-原肌球蛋白(Tm)：-tropomyosin肌联蛋白: titin,A simplified illustration of t,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 phospho

11、rylation sites.,Structure of a region of the o,Post-translational modifications of myofilament proteins,Post-translational modificatio,疾病蛋白质组学医学课件,Sample preparation,There are two commonly used myofilament protein-enrichment strategies. Both methods are compatible with 1-DE and 2-DE analysis：TFA (tr

12、ifluoroacetic 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 2019, 2, 978987.)Myofibril isolatio

13、n：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, 11371139.),Sample preparationThere are tw,Detection Methods for Protein modification,phosphorylation changes:1-D-IEF (phosphorylation significa

14、ntly 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,Detection Methods for Prot

15、ein,Immobilized metal affinity column (IMAC),Schematic of affinity binding of phosphopeptides to immobilized metal ion affinity columns.,Immobilized metal affinity col,Detection Methods for Protein modification,Protein degradation:1-D-gel separation followed by Western blot2-DE, 2-D DIGEdirect seque

16、ncing 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）,Detection Methods for Protein,文献阅读,Proteomics Clin. Appl. (2019)Chao Yuan, R. J

17、ohn 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),文献阅读Proteomics Clin. Appl. (20,2. Redox modifications in the cardiac proteome,Myocardial ischemia resu

18、lts 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

19、 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.,2. Redox modifications in the,Outline of potential consequences of oxidative stress in cell system,Oxidants can react with proteins

20、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

21、 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.,Outline of potential conseq

22、uen,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.,Mechanisms of ROS generation.,Diagram showing the production of NO and RNS, with their effects

23、 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 (NO

24、2), 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, nitr

25、ate reductase; RSNOs, S-nitrosothiols.,Diagram showing the production,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 alte

26、rations.,亚磺酸,磺酸,次磺酸,亚砜,亚硝基硫醇,羰基化,硝基化酪氨酸,Structure of common redox modi,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 sho

27、wn are those of free amino acids for convenience.,Commonly observed oxidative mo,List of the most utilized methods in redox proteomics,From: Journal of Experimental Botany, Vol. 59, No. 14, pp. 37813801, 2019,List of the most utilized meth,Biotin switch method A hypothetical protein is indicated wit

28、h 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 bio

29、tinylated 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 colu

30、mns or beads. PSH, protein sulphhydryl groups; PSNO, Snitrosated proteins.,Biotin switch method The biot,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 re

31、acts 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

32、. 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 betwee

33、n the two cell states,quantification of protein cysteineoxidation,Isotope Coded Affinity Tagging,List of cardiac proteins demonstrated to undergo oxidative modification,Ref: Proteomics Clin. Appl. 2019, 2, 823836,List of cardiac proteins demon,3. Cardiac biomarkers,Diagnosis of MI relies on the dete

34、ction 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 to cell death will help to allow even earlier intervention to save “

35、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.,3. Cardiac biomarkersDiagnosis,Current gold standard markers of CV distress,(i) electrop

36、hysiological 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, in

37、cluding C-reactive protein (CRP), （heart failure）.cardiac enzymes lactate dehydrogenase and creatine kinase (CK),Current gold standard markers,Several approaches currently used to quantitativelyprofile global proteomic expression patterns,fluorescence 2-D DIGE coupled to MS analysisProtein arraysin

38、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),Sever

39、al approaches currently u,疾病蛋白质组学医学课件,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 leadi

40、ng to a successful of determination of meaningful differentials.,Workflow for label-free dMS an,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 proteomic

41、s 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.

42、Proteomics can be applied to the characterization of these non-cellular components of the atherosclerotic microenvironment.,4. Secretory microvesiclesVasc,The limitations of plasma proteomics,plasma and serum are routinely used for biomarker discovery in proteomics.the high-abundance proteins, notab

43、ly 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: 3550 mg/mlthe limited ability of proteomics to detect low-abundance plasma proteins,T

44、he limitations of plasma prot,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 rele

45、ased 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 apo

46、ptotic 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 a

47、re late components of the endocytic pathway.,Proteomics of extracellular se,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 micr

48、oparticles 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 prost

49、acyclin 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,The critical patho-physiologic,Proteomics of mi

50、croparticles,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 pr