1、第四講第四講 疾病蛋白質(zhì)組學(xué)一疾病蛋白質(zhì)組學(xué)一 disease proteomics一、根本概念和總體研討概略一、根本概念和總體研討概略疾病蛋白質(zhì)組學(xué)疾病蛋白質(zhì)組學(xué)disease proteomics 運(yùn)用蛋白質(zhì)組學(xué)研討手段，經(jīng)過比較正常和病理情況下細(xì)運(yùn)用蛋白質(zhì)組學(xué)研討手段，經(jīng)過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)程度、表達(dá)位胞、組織或體液中蛋白質(zhì)在組成成分、表達(dá)程度、表達(dá)位置和修飾形狀上的差別，尋覓疾病診斷和預(yù)后的特異性蛋置和修飾形狀上的差別，尋覓疾病診斷和預(yù)后的特異性蛋白質(zhì)白質(zhì)( (群群) )，包括特異性抗原及相關(guān)抗原、受體、酶等，以，包括特異性抗原及相關(guān)抗原、受體

2、、酶等，以及藥物治療的靶標(biāo)等。經(jīng)過深化了解這些疾病特異性蛋白及藥物治療的靶標(biāo)等。經(jīng)過深化了解這些疾病特異性蛋白質(zhì)的構(gòu)造和功能，提示疾病過程中細(xì)胞內(nèi)全部蛋白質(zhì)的活質(zhì)的構(gòu)造和功能，提示疾病過程中細(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、開展機(jī)制的闡明和早期診斷及動(dòng)規(guī)律，為多種疾病發(fā)生、開展機(jī)制的闡明和早期診斷及治療提供實(shí)際根據(jù)和處理途徑。治療提供實(shí)際根據(jù)和處理途徑。研討進(jìn)展研討進(jìn)展腫瘤蛋白質(zhì)組：腫瘤蛋白質(zhì)組：研討細(xì)胞的增殖、分化、異常轉(zhuǎn)化、腫瘤構(gòu)成研討細(xì)胞的增殖、分化、異常轉(zhuǎn)化、腫瘤構(gòu)成白血病、乳腺癌、結(jié)腸癌、膀胱癌、前列腺癌、肺癌、腎癌、肝細(xì)胞白血病、乳腺癌、結(jié)腸癌、膀胱癌、前列腺癌、肺癌、腎

3、癌、肝細(xì)胞癌和神經(jīng)母細(xì)胞瘤等癌和神經(jīng)母細(xì)胞瘤等結(jié)合激光捕獲微切割技術(shù)結(jié)合激光捕獲微切割技術(shù)(Laser capture mierodisseetion，LCM)，直，直接從腫瘤組織中提取純腫瘤細(xì)胞，接從腫瘤組織中提取純腫瘤細(xì)胞， 以抑制組織內(nèi)異質(zhì)性的問題以抑制組織內(nèi)異質(zhì)性的問題 ，為，為腫瘤蛋白質(zhì)組研討提供了技術(shù)上的保證。腫瘤蛋白質(zhì)組研討提供了技術(shù)上的保證。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)后的判別提供了重要根據(jù)。預(yù)后的判別提供了重要根據(jù)。在心臟、肺部在心臟、肺部 、內(nèi)分泌系統(tǒng)、神經(jīng)系統(tǒng)疾病、藥物成癮性、內(nèi)

4、分泌系統(tǒng)、神經(jīng)系統(tǒng)疾病、藥物成癮性 、環(huán)境毒、環(huán)境毒理學(xué)理學(xué) 、傳染病、內(nèi)耳相關(guān)疾病等方面，蛋白質(zhì)組研討成果也為其提、傳染病、內(nèi)耳相關(guān)疾病等方面，蛋白質(zhì)組研討成果也為其提供了新的診療方向。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)傳染病等方面等方面存在問題和開展趨勢(shì)存在問題和開展趨勢(shì) 利用蛋白質(zhì)組研討的人類疾病的范圍雖然日趨擴(kuò)展，但利用蛋白質(zhì)組研討的人類疾病的范圍雖然日趨擴(kuò)展，但仍停留在初級(jí)比較階段。仍停留在初級(jí)比較階段。 進(jìn)一步鑒定、驗(yàn)證，開展成運(yùn)用于臨床的生物標(biāo)志物進(jìn)一步鑒定、驗(yàn)證，開展成運(yùn)用于臨床的

5、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)一步提高蛋白分別和鑒定的通量、靈敏度和規(guī)模；進(jìn)一步提高蛋白分別和鑒定的通量、靈敏度和規(guī)模； 提高生物信息學(xué)運(yùn)用范圍與準(zhǔn)確率，進(jìn)展信息綜合，準(zhǔn)提高生物信息學(xué)運(yùn)用范圍與準(zhǔn)確率，進(jìn)展信息綜合，準(zhǔn)確地分析蛋白質(zhì)的相互作用，界定相互作用連鎖群；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)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 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 肌絲肌絲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.肌球蛋白重鏈肌球蛋白重鏈(MHC):myosin heavy chain肌球蛋白輕鏈肌球蛋白輕鏈-1,2(MLC1,2): myosin light chain-1,2肌動(dòng)蛋白：肌動(dòng)蛋白：Actin肌球蛋白結(jié)合蛋白肌球蛋白結(jié)合蛋白C(MyBP-c): myosin binding protein C肌鈣蛋白肌鈣蛋白(TnT, TnI, TnC):troponin T, I ，C-原肌球蛋白原肌球蛋白(Tm)：-tropomyosin肌聯(lián)蛋白肌聯(lián)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 (傅里葉轉(zhuǎn)換離子盤旋共振質(zhì)譜傅里葉轉(zhuǎn)換離子盤旋共振質(zhì)譜文獻(xiàn)閱讀文獻(xiàn)閱讀 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.亞磺酸亞磺酸磺酸磺酸次磺酸次磺酸亞砜亞砜 亞硝基硫醇亞硝基硫醇 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 (順丁烯二酰亞胺順丁烯二酰亞胺) 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, “l(fā)abel-free differential (d)MS (無標(biāo)志的無標(biāo)志的質(zhì)譜定量方法質(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 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