2023年疾病蛋白质组学（教学课件）.ppt

第四讲第四讲 疾病蛋白质组学疾病蛋白质组学一一 disease proteomics 一、根本概念和总体研究概况一、根本概念和总体研究概况 疾病蛋白质组学疾病蛋白质组学 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、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、开展机制的说明和早期诊断及动规律，为多种疾病发生、开展机制的说明和早期诊断及治疗提供理论根据和解决途径。治疗提供理论根据和解决途径。研究进展研究进展 肿瘤蛋白质组：肿瘤蛋白质组：研究细胞的增殖、分化、异常转化、肿瘤形成研究细胞的增殖、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和神经母细胞瘤等 联合联合激光捕获微切割技术激光捕获微切割技术(Laser capture mierodisseetion，LCM)，直接从肿，直接从肿瘤组织中提取纯肿瘤细胞，瘤组织中提取纯肿瘤细胞，以克服组织内异质性的问题以克服组织内异质性的问题，为肿瘤蛋白质组研，为肿瘤蛋白质组研究提供了技术上的保障。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。判断提供了重要依据。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，蛋白质组研究成果也为其提、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。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 利用蛋白质组研究的人类疾病的范围虽然日趋扩大，但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。仍停留在初级比较阶段。进一步鉴定、验证，开展成应用于临床的生物标志物进一步鉴定、验证，开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 进一步提高蛋白别离和鉴定的通量、灵敏度和规模；进一步提高蛋白别离和鉴定的通量、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；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 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 1.The myofilament proteome.2.Redox modifications in the cardiac proteome.3.Cardiac biomarkers.4.Secretory microvesicles 5.Proteomics of the secretome 1.The myofilament proteome The myofilament 肌丝肌丝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: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 2002,2,978987.)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.2022,19,11371139.)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.(2