4疾病蛋白质组学-PPT文档资料

文献阅读
• Proteomics Clin. Appl. (2019)
– 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.
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。
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)
• PTMs of myofilaent proteins can directly impact on the contractility of the heart.
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
Detection Methods for Protein modification
phosphorylation changes:
– 1-D-IEF (phosphorylation significantly decreases protein pI values)
– Western blots with phosphorylation-site-specific antibodies
第四讲 疾病蛋白质组学（一） disease proteomics
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。
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.
Research Focus
1. The myofilament proteome. 2. Redox modifications in the cardiac
proteome. 3. Cardiac biomarkers. 4. Secretory microvesicles 5. Proteomics of the secretome
• A simplified illustration of the cardiac myofilament proteins. The thick filament proteins consist of myosin heavy chain (MHC), myosin-binding protein C (MyBPC), 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.
• The myofilament proteins are highly regulated by a number of specific post-translational modifications (PTMs) some of which have been discovered through proteomic studies.
– 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
1. 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.
Immobilized metal affinity column (IMAC)
Schematic of affinity binding of phosphopeptides to immobilized metal ion affinity columns.
Detection Methods for Protein modification
– 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 2019, 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. 2019, 19, 1137–1139.)
Post-translational modifications of myofilament proteins
Sample preparation
• There are two commonly used myofilament proteinenrichment strategies. Both methods are compatible with 1-DE and 2-DE analysis：
• 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.
• 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。