《疾病蛋白質(zhì)組學(xué)》ppt課件-文庫吧
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【正文】 Protein modification phosphorylation changes: – 1DIEF (phosphorylation significantly decreases protein pI values) – Western blots with phosphorylationsitespecific antibodies – MS analysis: ? MALDITOF 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: – 1Dgel separation followed by Western blot – 2DE, 2D DIGE – direct sequencing from the N terminus or MS (exact site of degradation) oxidation and nitrosylation: – gel electrophoresis(change apparent MWand pI values ) – nanoESI LC/MS/MS (identify nitrotyrosine residues) – “topdown” MS (傅里葉轉(zhuǎn)換離子回旋共振質(zhì)譜） 文獻(xiàn)閱讀 ? Proteomics Clin. Appl. (2022) – Chao Yuan, R. John Solaro. Myofilament proteins: From cardiac disorders to proteomic changes (p 788799) – Wenhai Jin, Anna T. Brown, Anne M. Murphy. Cardiac myofilaments: from proteome to pathophysiology (p 800810) 2. Redox modifications in the cardiac proteome ? Myocardial ischemia results 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 cellular response in acute and chronic disease settings. ? New approaches in the proteomics can help identify and define important pathway of nitric oxideinduced PTMs. Outline of potential consequences of oxidative stress in cell system ? Oxidants can react with proteins to cause one of two broad consequences. – They can oxidise cellular ponents 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 contrast, oxidants can induce regulatory posttranslational 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. (a) Mechanisms of ROS generation. Sequential reduction of molecular oxygen to generate superoxide, hydrogen peroxide and then hydroxyl radical. (b) 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 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 iron–sulphur centres in proteins are the main targets. NiNOR, nitrite:nitric oxide reductase。 Ni, nitrite reductase。 NOS, nitric oxide synthase。 NR, nitrate reductase。 RSNOs, Snitrosothiols. Structure of mon 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 alterations. 亞磺酸 磺酸 次磺酸 亞砜 亞硝基硫醇 羰基化 硝基化酪氨酸 Commonly observed oxidative modifications of protein amino acids (A) cysteine。 (B) methionine。 (C) tyrosi
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