Core A-PROTEOMIC CORE

核心A-蛋白质组核心

• 批准号: 7644320
• 负责人: Thomas M. Vondriska
• 金额: $ 45.41万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7644320
• 关键词: Accounting; Acetonitriles; Affinity; Affinity Chromatography; Aliquot; Alkylation; Aminocaproic Acids; Ampholytes; Antibodies; Area; Binding; Biochemical; Biochemistry; Bioinformatics; Biological Assay; Biology; Bistris; Blood capillaries; Boxing; Buffers; Carboxylic Acids; Cardiac; Cells; Centrifugation; Charge; Citric Acid Cycle; Collaborations; Column Chromatography; Commit; Complement; Complex; Complex Mixtures; Computer software; Condition; Confocal Microscopy; Control Groups; Cyclotrons; Cytochromes; Cytosol; Data; Databases; Detergents; Digestion; Dimensions; Dust; Edetic Acid; Electrophoresis; Electrospray Ionization; End Point; Endopeptidases; Ensure; Enzymes; Epitopes; Esters; Event; Excision; Ferritin; Formic Acids; Fourier Transform; Fractionation; Future; Gel; Gel Chromatography; Glutathione S-Transferase; Glycerol; Heart; High Pressure Liquid Chromatography; Housing; Human; Image; Immunoblotting; Immunoglobulin G; Immunoprecipitation; Incubated; Individual; Infusion procedures; Injection of therapeutic agent; Inner mitochondrial membrane; Investigation; Iodoacetamide; Ions; Isocitrate Dehydrogenase; Isoelectric Focusing; Jupiter; Laboratories; Laemmli buffer; Left; MAPK14 gene; Maps; Mass Spectrum Analysis; Measurement; Membrane; Membrane Proteins; Metals; Methodology; Methods; Minor; Mitochondria; Mitochondrial Membrane Protein; Mitochondrial Proteins; Modeling; Modification; Molecular; Molecular Weight; Monoclonal Antibodies; Multiprotein Complexes; Mus; Myocardial; Myocardial Ischemia; Myocardium; Nuclear; Numbers; Organelles; Outer Mitochondrial Membrane; Parents; Pathology; Pattern; Peptide Hydrolases; Peptide Mapping; Peptides; Phosphopeptides; Phosphoproteins; Phosphorylated Peptide; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Preparation; Principal Investigator; Procedures; Process; Promega; Property; Protease Inhibitor; Protein Analysis; Protein Databases; Proteins; Proteome; Proteomics; Protocols documentation; Pump; Range; Rate; Recombinant Proteins; Regulation; Relative (related person); Reproducibility; Research Personnel; Resolution; Resources; Respiratory Chain; Role; Room, Clean; Running; SLC25A4 gene; Sampling; Scanning; Score; Serum; Services; Signal Transduction; Silver; Silver Staining; Sodium Dodecyl Sulfate-PAGE; Solutions; Sonication; Source; Speed; Spottings; Staining method; Stains; Standards of Weights and Measures; Statistical Methods; Stream; Subcellular Fractions; Sucrose; Sum; Surveys; Syringes; System; Techniques; Technology; Temperature; Testing; Text; Thiourea; Thyroglobulin; Time; Trypsin; Ubiquitin; Ubiquitin C; United States Food and Drug Administration; Urea; VDAC1 gene; Validation; Vent; Voltage-Dependent Anion Channel; Western Blotting; Work; Yang; ammonium bicarbonate; austin; base; capillary; catalase; coomassie Brilliant Blue; data integration; desire; experience; formic acid; gel electrophoresis; genetic manipulation; in vivo; inhibitor/antagonist; instrument; mass spectrometer; member; microbial alkaline proteinase inhibitor; mitochondrial membrane; nano; nanoscale; novel; polyacrylamide gels; pressure; prevent; programs; protein protein interaction; reconstitution; research study; two-dimensional; voltage

Having identified the p38a subproteome, we will next undertake analysis of phosphoproteins within this subproteome using a combination of 1DE/2DE, IMAC and selective ion scanning mass spectrometry methods. Specifically, p38a-associated proteins will be isolated by Flag pull-down, separated by electrophoresis, and digested with trypsin. These tryptic peptides will either be subjected directly to precursor ion/neutral loss scan analysis with mass spectrometry, or will first undergo a phosphopeptide enrichment step using IMAC. Putative phosphoprotein identifications will be verified by western immunoblotting. Detailed procedures for IMAC and precursor/neutral loss ion scanning are listed below. II.D. Collaboration with Project 4 The general approach to characterize the Cdk2 subproteome will proceed via three steps (parallel to the analysis of p38a described above). First we will identify members of the subproteome by immunoprecipitation, electrophoresis and LC/MS/MS. Second, we will determine quantitative changes in this subproteome in the ischemic heart using DIGE and ICAT technologies. Third, we will examine phosphorylated members of this subproteome using IMAC and selective ion scanning methods. Aim 1 of Project 4 will examine proteins associated with Cdk2 in the normal and ischemic myocardium using a combination of immunoprecipitation (IP), 1DE/2DE, and LC/MS/MS. The mitochondrial fraction will be resuspended in sucrose buffer, sonicated briefly, and treated with 0.1% DDM (see Sample Preparation below for specific methodology). The mixture will be centrifuged again to pellet mitochondrial membranes, and released mitochondrial proteins in the supernatant will be used for IP. Myocardial cytosolic or mitochondrial fractions will be pre-cleared with protein A/G beads and Cdk2-associated proteins will be isolated by IP with anti-Cdk2 monoclonal antibodies (Santa Cruz, sc-6248). IgG will be used in place of anti-Cdk2 antibody as a negative control. Cdk2- associated proteins, retained on the beads after washing to remove non-specific interactions, will be eluted from the beads and separated by 1DE. Proteins will be excised from the gel, trypsinized and subjected to LC/MS/MS analysis. [Note: The members of Project 4 have worked closely with the Proteomic Core to optimize the purification of Cdk2 complexes by IP and the running of 1DE/2DE gels and will continue to do so for the duration of these studies.] Together, these techniques will provide the first map of the Cdk2 subproteome in the heart. Detailed procedures for 1DE and LC/MS/MS are described below. See Figure 2 for a general overview of the experimental strategy for Projects 3 and 4. Like p38a, Cdk2 is a soluble kinase, and thus we may be able to gain significant information from 2DE separation of Cdk2 complexes, especially those isolated from the cytosol. Also analogous to p38a, we will implement DIGE and ICAT technologies for the quantitation of protein abundance changes within the Cdk2 subproteome. We will also utilize IMAC and selective ion scanning to determine phosphorytation events within the Cdk2 subproteome and to track changes in these modifications in the ischemic myocardium. Extensive methodology for these techniques is provided below. PHS 398 (Rev. 05/01) Page 321 Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. CONTINUATION PAGE Principal Investigator/Program Director (Last, First, Middle): Ping, Peipei (Vondriska, ProteomJC Core) Note: Although the initial analyses of the p38a and Cdk2 subproteomes will employ denaturing techniques on the front end (1DE and 2DE), we are committed to comprehensive characterization of these subproteomes using nondenaturing approaches (such as BN PAGE) as described in Project 1 with VDAC. After the initial characterization of the p38a and Cdk2 subproteomes in years 1and 2 of the PPG, we will analyze native complexes formed by these molecules with a more directed mass spectrometry and immunoblotting approaches in years 3-5 of the PPG. III. VALIDATION STEPS III.A. Quantitative Validation of Immunoprecipitations and Affinity Pull-Downs and Necessary Controls We will perform western immunoblotting experiments to ensure that the pull-downs to isolate the subproteomes of all molecules are quantitatively consistent. Specifically, we will immunoblot for the target of isolation to ensure that the same amount of protein is used under all conditions to isolate protein complexes. For example, in Project 3, we will western blot for p38a on equal-volume amounts of all the Flag pull-downs from different isolations to be certain that changes in protein association with p38a cannot be attributed to changes in the amount of p38a isolated (i.e. the amount of Flag-p38a pulled down in each experiment will be constant). Likewise in Project 4, we will immunoblot for Cdk2 following immunoprecipitation of Cdk2 based on the same principle. In this regard, we can be sure that the amount of Cdk2 isolated in different experiments, and between different treatment groups, is equal, and thus changes in the associating proteins cannot be attributed to changes in the amount of target protein isolated. A second issue of considerable importance is the use of essential control groups for isolation of protein complexes by immunoprecipitation or affinity pull-down. In both cases, proteins samples are incubated with beads alone, prior to addition of antibody or recombinant protein, to "preclear" the lysate of non-specific interactions between sample proteins and the beads. In the case of immunoprecipitation, a second control that is always performed is substitution of the IgG for the antibody against the target of isolation (e.g. when Cdk2-associated proteins are isolated in Project 4, we will perform parallel experiments using mouse IgG in place of the Cdk2 antibody). This step is required to exclude proteins that associate with the nonspecific region of the antibody. For affinity pull-down experiments (e.g. isolation of VDAC native complexes in Project 1), the second control in addition to preclearing with beads is the substitution of HIS-null proteins for HIS-VDAC. This step helps exclude from analysis proteins that associate with the HIS epitope tag. III.B. Functional Validation of Subproteome Candidates: Collaboration with Heart Biology Core In addition to interacting with the individual Projects to perform proteomics experiments, the Proteomic Core will also interact extensively with the Heart Biology Core to facilitate verification of candidate members of the subproteomes. The validation process involves a battery of classical biochemistry, cell and organelle biology and histo-pathology approaches to determine the functional role of the individual molecules in the context of the entire subproteome in vivo. It is important to highlight the close interaction between the Heart Biology Core and the Proteomic Core that will persist throughout this PPG. In Project 1, we will identify the PKCe-VDAC and PP2CK-VDAC associated proteins and generate a bioinformatic map of these subproteomes in the context of the MPT pore as a whole. Once we have identified the members of these subproteomes, we will confirm these findings by testing for co-localization of the candidate molecules with VDAC and other core components of the MPT pore (such as ANT) in the heart using confocal microscopy in collaboration with the Heart Biology Core. When available, we will also use inhibitors and/or activators of these molecules to examine the effects on mitochondrial function. In Project 2, we will determine residues on ANT1 and VDAC that are targeted for phosphorylation in the protected myocardium. Once these residues have been identified, we will collaborate with the Heart Biology Core and Project 2 to test the functional role of these residues using reconstitution assays and genetic manipulation. In Projects 3 and 4, we will identify p38a- and Cdk2- associated proteins, respectively, and will incorporate this subproteomic data with the PKCe-VDAC/PP2Cic-VDAC bioinformatic map being generated in Project 1. Because we will also map cytosolic and mitochondrial complexes in Projects 3 and 4, the findings will further flesh out any role for p38a- or Cdk2-associated proteins in the regulation of the mitochondria from an "inside-out" and "outside-in" perspective. As with Project 1, the Proteomic Core will collaborate heavily with the Heart Biology Core in Projects 3 and 4 to confirm the localization of p38a- or Cdk2- associated proteins to the mitochondria, and to examine colocalization of these molecules with the components of PHS 398 (Rev. 05/01) Page 322 Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. CONTINUATION PAGE Principal Investigator/Program Director (Last, First, Middle): Ping, Peipei (Vondciska, PrOteOmJC Core) the MPT pore (such as VDAC and ANT) using confocal microscopy. When available, we will also use inhibitors and/or activators of these molecules to examine the effects on mitochondrial function. Overall, there is a bidirectional flow of information and collaboration between the two Cores in this Program Project. As the Proteomic Core unveils novel members of a subproteome, the Heart Biology Core will provide the resources to the individual Projects to test the functional role of these candidates in the cardiac cell. Likewise, as the Heart Biology Core and the individual Projects decipher new information about the functional aspects of these subproteomes, the Proteomic Core will investigate novel protein complexes to further expand our understanding of the signal network. In this regard, the interaction between the Proteomic Core and the Heart Biology Core will aid execution of the experiments proposed in Projects 1-4, however, this interaction will also generate novel hypotheses about the cardioprotective signaling network to be tested in the future.

确定了p38a次蛋白质组后，我们下次将对此进行磷蛋白的分析 使用1DE/2DE，IMAC和选择性离子扫描质谱法的组合组。 具体而言，p38a相关蛋白将通过标志下拉分离，被电泳分离并消化 与胰蛋白酶。这些胰蛋白酶肽要么直接接受前体离子/中性损失扫描分析 质谱法，或首先使用iMac进行磷酸肽富集步骤。推定的磷蛋白 西方免疫印迹将验证识别。 iMac和前体/中性损失的详细程序 离子扫描在下面列出。 II.D.与项目4合作 表征CDK2亚蛋白质组的一般方法将通过三个步骤进行（平行于 上述p38a的分析）。首先，我们将通过免疫沉淀确定次蛋白质的成员， 电泳和LC/MS/MS。其次，我们将确定缺血性的该亚蛋白质的定量变化 使用DIGE和ICAT技术的心脏。第三，我们将检查该亚蛋白质组的磷酸化成员 使用iMac和选择性离子扫描方法。 项目4的目标1将使用正常和缺血性心肌中与CDK2相关的蛋白质使用 免疫沉淀（IP），1DE/2DE和LC/MS/MS的组合。线粒体分数将是 重悬于蔗糖缓冲液中，短暂超声，并用0.1％DDM处理（请参阅下面的样本准备 特定方法）。该混合物将再次离心到颗粒线粒体膜，并释放 上清液中的线粒体蛋白将用于IP。心肌胞质或线粒体分数将是 用抗CDK2单克隆的IP分离与蛋白A/G珠和CDK2相关蛋白的预先清除 抗体（Santa Cruz，SC-6248）。 IgG将代替抗CDK2抗体作为阴性对照。 CDK2- 洗涤后保留在珠子上以消除非特异性相互作用的相关蛋白质将从 珠子，以1DE分离。蛋白质将从凝胶中切除，胰蛋白酶化并受到LC/MS/MS的影响 分析。 [注意：项目4的成员与蛋白质组学核心紧密合作以优化纯化 通过IP和1DE/2DE凝胶的运行CDK2复合物的仪表，并将在这些过程中继续这样做 研究。详细的 1DE和LC/MS/MS的程序如下所述。有关实验的一般概述，请参见图2 项目3和4的策略。 像p38a一样，CDK2是一种可溶性激酶，因此我们可能能够从2DE中获得重要信息 CDK2复合物的分离，尤其是那些从细胞质分离的复合物。也类似于p38a，我们将 实施DIGE和ICAT技术，用于定量CDK2内蛋白质丰度变化 次蛋白酶。我们还将利用iMac和选择性离子扫描来确定磷酸化事件 CDK2亚蛋白酶并跟踪缺血性心肌中这些修饰的变化。广泛的方法论 对于这些技术，下面提供了这些技术。 PHS 398（修订版05/01）第321页 整个应用程序中的底部连续数字页面。请勿使用3a，3b之类的后缀。 延续页面首席调查员/计划主管（最后，第一，中间）：ping，peipei（Vondriska，proteomjc core） 注意：尽管p38a和CDK2亚蛋白质体的初始分析将采用变性技术 在前端（1DE和2DE），我们致力于使用这些亚蛋白质体的全面表征 如项目1中所述的Nondenaturatis方法（例如BN页面）。初始之后 p38a和CDK2亚蛋白质体的表征在PPG的第1年和2年内，我们将分析天然复合物 由这些分子形成，具有更直接的质谱法和免疫印迹方法。 PPG。 iii。验证步骤 III.A.免疫沉淀和亲和力下拉和必要控制的定量验证 我们将进行西部免疫印迹实验，以确保下拉以分离 所有分子的亚蛋白酶均定量一致。具体来说，我们将免疫印迹 分离以确保在所有条件下使用相同量的蛋白质来分离蛋白质复合物。为了 例如，在项目3中，我们将以相等体积的所有旗帜下拉的质量为p38a的Western blot 不同的隔离确定蛋白质与p38a的变化不能归因于变化 分离的p38a量（即，在每个实验中拉下的FLAG-P38A的量将是恒定的）。同样 项目4，我们将根据相同原理对CDK2进行免疫沉淀后进行免疫印迹。在这个 考虑到，我们可以确定在不同的实验中以及不同处理之间分离的CDK2量 组相等，因此关联蛋白的变化不能归因于数量的变化 靶蛋白分离。 第二个非常重要的问题是使用基本控制组隔离蛋白质 通过免疫沉淀或亲和力下拉的复合物。在这两种情况下，蛋白质样品都与珠一起孵育 单独在添加抗体或重组蛋白之前，将非特异性相互作用的裂解物 在样品蛋白和珠子之间。在免疫沉淀的情况下，第二个控制始终是 执行的是IgG代替抗分离靶的抗体（例如，当CDK2相关时 蛋白质在项目4中分离出来，我们将使用小鼠IgG代替CDK2进行并行实验 抗体）。需要此步骤才能排除与抗体非特异性区域相关的蛋白质。为了 亲和力下拉实验（例如，项目1中VDAC本地复合物的隔离），第二个控制 用珠子预先清除是用his-null蛋白代替His-VDAC。此步骤有助于排除 与他的表位标签相关的分析蛋白。 III.B.亚蛋白酶候选人的功能验证：与心脏生物学核心合作 除了与单个项目进行蛋白质组学实验的互动外，蛋白质组学核心 还将与心脏生物学核心进行广泛相互作用，以促进验证候选人的成员 亚蛋白酶。验证过程涉及一系列经典的生物化学，细胞和细胞器生物学以及 历史病理学方法是在整个背景下确定单个分子的功能作用 体内次蛋白酶。重要的是要强调心脏生物学核心与 蛋白质组学核心将在整个PPG中持续存在。 在项目1中，我们将确定PKCE-VDAC和PP2CK-VDAC相关蛋白质并生成一个 这些亚蛋白质体的生物信息学在整个MPT孔的上下文中。一旦我们确定了 这些亚蛋白质体的成员，我们将通过测试候选人的共定位来确认这些发现 使用共焦 显微镜与心脏生物学核心合作。如果有的话，我们还将使用抑制剂和/或激活剂 这些分子检查对线粒体功能的影响。在项目2中，我们将确定残留物 ANT1和VDAC针对受保护心肌的磷酸化。一旦这些残留物 已确定，我们将与心脏生物学核心和项目2合作测试这些功能 使用重构测定和基因操纵的残留物。在项目3和4中，我们将确定P38A和CDK2- 相关的蛋白质分别将此亚蛋白质数据与PKCE-VDAC/PP2CIC-VDAC合并 在项目1中生成的生物信息学图。因为我们还将在 项目3和4，这些发现将进一步充实P38A或CDK2相关蛋白在调节中的任何作用 线粒体从“内而外”和“外部”视角。与项目1一样，蛋白质组学核心将 在项目3和4中与心脏生物学核心进行大量合作，以确认p38a-或cdk2-的本地化。 相关蛋白与线粒体相关，并检查这些分子与成分的共定位 PHS 398（修订版05/01）第322页 整个应用程序中的底部连续数字页面。请勿使用3a，3b之类的后缀。 延续页面首席调查员/计划主管（最后，第一，中间）：ping，peipei（Vondciska，proteomjc core） 使用共聚焦显微镜的MPT孔（例如VDAC和ANT）。如果有的话，我们还将使用抑制剂 和/或这些分子的激活剂检查对线粒体功能的影响。 总体而言，该计划中两个核心之间的信息和协作的双向流动 项目。随着蛋白质组学核心揭示了次蛋白质组的新成员，心脏生物学核心将提供 为各个项目的资源来测试这些候选者在心脏细胞中的功能作用。同样，作为 心脏生物学核心和单个项目解密有关这些功能方面的新信息 亚蛋白质组学核心将研究新型蛋白质复合物，以进一步扩展我们对 信号网络。在这方面，蛋白质组学核与心脏生物学核心之间的相互作用将 但是，在项目1-4中提出的实验的辅助执行，但是，这种相互作用也将产生新颖 关于将来要测试的心脏保护信号网络的假设。