Quantitative Proteomics

定量蛋白质组学

• 批准号: 9143509
• 负责人: Leesa Deterding
• 金额: $ 81.54万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 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/9143509
• 关键词: Acids; Acrylamides; Adrenal Cortex Hormones; Adult Respiratory Distress Syndrome; Androgens; Animal Model; Animals; Appearance; Aqueous Humor; Arachidonic Acids; Biological; Bronchoalveolar Lavage; Buffers; Buprenorphine; Cell Culture Techniques; Cell Death; Cells; Charge; Chemicals; Cholesterol; Collaborations; Colon; Complex; Computer software; Concentration measurement; DNA Damage; Data; Data Analyses; Detection; Diagnosis; Diethylstilbestrol; Dinoprost; Disease; Doxorubicin; Drops; Drug Formulations; Early Diagnosis; Eicosanoids; Embryo; Epigenetic Process; Epithelial Cells; Estrogens; Extramural Activities; Eye; Family; Family Study; Fatty Acids; Feces; Fibroblasts; Freezing; Gases; Gel; Generations; Genes; Glaucoma; Glucuronides; Goals; Hour; Human; Individual; Inflammation; Institutes; Investigation; Ions; Isoprostanes; Knockout Mice; Knowledge; Label; Laboratories; Link; Lipid Peroxidation; Lipids; Liquid Chromatography; Liquid substance; Lysophospholipids; Mass Spectrum Analysis; Measurement; Measures; Metabolic; Metabolism; Methodology; Methods; Molecular Target; Myositis; National Institute of Environmental Health Sciences; Nature; Obesity; Opiates; Orphan; Oxidative Stress; PTGS2 gene; Pain management; Peptides; Phase; Physiologic Intraocular Pressure; Plasma; Plasma Cells; Pluronics; Process; Prostaglandin-Endoperoxide Synthase; Prostaglandins; Protein p53; Proteins; Proteolysis; Proteomics; Protocols documentation; Rattus; Regulation; Research Personnel; Respiratory distress; Retinoids; Rheumatism; Rodent; Role; Sampling; Scanning; Science; Serum; Serum Proteins; Shapes; Siblings; Signal Transduction; Signaling Molecule; Source; Speed; Steam; Sterilization; Steroids; Subcutaneous Injections; Technology; Therapeutic; Time; Tissue Sample; Tissues; Transcriptional Regulation; Travel; Twin Multiple Birth; Urine; Volatile Fatty Acids; Work; base; biological systems; brain tissue; cardiovascular health; cyclooxygenase 1; data acquisition; experience; feeding; glycidamide; improved; inhibitor/antagonist; instrument; instrumentation; interest; ion mobility; ionization; lysophosphatidic acid; method development; neoplastic cell; novel; orphan nuclear receptor ROR-gamma; outcome forecast; pressure; protein complex; protein expression; receptor; research study; sample fixation; small molecule; stereochemistry; steroid hormone; tandem mass spectrometry; theories; tool

We have analyzed a variety of molecules obtained from various sources to get the quantitative information. Additionally, we are developing methods to improve the quantitative information that can be gained. 1. Method Development. The use of label-free approaches for quantitative proteomics studies has been implemented. This methodology involves data-independent analyses (MSe) and an online UPLC method suitable for the analysis of proteins from complex samples. In theory, MSe can rapidly acquire accurate mass precursor and fragment ion information while simultaneously obtaining fairly accurate quantitative profiles from every detectable component in the sample. The data obtained from depleted serum (depletion of the 14 most abundant proteins) followed by six different sample treatments has been acquired. The results obtained by data-independent analyses and by data-dependent analyses are being evaluated. Additionally, we are incorporating ion mobility methods in an effort to gain further information. Ion mobility is a useful tool to aid in mass spectrometry applications because it allows for the measurement of the collisional cross section of a molecule and gives information about the three-dimensional shape of a compound in the gas phase. Ion mobility separates ions based on their differential mobility through a buffer gas based on the ions shape, charge, and mass. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions and thus travel more slowly than those ions that comprise a smaller cross-section. Thus, ion mobility serves as a useful means of orthogonal separation in unrelated molecules as well. This is a real benefit because the ability to identify and quantify proteins is directly linked to the power of chromatographic separations. 2. Myositis Study. A differential proteomics project comparing the quantitation of proteins from sera of healthy individuals to individuals diagnosed with a rheumatic disease is underway. This work is in collaboration with F. Miller (EAG) as part of the EAGs study of families with twins or siblings discordant for systemic rheumatic disorders. The goal of this project is to determine whether a protein or a suite of proteins can be identified that would allow for the early diagnosis, prognosis, and treatment of these diseases. Sera samples have been depleted and digested, data has been acquired, and the results are being processed. 3. Tissue Study. A protein expression study of LCM tissue is underway. Frozen rat brain tissue has been evaluated for protein extraction using various extraction protocols. The best results gave an MS identity of about 300 proteins. A study comparing the extraction efficacy of proteins from PAXgene fixation versus traditional fixation is under investigation. 5. Acrylamide Study. The effects of sterilization parameters on rodent feed have been determined by measuring the amount of acrylamide that is produced in the feed with different sterilization parameters, primarily different steam autoclaving times and number of cycles. Analysis of acrylamide and its genotoxic metabolite glycidamide in rat plasma and urine is currently ongoing. Mass spectral analysis of these low mass polar analytes in complex matrices is challenging. We have developed a novel derivatization method to aid in the extraction, separation, and ionization of these target analytes in biological samples. 6. Isoprostane Study. Isoprostanes are a family of compounds that are identical to their corresponding prostaglandins except for differences in stereochemistry. Because isoprostanes can be formed nonenzymatically, they are often studied as markers of oxidative stress. The generation of the most often measured isoprostane, 8-iso-PGF2alpha; by prostaglandin-endoperoxide synthase and arachidonic acid even with low enzymatic activity has been investigated. From our studies, an increase in the concentration of 8-iso-PGF2alpha; does not necessarily reflect an increase in chemical lipid peroxidation. We recommend using the 8-iso-PGF2alpha;/PGF2alpha; ratio to quantitatively distinguish between increases in 8-iso-PGF2alpha; by enzymatic and/or chemical lipid peroxidation. 7. Lipid/Fatty Acid Studies. A) We have quantified cholesterol metabolites in human serum and lung lavage as well as samples from animal and cell culture experiments. From initial studies, a correlation between the levels of these compounds and individuals with Acute Respiratory Distress Syndrome has been observed. Additional samples are currently being interrogated to assess the usefulness of these compounds as markers of respiratory distress. B) The autotaxin (ATX)-lysophosphatidic acid (LPA) signaling axis is being investigated as a molecular target for lowering intraocular pressure (IOP) in animal models of glaucoma. Using ATX inhibitors, the role of autotaxin in regulation of intraocular pressure is characterized with the detection of LPA and LPC in the aqueous humor of eye. C) The role of COX-2 as an upstream regulator of p53 levels was investigated. DNA damage-induced increase in p53 protein levels was greatly reduced in COX-2 knockout (KO) mouse embryonic fibroblasts (MEFs) compared to that in wild type or COX-1 KO MEFs. Furthermore, a COX-2 specific inhibitor suppressed doxorubicin-induced p53 accumulation and cell death. D) The role of the retinoid acid-related orphan receptor gamma, ROR gamma, in the transcriptional regulation of lipid metabolic genes was determined by the quantitation of fatty acids. E) We are looking at a comparison of short chain fatty acids in feces to determine if obesity has an effect on the metabolism of cells. It was shown previously that obesity can result in some epigenetic changes in colon epithelial cells and make them resemble tumor cells. 8. Eicosanoid Studies. Eicosanoids and related fatty acid metabolites serve as signaling molecules and are intricately involved in inflammation and cardiovascular health. The level of eicosanoids and eicosanoid metabolites are thought to be involved in many diseases. We are involved in a variety of projects measuring these compounds using mass spectrometry. We use liquid chromatography tandem mass spectrometry to analyze a panel of 49 of these molecules which has allowed us to collaborate with several intramural and extramural researchers. 9. Buprenorphine Study. Buprenorphine is an opiate which is used to control pain in experimental animals by our CMB. Plasma concentrations of buprenorphine drop below therapeutic levels between 2 and 4 hours after subcutaneous injection. To achieve a longer therapeutic window, sustained release formulations and pluronic gel formulations are being investigated. Buprenorphine, norbuprenorphine, and their glucuronides in plasma have been quantified from initial formulations. 10. Steroid Studies. Steroid hormones are widely distributed in nature and are potent signaling molecules. As such, they are of interest to several researchers within the institute. We have done a comparison of the MS sensitivity for a variety of steroids on different instruments in our laboratory. A supported liquid extraction protocol followed by chemical derivatization of estrogens has been developed to allow the simultaneous quantification of androgens, estrogens, and the estrogenic compound diethylstilbestrol in plasma. We are currently evaluating the expansion of the methodology to include corticosteroids and bisphenols.

我们分析了从各种来源获得的各种分子以获得定量信息。 此外，我们正在开发方法来改进可以获得的定量信息。 1. 方法开发。 使用无标记方法进行定量蛋白质组学研究已经实施。 该方法涉及数据独立分析 (MSe) 和适用于分析复杂样品中的蛋白质的在线 UPLC 方法。 理论上，MSe 可以快速获取准确的质量母离子和碎片离子信息，同时从样品中的每个可检测成分获得相当准确的定量分布。 已获得从去除血清（去除 14 种最丰富的蛋白质）和六种不同样品处理后获得的数据。 正在评估通过数据独立分析和数据依赖分析获得的结果。 此外，我们正在结合离子淌度方法来获取更多信息。离子淌度是一种有助于质谱应用的有用工具，因为它可以测量分子的碰撞截面，并提供有关气相化合物的三维形状的信息。 离子淌度根据离子形状、电荷和质量通过缓冲气体的不同迁移率来分离离子。离子穿过漂移区域的速度取决于其尺寸：大离子将经历更多次数的碰撞，因此比那些具有较小横截面的离子移动得更慢。因此，离子淌度也可作为不相关分子正交分离的有用手段。 这是一个真正的好处，因为识别和定量蛋白质的能力与色谱分离的能力直接相关。 2.肌炎研究。一项差异蛋白质组学项目正在对健康个体和诊断患有风湿病的个体血清中的蛋白质进行定量比较。这项工作是与 F. Miller (EAG) 合作进行的，是针对患有系统性风湿性疾病的双胞胎或兄弟姐妹的家庭进行 EAG 研究的一部分。 该项目的目标是确定是否可以鉴定出一种蛋白质或一组蛋白质，以便对这些疾病进行早期诊断、预后和治疗。 血清样本已被耗尽和消化，数据已获取，结果正在处理中。 3.组织研究。 LCM 组织的蛋白质表达研究正在进行中。 已使用各种提取方案对冷冻大鼠脑组织的蛋白质提取进行了评估。 最好的结果给出了约 300 种蛋白质的 MS 鉴定。 一项比较 PAXgene 固定与传统固定的蛋白质提取效率的研究正在进行中。 5.丙烯酰胺研究。通过测量不同灭菌参数（主要是不同蒸汽高压灭菌时间和循环次数）的饲料中产生的丙烯酰胺量，确定了灭菌参数对啮齿动物饲料的影响。 目前正在对大鼠血浆和尿液中的丙烯酰胺及其遗传毒性代谢物缩水甘油酰胺进行分析。对复杂基质中这些低质量极性分析物进行质谱分析具有挑战性。我们开发了一种新颖的衍生化方法，有助于生物样品中这些目标分析物的提取、分离和电离。 6.异前列烷研究。异前列腺素是一类化合物，除了立体化学差异外，与相应的前列腺素相同。 由于异前列腺素可以非酶促形成，因此它们经常被作为氧化应激的标志物进行研究。 最常测量的异前列腺素 8-iso-PGF2alpha 的生成；即使酶活性较低，也已研究了前列腺素内过氧化物合酶和花生四烯酸的作用。根据我们的研究，8-iso-PGF2α 浓度增加；不一定反映化学脂质过氧化的增加。我们建议使用 8-iso-PGF2alpha;/PGF2alpha;定量区分 8-iso-PGF2α 增加的比率；通过酶促和/或化学脂质过氧化。 7.脂质/脂肪酸研究。 A) 我们对人血清和肺灌洗液以及动物和细胞培养实验样本中的胆固醇代谢物进行了定量。 从初步研究来看，已经观察到这些化合物的水平与患有急性呼吸窘迫综合征的个体之间存在相关性。 目前正在研究更多样品，以评估这些化合物作为呼吸窘迫标志物的有用性。 B) 正在研究自分泌运动因子 (ATX)-溶血磷脂酸 (LPA) 信号轴作为青光眼动物模型中降低眼内压 (IOP) 的分子靶标。 使用ATX抑制剂，通过检测眼房水中的LPA和LPC来表征自分泌运动因子在眼内压调节中的作用。 C) 研究了 COX-2 作为 p53 水平上游调节剂的作用。与野生型或 COX-1 KO MEF 相比，COX-2 敲除 (KO) 小鼠胚胎成纤维细胞 (MEF) 中 DNA 损伤诱导的 p53 蛋白水平大大降低。此外，COX-2 特异性抑制剂抑制阿霉素诱导的 p53 积累和细胞死亡。 D)通过脂肪酸定量确定视黄酸相关孤儿受体γ（RORγ）在脂质代谢基因转录调节中的作用。 E) 我们正在比较粪便中的短链脂肪酸，以确定肥胖是否对细胞的代谢有影响。 之前的研究表明，肥胖会导致结肠上皮细胞发生一些表观遗传变化，并使它们类似于肿瘤细胞。 8.类二十烷酸研究。类二十烷酸和相关脂肪酸代谢物作为信号分子，与炎症和心血管健康密切相关。类二十烷酸和类二十烷酸代谢物的水平被认为与许多疾病有关。 我们参与了利用质谱法测量这些化合物的各种项目。 我们使用液相色谱串联质谱分析了 49 种分子，这使我们能够与几位校内和校外研究人员合作。 9. 丁丙诺啡研究。丁丙诺啡是一种阿片类药物，我们的 CMB 用于控制实验动物的疼痛。皮下注射后 2 至 4 小时内，丁丙诺啡的血浆浓度降至治疗水平以下。为了实现更长的治疗窗口，正在研究缓释制剂和普朗尼克凝胶制剂。血浆中的丁丙诺啡、去甲丁丙诺啡及其葡萄糖醛酸苷已从初始制剂中定量。 10.类固醇研究。 类固醇激素在自然界中广泛分布，是有效的信号分子。 因此，研究所内的几位研究人员对它们感兴趣。我们在实验室的不同仪器上对各种类固醇的 MS 灵敏度进行了比较。已经开发出一种支持液体提取方案，然后进行雌激素化学衍生化，以允许同时定量血浆中的雄激素、雌激素和雌激素化合物己烯雌酚。我们目前正在评估该方法的扩展，以包括皮质类固醇和双酚。