Mass Spectrometry Quantitation

质谱定量

• 批准号: 10925018
• 负责人: Leesa Deterding
• 金额: $ 189.84万
• 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/10925018
• 关键词: Acrylamides; Adenosine; Adopted; Adrenalectomy; Aging; Animal Experiments; Arachidonic Acids; Area; Area Under Curve; Beds; Bile Acids; Biological; Biological Assay; Bleomycin; Blood Platelets; Buprenorphine; Cells; Chromatography; Circulation; Collaborations; Computer software; Consumption; Custom; DNA; DNA Repair; Data; Development; Dexamethasone; Diet; Disease; Drops; Dryness; Eicosanoids; Environment; Environmental Exposure; Enzymes; Evaluation; Extramural Activities; Fatty Acids; Fibroblasts; Formulation; Goals; Growth; Harvest; Heterogeneity; Hour; Immunoglobulin A; Individual; Inflammation; Injections; Ions; Isomerism; Knockout Mice; Label; Laboratories; Lipids; Liquid Chromatography; Liquid substance; Liver; London; Lung; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Membrane Lipids; Metabolic; Metabolic Diseases; Metabolism; Methionine; Methods; Microsomes; Molecular; Mus; National Institute of Drug Abuse; National Institute of Environmental Health Sciences; Nature; Neuroblastoma; Nucleosides; Nutritional; Opioid; Output; PDGFRB gene; Pain management; Palmitates; Pathology; Patients; Pentas; Peptides; Phosphorylation; Plasma; Plasma Proteins; Play; Preparation; Protein Analysis; Proteins; Proteome; Proteomics; Protocols documentation; Publications; Reaction; Reagent; Recommendation; Regimen; Reporting; Repression; Research Personnel; Research Project Grants; Ribonucleosides; Role; Running; Sampling; Scanning; Science; Signaling Molecule; Source; Stable Isotope Labeling; Statistical Data Interpretation; Statistical Methods; Sterilization; Subcutaneous Injections; Sulfur Metabolism Pathway; Sum; Techniques; Therapeutic; Time; Tissues; bile acid metabolism; cancer cell; cancer therapy; cardiovascular health; cell injury; cell type; cofactor; comparative; cost efficient; data acquisition; deprivation; dietary; experimental study; exposure to cigarette smoke; improved; knockout gene; lipidomics; liquid chromatography mass spectrometry; manufacture; mesangial cell; method development; novel; protein complex; protein expression; response; tandem mass spectrometry; 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. We have put effort into the development of lipidomics analyses. This project involves the identification as well as the quantitation of compounds; thus, this project is included in both the identification/characterization project and the quantitation project. Currently, we have the framework for a turn-key analysis from sample to data output. In collaboration with the Harvey lab at NIDA, this was utilized for the analysis of membrane lipid composition following treatment of neuroblastoma SH-SY5Y cells with palmitate. We were able to show a change in the PC:PE ratio upon treatment. We currently are able to distinguish and annotate many hundreds of lipids, however the isomeric nature of lipids limits the exactness of the assay due to this theoretical possibility. We have been working with Alan Jarmusch to implement statistical methods to deal with the large amounts of data that mass spec can provide. These are novel custom-built tools, not relying on software packages, for in-house statistical analysis. This allows us to provide relative, semi-quantitative measurements for several hundred or more individual features. 2. 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 75 of these molecules which has allowed us to collaborate with several intramural and extramural researchers. 3. Bile acid studies. In collaboration with the Zeldin laboratory, we have profiled bile acids in mice. For this project, we examined cyp450 enzymes for bile acid metabolism. Positive control cyp2C70 formed alpha-MCA from CDCA but 2c44 and 2c29 did not. 2c44 microsome activity was confirmed which showed arachidonic acid being converted to oxylipin species. 4. NAD Studies. NAD is a cofactor for hundreds of metabolic reactions in all cell types, and plays an essential role in metabolism, DNA repair, and aging. How NAD metabolism is impacted by the environment remains unclear. In the continuing collaboration with the Li laboratory, the MSRSG has extended these studies to gene knockout mice as well additional compounds and flux analyses. 5. Comparative Proteomics Studies. The MSRSG has undertaken several collaborative studies that involve both identification and relative quantification of proteins in samples. While some studies may employ tandem mass tags (TMT), most of these studies have relied on label-free quantification (LFQ). As example, in collaboration with the Cidlowski laboratory, we have performed a study evaluating the proteome changes that occur in murine platelets upon adrenalectomy and dexamethasone treatments. Similarly, in collaboration with Carol Trempus and Dr. S. Garantziotis, we have performed a relative quantification of proteins from the PDGFRalpha positive fibroblasts harvested from the lungs of PBS and bleomycin treated mice. Additionally, in collaboration with Drs. Y Liu and P. Wade, we have performed proteomic studies to evaluate the protein expression profiles in the livers of mice that have been fed a normal diet versus mice that have undergone methionine deprivation. Finally, in collaboration with Drs. E. Haque and S. London, we have evaluated protein expression profiles in the lungs of mice exposed to cigarette smoke versus mice that have not undergone such treatment. 6. TMT studies. This well-established method utilizes stable isotope labels which are incorporated within the peptides, introducing an expectable mass difference within two or more experimental conditions. The strategy considerably increases sample throughput and enables relative quantitation of proteins in samples derived from cells, tissues or biological fluids, thus saving time by reducing the number of mass spectrometry runs required for analysis. We have recently used the approach to quantify IgA-containing protein complexes in circulation of IgAN patients and controls. IgA protein complexes were enzymatically digested, and the resulting peptides were labelled with TMT-11 reagent. Differences in IgA-protein complex abundance were quantified by combining TMT-labeled peptides from patients and controls and analyzing the peptide simultaneously by LC-MS/MS. A minimalistic sample preparation strategy was chosen to streamline TMT sample preparation. The protocol consumes only one-eighth of the starting material (12.5g plasma proteins) recommended by the company that manufactures TMT, and thus used the proportional reduced amount of the labeling reagent. Each TMT-11 set included 1 pooled control sample for across TMT-plex normalizations, allowing up to 10 biological samples to be analyzed in a single mass spectrometry experiment (Figure 1). In total, 116 samples (12 injections), including replicates and triplicates, were analyzed. 195 proteins were identified, and 151 proteins were robustly quantified, in the tryptic digest of the 2 types of samples, including one significantly up regulated protein in the patient samples that, according to a recent publication, showed strong positive correlation with mesangial cell injury parameters in IgAN patients. 7. Methionine deprivation studies. Methionine restriction, a dietary regimen that protects against metabolic diseases and aging, represses cancer growth and improves cancer therapy. However, the response of different cancer cells to this nutritional manipulation is highly variable, and the molecular determinants of this heterogeneity remain poorly understood. In collaboration with the Wade lab, we have measured the effect of methionine restriction on sulfur metabolism in mice. 8. Nucleosides. The nucleoside methods have been available for some time for non-phosphorylated ribonucleosides, but this year we have expanded the assay to include some modified forms of adenosine. We have performed analyses looking at N-6-methyldeoxyadenosine incorporation into DNA for the Wade lab. A new chromatographic regime was employed for this assay, using a mixed bed C18-Penta-flouro phenyl (PFP) column. New chromatographic methods will explore the feasibility of assaying phosphorylated nucleoside and deoxynucleoside species in an effort to expand and increase the sensitivity of the assay. 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. We initially reported the results from extended-release formulations out to 24 hours. We recently acquired data from samples out to 48 hours. In addition, we are also acquiring data to compare bup SR-LAB to the new extended release product Zorbium. 10. Acrylamide studies. We recently obtained acrylamide levels in feed resulting from dry heat sterilization to compare to our previously obtained unsterilized, irradiated, and autoclaved feed. We determined that sterilizing dry heat conditions created an order of magnitude increase in acrylamide content compared to autoclave sterilization. Additionally, the MSRSG has developed specialized sample preparation and mass spectrometric techniques to successfully detect and quantitate a variety of compounds in multiple research projects (e.g. ppGpp, acylCoAs, etc).

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。我们致力于脂质组学分析的开发。 该项目涉及化合物的鉴定和定量；因此，该项目既包含在鉴定/表征项目中，也包含在定量项目中。目前，我们拥有从样本到数据输出的交钥匙分析框架。 与 NIDA 的 Harvey 实验室合作，将其用于分析用棕榈酸酯处理神经母细胞瘤 SH-SY5Y 细胞后的膜脂成分。 我们能够显示治疗后 PC:PE 比率的变化。 目前，我们能够区分和注释数百种脂质，然而，由于这种理论上的可能性，脂质的异构性质限制了测定的准确性。我们一直与 Alan Jarmusch 合作实施统计方法来处理质谱可以提供的大量数据。 这些是新颖的定制工具，不依赖于软件包，用于内部统计分析。 这使我们能够为数百个或更多单独的特征提供相对的半定量测量。 2.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 75 种分子，这使我们能够与几位校内和校外研究人员合作。 3.胆汁酸研究。我们与 Zeldin 实验室合作，对小鼠胆汁酸进行了分析。在这个项目中，我们检查了 cyp450 酶对胆汁酸代谢的影响。阳性对照 cyp2C70 从 CDCA 形成 α-MCA，但 2c44 和 2c29 则不然。 2c44 微粒体活性得到证实，表明花生四烯酸转化为氧脂质物质。 4.NAD研究。 NAD 是所有细胞类型中数百种代谢反应的辅助因子，在新陈代谢、DNA 修复和衰老中发挥着重要作用。 NAD 代谢如何受环境影响仍不清楚。 在与 Li 实验室的持续合作中，MSRSG 将这些研究扩展到基因敲除小鼠以及其他化合物和通量分析。 5.比较蛋白质组学研究。 MSRSG 开展了多项合作研究，涉及样品中蛋白质的鉴定和相对定量。 虽然一些研究可能采用串联质量标签 (TMT)，但大多数研究依赖于无标记定量 (LFQ)。例如，我们与 Cidlowski 实验室合作进行了一项研究，评估肾上腺切除术和地塞米松治疗后小鼠血小板中发生的蛋白质组变化。 同样，我们与 Carol Trempus 和 S. Garantziotis 博士合作，对从 PBS 和博来霉素处理的小鼠肺部采集的 PDGFRalpha 阳性成纤维细胞的蛋白质进行了相对定量。 此外，与博士合作。 Y Liu 和 P. Wade，我们进行了蛋白质组学研究，以评估正常饮食小鼠与蛋氨酸剥夺小鼠肝脏中的蛋白质表达谱。 最后，与博士合作。 E. Haque 和 S. London，我们评估了暴露于香烟烟雾的小鼠与未接受此类治疗的小鼠肺部的蛋白质表达谱。 6.TMT研究。这种成熟的方法利用稳定同位素标记，将其掺入肽中，在两个或多个实验条件下引入预期的质量差异。该策略显着提高了样品通量，并能够对细胞、组织或生物液体样品中的蛋白质进行相对定量，从而通过减少分析所需的质谱运行次数来节省时间。我们最近使用该方法来量化 IgAN 患者和对照循环中含有 IgA 的蛋白质复合物。 IgA 蛋白复合物经过酶消化，所得肽用 TMT-11 试剂标记。通过结合来自患者和对照的 TMT 标记肽并通过 LC-MS/MS 同时分析肽来量化 IgA-蛋白质复合物丰度的差异。选择简约的样品制备策略来简化 TMT 样品制备。该方案仅消耗 TMT 制造公司推荐的起始材料（12.5g 血浆蛋白）的八分之一，因此标记试剂的使用量也相应减少。每个 TMT-11 组包括 1 个用于跨 TMT-plex 归一化的混合对照样本，允许在单个质谱实验中分析多达 10 个生物样本（图 1）。总共分析了 116 个样品（12 次进样），包括重复样品和三次重复样品。在 2 类样品的胰蛋白酶消化中，鉴定出 195 种蛋白质，并对 151 种蛋白质进行了稳健定量，其中包括患者样品中一种显着上调的蛋白质，根据最近的出版物，该蛋白质与系膜细胞损伤参数显示出强正相关性IgAN 患者。 7. 蛋氨酸剥夺研究。 限制蛋氨酸是一种预防代谢疾病和衰老、抑制癌症生长并改善癌症治疗的饮食方案。然而，不同癌细胞对这种营养操作的反应差异很大，而且这种异质性的分子决定因素仍然知之甚少。我们与韦德实验室合作，测量了蛋氨酸限制对小鼠硫代谢的影响。 8.核苷。 核苷方法用于非磷酸化核糖核苷已经有一段时间了，但今年我们扩大了检测范围，包括一些修饰形式的腺苷。 我们为 Wade 实验室进行了 N-6-甲基脱氧腺苷掺入 DNA 的分析。 该测定采用了新的色谱方案，使用混合床 C18-五氟苯基 (PFP​​) 柱。新的色谱方法将探索测定磷酸化核苷和脱氧核苷种类的可行性，以扩大和提高测定的灵敏度。 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。我们最初报告了 24 小时缓释制剂的结果。 我们最近从 48 小时内的样本中获取了数据。 此外，我们还正在获取数据以将 bup SR-LAB 与新的缓释产品 Zorbium 进行比较。 10.丙烯酰胺研究。 我们最近获得了干热灭菌饲料中丙烯酰胺的含量，以与我们之前获得的未经灭菌、辐照和高压灭菌的饲料进行比较。 我们确定，与高压灭菌器相比，干热灭菌条件下丙烯酰胺含量增加了一个数量级。 此外，MSRSG 还开发了专门的样品制备和质谱技术，可成功检测和定量多个研究项目中的各种化合物（例如 ppGpp、酰基辅酶 As 等）。