Core N: MOLECULAR PROFILING

核心 N：分子谱分析

• 批准号: 8126787
• 负责人: Ian Alexander Blair
• 金额: $ 31.14万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8126787
• 关键词: Biological Assay; Biological Markers; Brain; Cancer Model; Cancer Patient; Cancer cell line; Caring; Cells; Chemicals; Chemistry; Clinical; Colon Carcinoma; Conditioned Culture Media; Coupled; DNA Adducts; Development; Diagnostic; Discrimination; Disease; Ensure; Folate; Glucuronides; Glutathione; Individual; Inorganic Sulfates; Ions; Isotope Labeling; Label; Liquid substance; Lysine; Malignant neoplasm of pancreas; Metabolic; Methodology; Molecular Profiling; Mus; Non-Steroidal Anti-Inflammatory Agents; Parents; Patients; Peptides; Pharmaceutical Preparations; Premature Birth; Preparation; Procedures; Process; Proteins; Proteomics; Quality Control; Reagent; Risk; Sampling; Schizophrenia; Serum; Source; Staging; Steroids; Sulfhydryl Compounds; Synapses; Techniques; Technology; Treatment Efficacy; Unspecified or Sulfate Ion Sulfates; Validation; adduct; amino group; density; pancreatic cancer cells; protein expression; research study; response; stable isotope; validation studies

Quantitative proteomics studies are accomplished by the use of LC-MS analysis of tryptic peptides coupled with tagging with chemical reagents or metabolic labeling of cells in culture. The majority of current chemical tagging techniques label peptides on the N-terminus and epsilon-amino group of lysine using Nhydroxysuccinimide chemistry. Isobaric mass tags are particularly popular for biomarker discovery experiments since the mass difference between labeled forms occur on fragment ions rather than on the parent analyte (3). A limitation to this application, however, is that the isotope label is introduced post-protein extraction, meaning extreme care must be taken to ensure error is not introduced during sample preparation. We have shown the utility of using the SILAC labeled proteins as SILAP standards which can be applied to any source material (1). Proof of principle for the utility of this approach was obtained for cells in culture (4;5) and for serum samples from pancreatic cancer patients (6). In this latter study, the CAPAN-2 pancreatic cancer cell line was labeled by SILAC and secreted proteins from the conditioned media collected. The secreted labeled proteins were then used as a SILAP standard that was added to pooled pancreatic cancer or control serum Over one hundred differentially expressed biomarker candidates were identified. This approach was extended to study biomarkers in the Apcmin mouse, a colon cancer model (7), for cervicovaginal fluid samples in a preterm birth study (8), and differential protein expression in the post-synaptic density from schizophrenic compared with normal brain samples (9). The development of clinically useful biomarkers is a two-stage process. The initial bioanalytical validation stage uses control samples in appropriate biofluids to establish analytical limits of quantification and determine errors introduced by sample handling that are typically used in conventional bioanalytical validation studies (10). This stage is followed by clinical validation using patient samples to determine the assay¿s sensitivity to detect the drug response or discrimination of a particular disease. We have focused on the use of LC-MS methodology for the analysis of DNA-adducts, glutathione-adducts, thiols, steroids, folates, and glucuronide and sulfate metabolites because it is applicable to the analysis of a wider range of biomarkers (11-16). The analysis of NSAIDs requires rigorous validation and quality control procedures to ensure that levels are determined with high accuracy and precision. This is accomplished by the use of stable isotope dilution methodology coupled with the implementation of rigorous standard operating procedures (10).

定量蛋白质组学研究是通过使用胰蛋白酶肽偶联的LC-MS分析来完成的。 用化学试剂标记或代谢标记培养中的细胞。目前大多数化学品 标记技术使用N-羟基琥珀酰亚胺在赖氨酸的N-末端和ε-氨基上标记肽 化学.等压质量标签在生物标记物发现实验中特别受欢迎 因为标记形式之间的质量差异发生在碎片离子上而不是母体分析物（3）上。 然而，该应用的限制是同位素标记在蛋白质提取后引入，这意味着 必须极其小心以确保在样品制备期间不引入误差。我们展示了 使用SILAC标记的蛋白质作为可应用于任何源材料的SILAC标准品的效用（1）。 对于培养物中的细胞（4;5）和血清样品，获得了该方法的实用性的原理证明 胰腺癌患者（6）。在后一项研究中，CAPAN-2胰腺癌细胞系被标记为 SILAC和从条件培养基收集的分泌蛋白。然后将分泌的标记蛋白质 用作SIRECT标准品，添加到合并的胰腺癌或对照血清中 鉴定了差异表达的生物标记候选物。这种方法被扩展到研究 Apcmin小鼠（结肠癌模型）中的生物标志物（7），用于早产中的宫颈阴道液样本 研究（8），以及精神分裂症患者与对照组相比， 正常脑样品（9）。 临床上有用的生物标志物的开发是一个两阶段的过程。初始生物分析验证阶段 在适当的生物液体中使用对照样品，以建立定量分析限并确定误差 通过通常用于常规生物分析验证研究的样品处理引入（10）。这 阶段之后是使用患者样本进行临床验证，以确定检测试剂盒检测 药物反应或对特定疾病的歧视。我们专注于LC-MS方法的使用 用于分析DNA加合物、谷胱甘肽加合物、硫醇、类固醇、叶酸、葡糖苷酸和硫酸盐 代谢物，因为它适用于更广泛的生物标志物的分析（11-16）。分析 NSAIDs需要严格的验证和质量控制程序，以确保使用 准确度和精密度高。这是通过使用稳定同位素稀释方法结合 执行严格的标准作业程序（10）。