Subtle Modification of Isotope Ratio Proteomics (SMIRP)

同位素比蛋白质组学的微妙修改 (SMIRP)

• 批准号: 7347308
• 负责人: Julian P Whitelegge
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-22 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7347308
• 关键词: Analytical Chemistry; Biochemistry; Biological; Code; Condition; Cyclotrons; Development; Escherichia coli; Fourier Transform; Genome; Glycine; Growth; Human; Hybrids; Ions; Isotopes; Label; Lac Operon; Life; Mass Spectrum Analysis; Measurement; Measures; Methods; Modification; Monitor; Organism; Peptide Signal Sequences; Peptides; Prokaryotic Cells; Proteins; Proteomics; Protocols documentation; Rate; Rattus; Relative (related person); Repression; Sampling; System; Technology; Time; Variant; Work; cost; dalton; day; mass spectrometer; programs; protein degradation; protein expression; research study; stable isotope; statistics; tandem mass spectrometry; wasting

DESCRIPTION (provided by applicant): Proteomics seeks to monitor the flux of protein through a biological system under variable developmental and environmental influences as programmed by the genome but, if it is to deliver its true potential, it is critically important that our proteomics technologies deliver reproducible quantitation. Because of the variability of day-to-day measurements of protein quantities, a common feature of quantitative proteomics is the use of stable isotope coding to distinguish control and experimental samples in a mixture that can be profiled in a single experiment. Coding with stable isotopes can be achieved by growth of an organism in depleted/enriched media, or by chemically modifying proteins after extraction from the organism, though these later approaches are not useful for measurement of protein turnover rates. Current stable isotope strategies seek full isotope- exchange, that is, to swap all 14N for 15N, 12C for 13C, or 16O for 18O, such that a peptide's mass is altered by several Daltons. These methods are expensive, because of the need for high isotope purity, and it was estimated that the first 15N rat cost ~$10,000. Furthermore, the fact that two peptide isotopomer distributions replace one leads to a practical loss of separation space in the mass spectrometer demanding more efficient peptide separations. Moreover, the second isotopomer distribution may trigger MS-MS in automated proteomics experiments, wasting mass spectrometer time. If a particular protein has been massively up-or-down-regulated there is also the possibility that its presence will be ignored because there weren't any readily identified pairs of peptide signals in the mass spectrum. We propose here an inexpensive strategy, potentially applicable to expression proteomics and turnover measurements in living humans, that codes the quantitative differential expression information within a single isotopomer envelope.

描述（由申请人提供）：蛋白质组学旨在监测在基因组编程的可变发育和环境影响下通过生物系统的蛋白质通量，但是，如果要发挥其真正的潜力，我们的蛋白质组学技术提供可重现的定量至关重要。由于蛋白质量的日常测量的可变性，定量蛋白质组学的一个共同特征是使用稳定同位素编码来区分混合物中的对照和实验样品，该混合物可以在单个实验中进行分析。用稳定同位素编码可以通过在耗尽/富集培养基中生长生物体或通过在从生物体提取后化学修饰蛋白质来实现，尽管这些后一种方法对于测量蛋白质周转率没有用。目前的稳定同位素策略寻求完全同位素交换，即，将所有14 N交换为15 N，将12 C交换为13 C，或将16 O交换为18 O，使得肽的质量改变几个道尔顿。这些方法是昂贵的，因为需要高同位素纯度，据估计，第一只15 N大鼠的成本约为10，000美元。此外，两个肽同位素异构体分布取代一个的事实导致质谱仪中分离空间的实际损失，需要更有效的肽分离。此外，第二同位素异构体分布可能在自动化蛋白质组学实验中触发MS-MS，浪费质谱仪时间。如果一种特定的蛋白质被大量上调或下调，那么它的存在也有可能被忽略，因为在质谱中没有任何容易识别的肽信号对。 我们在这里提出了一种廉价的策略，可能适用于表达蛋白质组学和营业额测量在活着的人，编码的定量差异表达信息在一个单一的同位素信封。