Subtle Modification of Isotope Ratio Proteomics (SMIRP)

描述(申请人提供)：蛋白质组学寻求监测在基因组编程的不同发育和环境影响下通过生物系统的蛋白质流量，但如果要发挥其真正的潜力，至关重要的是我们的蛋白质组学技术提供可重复的定量。由于蛋白质数量日常测量的可变性，定量蛋白质组学的一个共同特征是使用稳定的同位素编码来区分混合物中的对照样品和实验样品，这种混合物可以在单个实验中进行分析。编码稳定的同位素可以通过在枯竭/浓缩的介质中生长的有机体来实现，或者通过从有机体中提取蛋白质后对蛋白质进行化学修饰来实现，尽管这些后一种方法对于测量蛋白质周转率并不有用。目前的稳定同位素策略寻求完全的同位素交换，即用所有的14N交换15N，用12C交换13C，或者用16O交换18O，这样一个肽的质量就会被几个道尔顿改变。这些方法价格昂贵，因为需要高同位素纯度，估计第一只15N大鼠的成本约为10,000美元。此外，两个多肽同位异构体分布取代一个多肽同位异体分布的事实导致了要求更有效的多肽分离的质谱仪中分离空间的实际损失。此外，二次分布的同位素可能会在自动化蛋白质组学实验中触发MS-MS，浪费质谱仪的时间。如果一种特定的蛋白质被大量上调或下调，它的存在也有可能被忽视，因为在质谱图中没有任何容易识别的多肽信号对。 我们在这里提出了一种廉价的策略，可能适用于活人的表达蛋白质组学和周转测量，将定量差异表达信息编码在单个同位异构体包膜内。

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.