Subtle Modification of Isotope Ratio Proteomics (SMIRP)

描述（由申请人提供）：蛋白质组学旨在监测蛋白质在基因组编程的可变发育和环境影响下通过生物系统的通量，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复的定量是至关重要的。由于日常蛋白质量测量的可变性，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样品和实验样品，这些样品可以在一次实验中进行分析。稳定同位素编码可以通过生物体在贫/富培养基中生长，或通过从生物体中提取蛋白质后进行化学修饰来实现，尽管这些方法对蛋白质周转率的测量并不有用。目前的稳定同位素策略寻求完全的同位素交换，即把所有的14N换成15N，把12C换成13C，或者把16O换成18O，这样肽的质量就会被几个道尔顿改变。由于需要较高的同位素纯度，这些方法都很昂贵，据估计，第一个15N大鼠的成本约为1万美元。此外，两个肽同位素体的分布取代一个的事实导致了质谱仪中分离空间的实际损失，需要更有效的肽分离。此外，在自动化蛋白质组学实验中，第二同位素分布可能会触发MS-MS，浪费质谱仪时间。如果一种特定的蛋白质被大规模上调或下调，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。

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.