Multidimensional Separation Tools for Proteomics and Methods to Determine Metabolic Capacity

蛋白质是名副其实的生命构件，具有多种功能，是体内最重要、最多样的生物物种。有成千上万种不同的蛋白质，每一种都发挥着自己的特殊功能，同时在健康人体内保持着谨慎的平衡。为了应对生活中的压力、药物、感染、青春期、疾病、伤害、怀孕和其他事件，身体会改变蛋白质的类型和浓度，以更好地执行特定的任务。显然，识别和测量所有这些蛋白质对于推进我们对蛋白质及其相互作用如何驱动生命过程的理解至关重要。为了回答生物化学的基本问题，我们需要能够直接测量和表征复杂生物样品中的蛋白质的分析工具。这项提议有两个主要的研究途径。首先，我们提出建立一个能够分辨和测量数千种蛋白质的多维分离系统。与目前只能分解数百种蛋白质的系统相比，这将是一个巨大的进步。我们将使用这个系统来定位存在于不同生物样本中的蛋白质，如尿液、羊水和血清。与分离系统携手，我们将开发一种仪器，使我们能够从图谱中选择特定的蛋白质，并将它们分解成其组成的多肽进行鉴定。*这项建议的第二个研究途径是开发蛋白质组学策略，以量化法医样本中位于肝脏的细胞色素P450酶。这些酶负责分解体内的药物。在药物中毒/过量的情况下，法医毒物学家需要能够测量这些关键酶的浓度。它们是不是非常低，并允许药物积累到有毒水平？它们是不是太高了，把有益的药物转化为有毒的副产品的速度比人体清除它的速度快？受害者是否有遗传缺陷，使他们容易受到药物中毒的影响？要有工具来回答这些问题是具有挑战性的，因为在死亡时，肝组织开始分解，微生物入侵肝脏。我们将确定可以确定这些酶的死后窗口，开发一种定量方法来测量它们，确定受害者是否有异常基因突变，并最终对受害者的新陈代谢能力进行分类。有了这些信息，法医毒理学家可以确定药物在死亡中的作用。

Proteins, the veritable building blocks of life, perform a multitude of functions and are the most important and diverse biological species in the body. There are tens of thousands of different proteins, each performing its specialized function while being held in careful balance in a healthy person. In response to stress, drugs, infection, puberty, disease, injury, pregnancy and other events in life, the body alters the types and concentrations of proteins to better perform specific tasks. Clearly, identifying and measuring all these proteins is critical to advancing our understanding of how proteins, and their interactions, drive the life process. To answer fundamental questions of biochemistry, we need analytical tools that can directly measure and characterize proteins from complex biological samples. This proposal has two main avenues of research. In the first, we propose building a multidimensional separation system capable of resolving and measuring thousands of proteins. Compared to current systems that can only resolve hundreds of proteins, this will be a huge step forward. We will use this system to map the proteins present in diverse biological samples such as urine, amniotic fluid and serum. Hand-in-hand with the separation system we will develop an instrument that will allow us to select specific proteins from the map and break them down into their component peptides for identification.***The second avenue of research in this proposal is to develop proteomic strategies to quantitate liver-localized cytochrome P450 enzymes in forensic samples. These enzymes are responsible for breaking-down drugs in the body. In cases of drug toxicity/overdose forensic toxicologists need to be able to measure the concentration of these key enzymes. Were they critically low and allowed the drug to build-up to toxic levels? Are they too high and converted a beneficial pharmaceutical into a toxic by-product faster than the body could clear it? Does the victim have a genetic fault that makes them vulnerable to drug toxicity? Having the tools to answer these questions is challenging because, at death, the liver tissue begins to breakdown and microbes invade the liver. We will determine the postmortem window in which these enzymes can be determined, develop a quantitative method to measure them, determine if the victim had unusual genetic mutations and ultimately classify the victim's metabolic capacity. With these pieces of information, the forensic toxicologist can determine the role of drugs in the death.