Multidimensional Separation Tools for Proteomics and Methods to Determine Metabolic Capacity

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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.