Since its inception in 1999, the NIH-funded Research Resource on the biomedical applications of accelerator mass spectrometry (AMS) has performed numerous groundbreaking studies in many areas including biomolecule turnover, quantitative metabolism of endogenous compounds and xenobiotics, and analysis of potentially mutagenic adducts. These studies were made possible by the high sensitivity, accuracy, and precision of AMS. The work proposed in this TR&D core will build on this legacy of successful, high impact research and will extend the applicability of current AMS experimental and analytical methods to a wider set of scientific and technical challenges. The focus for this core i the development and routine implementation of methods for absolute quantitation of proteins and other biological macromolecules. This core will support several driving biomedical projects, including: 1) Characterization of turnover rates of tissues and cell types labeled as a result of anthropogenic atmospheric 14C release (bomb-pulse biology); 2) Absolute quantitation of protein post-translational modifications; 3) Identification of receptors based on 14C-ligand binding; and 4) Development of labeling strategies, separation techniques, and analytical procedures for quantitation of computationally-designed synthetic protein drugs in biological matrices, in support of animal dosing experiments and human microdosing studies. Biological macromolecules play a wide variety of essential roles in cells, tissues, and organisms. Proteins are the basic building blocks of all living organisms, and proteinaceous enzymes catalyze the chemical reactions responsible for life. Nucleic acids provide the genetic material containing information for building, maintaining, and regulating living organisms. Because biological macromolecules play such essential and diverse roles, improvements in our ability to quantitatively trace macromolecules and their modifications will enable important discoveries that will significantly advance our understanding of biological processes. In a recent paper, Hanke et al (Hanke et al., 2008) wrote: Ultimately, it would be highly desirable to obtain exact quantitative values of each protein in a system, e.g., their copy number per cell or their concentration in nanogram per milliliter of body fluid. While this kind of basic information about a protein is already per se valuable for the biologist, systems biology even requires it as input for modeling. In a medical context, knowing the exact amounts of certain proteins in blood or other common sources of biomarkers can provide diagnostically relevant information for patient treatment. Analytical measurement of proteins and other biological macromolecules presents a number of significant technical challenges, particularly when quantitation is required. Macromolecule extraction from biological matrices is typically more complex than the extraction of small molecules, and extracts are more prone to contamination and degradation. Macromolecules such as proteins and nucleic acids are heterogeneous molecules, and may be chemically modified as a result of enzyme-mediated reactions (e.g., post-translational modifications) or as a result of tissue processing, as in the case of formalin-fixed tissue. In addition, biological macromolecules may be susceptible to cleavage by physical or chemical means. While analytical standards can usually be purchased or synthesized for small molecules of interest, similar standards are rarely available for biological macromolecules. The ability to measure absolute quantities of biological macromolecules has broad relevance for biology and medicine, as there currently is no standardized, universal analytical method capable of making these measurements. The major focus of this core is to develop methods for applying AMS technology, especially in conjunction with the new liquid sample AMS interface, to overcome these major challenges of biological macromolecule analysis. To this end, we propose to interact with collaborating researchers to solve a variety of important biological problems.
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