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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Biotechnology Resource Grants (P41)
Project #
2P41GM103483-16
Application #
8666887
Study Section
Special Emphasis Panel (ZRG1-BCMB-H (40))
Program Officer
Sheeley, Douglas
Project Start
2000-09-01
Project End
2019-05-31
Budget Start
2014-08-15
Budget End
2015-05-31
Support Year
16
Fiscal Year
2014
Total Cost
$1,966,400
Indirect Cost
$591,617
Name
Lawrence Livermore National Laboratory
Department
Biology
Type
Organized Research Units
DUNS #
827171463
City
Livermore
State
CA
Country
United States
Zip Code
94550
Madeen, Erin; Corley, Richard A; Crowell, Susan et al. (2015) Human in Vivo Pharmacokinetics of [(14)C]Dibenzo[def,p]chrysene by Accelerator Mass Spectrometry Following Oral Microdosing. Chem Res Toxicol 28:126-34
Zhao, Chunqing; Hwang, Sung Hee; Buchholz, Bruce A et al. (2014) GABAA receptor target of tetramethylenedisulfotetramine. Proc Natl Acad Sci U S A 111:8607-12
Malfatti, Michael A; Lao, Victoria; Ramos, Courtney L et al. (2014) Use of microdosing and accelerator mass spectrometry to evaluate the pharmacokinetic linearity of a novel tricyclic GyrB/ParE inhibitor in rats. Antimicrob Agents Chemother 58:6477-83
Dingley, Karen H; Ubick, Esther A; Vogel, John S et al. (2014) DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry. Methods Mol Biol 1105:147-57
Cappuccio, Jenny A; Falso, Miranda J Sarachine; Kashgarian, Michaele et al. (2014) 14C Analysis of protein extracts from Bacillus spores. Forensic Sci Int 240:54-60
Etminan, Nima; Dreier, Rita; Buchholz, Bruce A et al. (2014) Age of collagen in intracranial saccular aneurysms. Stroke 45:1757-63
Etminan, Nima; Buchholz, Bruce A; Dreier, Rita et al. (2014) Cerebral aneurysms: formation, progression, and developmental chronology. Transl Stroke Res 5:167-73
Thomas, Avi T; Stewart, Benjamin J; Ognibene, Ted J et al. (2013) Directly coupled high-performance liquid chromatography-accelerator mass spectrometry measurement of chemically modified protein and peptides. Anal Chem 85:3644-50
Sarachine Falso, Miranda J; Buchholz, Bruce A (2013) Bomb Pulse Biology. Nucl Instrum Methods Phys Res B 294:666-670
Alkass, Kanar; Saitoh, Hisako; Buchholz, Bruce A et al. (2013) Analysis of radiocarbon, stable isotopes and DNA in teeth to facilitate identification of unknown decedents. PLoS One 8:e69597

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