Mass Spectrometry Instrument for Mass Determination of Protein and Nucleic Acid Conjugates
Abstract
This DURIP award provides funds for the Principal Investigator to purchase a quadrupole time-of-flight mass spectrometer to place within the Center for Molecular Design and Biomimetics in the Biodesign Institute at Arizona State University, for the mass determination of proteins, peptides, DNA, and their respective conjugates. Two current DoD funded efforts led by the Principal Investigator will benefit from the acquisition of this instrument as will the research programs of two new junior faculty members within the Center for Molecular Design and Biomimetics, Drs. Nicholas Stephanopoulos and Jeremy Mills, and the research programs of eight other faculty members at Arizona State University. Specifically, the Principal Investigator~s Army Research Office (ARO) sponsored Multidisciplinary Research Program of the University Research Initiatives (MURI) project titled ~Translating Biochemical Pathways to Non-cellular Environments~, and his Office of Naval Research project titled ~Self-replicating DNA Nanostructures~ (Award number N00014-15-1-2689), will utilize the proposed equipment. Three projects within the ARO MURI involve protein~DNA hybrids to either immobilize enzymes on a DNA scaffold or incorporate dyes using branched DNA components. The requested mass spectrometry system will be used to accurately determine the mass of the proteins and characterize the synthetic conjugates in that effort. The ONR project involves generating self-replicating single-stranded DNA origami nanostructures and then evaluating the ability of those single-stranded DNA nanostructures to be decorated with other molecules for addressability. Specifically, a DNA stem-loop and a half PX tile will be examined for their ability to anchor DNA-modified proteins, and then mass determination of the created DNA-protein conjugates will be interrogated as in the MURI project. A second aim of the ONR project is the development of synthetic antibody mimics, with single-stranded DNA loops for binding to a target protein. Large DNA libraries for these loops will be screened and successful ~hits~ will be amplified and mutated. However, a key challenge will be the elucidation of loop binding to the target protein. To achieve that, the synthetic antibody will be chemically cross-linked to the target, followed by proteolytic digestion and mass spectrometry-mass spectrometry analysis to determine the exact site of conjugation. Mass spectrometry is the only methodology that can address this challenge, thereby allowing for more precise design and optimization of next generation antibodies. Dr. Stephanolpoulos~ research program involves the construction of arbitrarily shaped two-and three-dimensional nanomaterials using peptide-DNA nanomaterial building blocks, and the site-selective modification of proteins with multiple DNA handles for control of protein orientation on a DNA-based nanostructure. Dr. Mills~ research program aims to develop a fundamental understanding of how computational protein design methods may be used to carry out protein engineering in the context of an expanded genetic code and to leverage that knowledge to engineer novel enzymes, protein therapeutics, and diagnostic tools. The proposed mass spectrometry system is invaluable to both of their research efforts. Beyond the value to the specific research efforts in which it will be used, the proposed equipment will also play an important role in the science and technology education of the undergraduate students, graduate students, and postdoctoral fellows performing the research activities with the equipment. Those trainees will need to develop new methods on the proposed instrument for protein-DNA hybrids and other novel hybrid molecules.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Sep 23, 2016
- Source ID
- N000141613045
Entities
People
- Hao Yan
Organizations
- Arizona State University
- Office of Naval Research
- United States Navy