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Joshua J. Coon

Joshua J. Coon
Program
Beckman Young Investigators

Award Year
2007

Institution
University of Wisconsin Madison

Email:
jcoon@chem.wisc.edu

Website:

Research Title:
Gas-phase coordination chemistry for rapid, robust whole portein sequence analysis

Abstract:
Technologies for protein characterization are necessary to virtually all areas of the biological sciences. The field of proteomics aims to characterize the many isoforms and modification states of complex protein mixtures from a collection of cells. Mass spectrometry-based (MS) peptide sequencing strategies (~bottom-up") have provided a unique window to view the proteorne, monitor post-translational modifications (PTMs), and to quantitate their changes. These methods rely on enzymatic protein digestion to create a collection of peptides of suitable size for successful sequencing; however, many investigators now realize this approach has significant limitations: First, PTMs on multi-domain proteins work in concert; to determine their biological relevance, these patterns must be detected within the context of one another (across the whole protein). Second, transcriptional editing processes are pervasive in higher eukaryotes and difficult to predict, even with a completely sequenced genome. Skipped codons, frameshifting, gene fusion, and single nucleotide polymorphisms {SNPs) also occur. Top-down protein sequencing approaches (i.e., whole protein) allow observation of the all the processes above, but are not routine for proteins whose mass exceeds - 25 kDa. How does one successfully characterize the many isoforms and modified versions of each gene product generated within the context of a living cell? We must remove the mass limitation presently restricting "top-down" sequencing methodologies by developing novel chemistries for the systematic disassembly of a gas-phase protein cation. we propose to react a population of protein cations with small molecule anions that covalently bind to selected sites (e.g., specific amino acid side-chains). The bound anionic reagents will then serve as scaffolds to harbor site-specific backbone cleavage, with enzyme-like specificity. The resulting peptides are then individually interrogated and sequenced with conventional peptide dissociation techniques. By incorporating intact protein mass measurement with subsequent "gas-phase digestion", we shall eliminate the upper mass limit currently limiting the widespread implementation of "top-down" proteomics.

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