EXCEED THE SPACE PROVIDED. With the human and other important genomes now sequenced, a key challenge is the separation, identification, and structural characterization of the genome' s expressed proteins. Proteolysis of the separated protein, with MS and MS/MS of its peptides, is now a powerful tool for the protein's identification, but is far less useful for structural characterization of posttranslational modifications or DNA sequence errors. Our 'top down' approach appears highly promising for this: the protein FTMS spectrum provides an accurate molecular weight, while MS/MS provides information on the sequence and its specific modifications. For this, the new technique Electron Capture Dissociation (ECD) has been improved to provide nearly complete information of this type on a 29 kDa protein. ECD is unique in selectively cleaving covalent backbone bonds; several excitation methods have now been developed to cleave noncovalent bonds in order to obtain ECD spectra of large proteins.. These will be applied to a variety of larger proteins with various posttranslational modifications, including those from important biomedical problems like the biosynthesis of thiamin and NAD, the proteomics ofArabidopsis thialiana, the quantitative analysis of multiple phosphorylation sites in the 50 kDa Lyn kinase, and enzyme function from direct MS/MS of enzyme/product noncovalent complexes. Picomolar sample limits for extensive top down characterization will be sought with automation for electrospray and computer algorithms. The ECD backbone fragmentation without disrupting the non-covalent bonding will be exploited to elucidate further the unfolding and folding of gaseous proteins, and to characterize protein intermolecular adducts in the gas phase. The nonergodic nature of ECD raises several unique mechanistic problems that will be explored further. PERFORMANCE SITE ========================================Section End===========================================

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-BECM (01))
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Edmonds, Charles G
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Cornell University
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Skinner, Owen S; Breuker, Kathrin; McLafferty, Fred W (2013) Charge site mass spectra: conformation-sensitive components of the electron capture dissociation spectrum of a protein. J Am Soc Mass Spectrom 24:807-10
Begley, Tadhg P; Ealick, Steven E; McLafferty, Fred W (2012) Thiamin biosynthesis: still yielding fascinating biological chemistry. Biochem Soc Trans 40:555-60
Breuker, Kathrin; Skinner, Owen S; McLafferty, Fred W (2012) Femtosecond laser vaporization that preserves protein-folded structure: an unproven idea. Proc Natl Acad Sci U S A 109:E206; author reply E207
Skinner, Owen S; McLafferty, Fred W; Breuker, Kathrin (2012) How ubiquitin unfolds after transfer into the gas phase. J Am Soc Mass Spectrom 23:1011-4
Kong, Xianglei; Lin, Cheng; Infusini, Giuseppe et al. (2009) Numerous isomers of serine octamer ions characterized by infrared photodissociation spectroscopy. Chemphyschem 10:2603-6
Han, Xuemei; Smith, Norah L; Sil, Dwaipayan et al. (2009) IgE receptor-mediated alteration of membrane-cytoskeleton interactions revealed by mass spectrometric analysis of detergent-resistant membranes. Biochemistry 48:6540-50
Steinberg, Michal Z; Elber, Ron; McLafferty, Fred W et al. (2008) Early structural evolution of native cytochrome c after solvent removal. Chembiochem 9:2417-23
Breuker, Kathrin; Jin, Mi; Han, Xuemei et al. (2008) Top-down identification and characterization of biomolecules by mass spectrometry. J Am Soc Mass Spectrom 19:1045-53
Breuker, Kathrin; McLafferty, Fred W (2008) Stepwise evolution of protein native structure with electrospray into the gas phase, 10(-12) to 10(2) s. Proc Natl Acad Sci U S A 105:18145-52
McLafferty, Fred W (2008) Mass spectrometry across the sciences. Proc Natl Acad Sci U S A 105:18088-9

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