The overall thrust of this research initiative is to develop new and improved means for the structural characterization of ions of biomedical relevance, with particular emphasis on those formed from peptides derived from enzymatic or chemical digestion, whole proteins, and protein complexes. The objective is to develop a new direction in tandem mass spectrometry that will enable a wide range of novel strategies to deal with biomolecule identification/characterization problems. The vast majority of tandem mass spectrometry experiments rely on fragmentation as the key structurally informative reaction. In this work, we seek to add the capability for selective covalent or non-covalent derivatization of gaseous ions within the context of an MSn experiment. The development of specific gas-phase chemistries fundamentally enables the implementation of a very wide range of novel applications. Just as bio-conjugation chemistries in solution have become broadly used by the molecular biology research community in a wide range of measurement strategies, the ability to effect gas-phase bio-conjugation will enable many novel approaches to structural characterization. Among the interesting features of the gas-phase approach are that it is fast (i.e., tens of milliseconds timescale) and it completely avoids sample manipulation issues. The work will involve the development of novel selective reactions, many of which will be inspired by the rich bio-conjugation literature, the development of peptide ion-based applications, and the development of whole protein/protein complex ion-based applications, as reflected in the specific aims:
Specific Aim 1 : Develop top-down protein characterization methods based on selective enzyme-like fragmentation enabled by the functional group specific ion/ion reactions discovered in the early years of this project.
Specific Aim 2 : Develop applications for the use of selective ion/ion reactions in the Msn of peptide and protein ions for the identification/characterization of sub-stoichiometric post-translational modifications in both bottom-up and top-down proteomics scenarios.
Specific Aim 3 : Develop novel approaches for protein and protein complex characterization using ion/ion charge transfer strategies. These approaches will be applied to protein complexes and large biological complexes (e.g., viruses).

Public Health Relevance

The development of novel chemistries and methodologies based on selective gas-phase reactions is relevant to many areas of biomedical research by virtue of the role that tandem mass spectrometry already plays across the spectrum of health related science. The work in this proposal leads to major advances in the measurements that underlie the various 'omics' fields that rely heavily on mass spectrometry (e.g., proteomics, metabolomics, glycomics, lipidomics, etc.). The potential bio-medical impact for this work, therefore, is unusually broad.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Krepkiy, Dmitriy
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Purdue University
West Lafayette
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Foreman, David J; Betancourt, Stella K; Pilo, Alice L et al. (2018) Novel Peptide Ion Chemistry Associated with Gold (I) Cationization: Preferential Cleavage at Lysine Residues. Int J Mass Spectrom 427:114-122
Pitts-McCoy, Anthony M; Harrilal, Christopher P; McLuckey, Scott A (2018) Gas-Phase Ion/Ion Chemistry as a Probe for the Presence of Carboxylate Groups in Polypeptide Cations. J Am Soc Mass Spectrom :
Pilo, Alice L; Zhao, Feifei; McLuckey, Scott A (2017) Gas-Phase Oxidation via Ion/Ion Reactions: Pathways and Applications. J Am Soc Mass Spectrom 28:991-1004
Zhao, Feifei; Matt, Sarah M; Bu, Jiexun et al. (2017) Joule Heating and Thermal Denaturation of Proteins in Nano-ESI Theta Tips. J Am Soc Mass Spectrom 28:2001-2010
Betancourt, Stella K; Canez, Carlos R; Shields, Samuel W J et al. (2017) Trimethylation Enhancement Using 13C-Diazomethane: Gas-Phase Charge Inversion of Modified Phospholipid Cations for Enhanced Structural Characterization. Anal Chem 89:9452-9458
Peng, Zhou; Bu, Jiexun; McLuckey, Scott A (2017) The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages. J Am Soc Mass Spectrom 28:1765-1774
Bu, Jiexun; Peng, Zhou; Zhao, Feifei et al. (2017) Enhanced Reactivity in Nucleophilic Acyl Substitution Ion/Ion Reactions Using Triazole-Ester Reagents. J Am Soc Mass Spectrom 28:1254-1261
Pilo, Alice L; Peng, Zhou; McLuckey, Scott A (2016) The dehydroalanine effect in the fragmentation of ions derived from polypeptides. J Mass Spectrom 51:857-866
Pilo, Alice L; Zhao, Feifei; McLuckey, Scott A (2016) Selective Gas-Phase Oxidation and Localization of Alkylated Cysteine Residues in Polypeptide Ions via Ion/Ion Chemistry. J Proteome Res 15:3139-46
Pilo, Alice L; Bu, Jiexun; McLuckey, Scott A (2016) Gas-Phase Oxidation of Neutral Basic Residues in Polypeptide Cations by Periodate. J Am Soc Mass Spectrom 27:1979-1988

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