The overall thrust of this research initiative is to develop new and improved means for the structural characterization of polypeptide ions, including those formed from peptides derived from enzymatic or chemical digestion, whole proteins, and protein complexes. The near-term objective is to develop a new direction in tandem mass spectrometry that will enable a wide range of novel strategies to deal with protein 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 development of a very wide range of 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 time scale) 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 : Discover/develop functional group specific ion/ion reactions for reactive amino acid side chains, including, but not limited to, lysine (and N-termini), residues with acidic side-chains (i.e., glutamic and aspartic acids) and C-termini, arginine, and cysteine, as well as common post-translational modifications, such as phosphate and sulfate.
Specific Aim 2 : Develop applications for the use of selective ion/ion reactions in the MSn of peptide ions in support of peptide-based (i.e., bottom-up) approaches in protein identification/characterization.
Specific Aim 3 : Develop applications for the use of selective ion/ion reactions in the MSn of intact protein and protein complex ions in support of protein-based (i.e., top-down) approaches in protein identification/characterization. The development of novel chemistries and methodologies based on selective gas-phase reactions will be relevant to many areas of bio-medical research by virtue of the role that tandem mass spectrometry already plays across the spectrum of health related science. The work in this proposal must necessarily be focused, and will be directed to protein analysis. However, functional group-specific gas-phase reactions can also lead to new applications in other areas, such as metabolomics, glycomics, lipidomics, etc. The potential bio- medical impact for this work, therefore, is unusually broad.

Public Health Relevance

This project is devoted to establishing a new direction in tandem mass spectrometry that is aimed at improved protein identification/characterization via novel methods based on selective covalent and non-covalent chemistries that enable gas-phase bio-conjugation. The ability to identify and quantify proteins at the gene level, fully characteriz protein post-translational modification states, and identify interacting partners in protein complexes underlies much of fundamental biomedical research. These capabilities, which are provided by mass spectrometry, are also key to the identification of protein biomarkers and their correlation with disease.

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 (ZRG1)
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Krepkiy, Dmitriy
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Purdue University
Schools of Arts and Sciences
West Lafayette
United States
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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
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 :
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
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; McLuckey, Scott A (2016) Selective Gas-Phase Ion/Ion Reactions: Enabling Disulfide Mapping via Oxidation and Cleavage of Disulfide Bonds in Intermolecularly-Linked Polypeptide Ions. Anal Chem 88:8972-9
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
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; Peng, Zhou; McLuckey, Scott A (2016) The dehydroalanine effect in the fragmentation of ions derived from polypeptides. J Mass Spectrom 51:857-866
Peng, Zhou; Pilo, Alice L; Luongo, Carl A et al. (2015) Gas-Phase Amidation of Carboxylic Acids with Woodward's Reagent K Ions. J Am Soc Mass Spectrom 26:1686-94

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