title: Mechanistic study of competitive releases of H2O, NH3and CO2from deprotonated aspartic and glutamic acids: Role of conformation.
abstract: The aims of this study were to highlight the impact of minor structural differences (e.g. an aminoacid side chain enlargement by one methylene group), on ion dissociation under collision-induced dissociation conditions, and to determine the underlying chemical mechanisms. Therefore, we compared fragmentations of deprotonated aspartic and glutamic acids generated in negative electrospray ionization. Energy-resolved mass spectrometry breakdown curves were recorded and MS3experiments performed on an Orbitrap Fusion for high-resolution and high-mass accuracy measurements. Activated fragmentations were performed using both the resonant and non-resonant excitation modes (i.e., CID and HCD, respectively) in order to get complementary information on the competitive and consecutive dissociative pathways. These experiments showed a specific loss of ammonia from the activated aspartate but not from the activated glutamate. We mainly focused on this specific observed loss from aspartate. Two different mechanisms based on intramolecular reactions (similar to those occurring in organic chemistry) were proposed, such as intramolecular elimination (i.e. Ei-like) and nucleophilic substitution (i.e. SNi-like) reactions, respectively, yielding anions as fumarate and α lactone from a particular conformation with the lowest steric hindrance (i.e. with antiperiplanar carboxyl groups). The detected deaminated aspartate anion can then release CO2as observed in the MS3experimental spectra. However, quantum calculations did not indicate the formation of such a deaminated aspartate product ion without loss of carbon dioxide. Actually, calculations displayed the double neutral (NH3+CO2) loss as a concomitant pathway (from a particular conformation) with relative high activation energy instead of a consecutive process. This disagreement is apparent since the concomitant pathway may be changed into consecutive dissociations according to the collision energy i.e., at higher collision energy and at lower excitation conditions, respectively. The latter takes place by stabilization of the deaminated aspartate solvated with two residual molecules of water (present in the collision cell). This desolvated anion formed is an α lactone substituted by a methylene carboxylate group. The vibrational excitation acquired by [(D-H)-NH3]-during its isolation is enough to allow its prompt decarboxylation with a barrier lower than 8.4kJ/mol. In addition, study of glutamic acid-like diastereomers constituted by a cyclopropane, hindering any side chain rotation, confirms the impact of the three-dimensional geometry on fragmentation pathways. A significant specific loss of water is only observed for one of these diastereomers. Other experiments, such as stable isotope labeling, need to be performed to elucidate all the observed losses from activated aspartate and glutamate anions. These first mechanistic interpretations enhance understanding of this dissociative pathway and underline the necessity of studying fragmentation of a large number of various compounds to implement properly new algorithms for de novo elucidation of unknown metabolites.
authors: CEA, iBiTec-S, SPI, LEMM, Metabohub Gif Sur Yvette, France.
authors: Pierre
authors: P
authors: Barbier Saint Hilaire
authors: CEA, iBiTec-S, SPI, LEMM, Metabohub Gif Sur Yvette, France; Université Paris VI (UPMC), CNRS UMR 7201, Paris Cedex 05, France. Electronic address: anna.warnet@upmc.fr.
authors: Anna
authors: A
authors: Warnet
authors: Université Grenoble Alpes et CNRS, DCM UMR 5250, Grenoble, France.
authors: Yves
authors: Y
authors: Gimbert
authors: CEA, iBiTec-S, SPI, LEMM, Metabohub Gif Sur Yvette, France.
authors: Ulli Martin
authors: UM
authors: Hohenester
authors: University of Siena, Department of Biotechnology, Chemistry and Pharmacy, Via Aldo Moro, I-53100 Siena, Italy.
authors: Gianluca
authors: G
authors: Giorgi
authors: CEA, iBiTec-S, SPI, LEMM, Metabohub Gif Sur Yvette, France.
authors: Marie-Françoise
authors: MF
authors: Olivier
authors: CEA, iBiTec-S, SPI, LEMM, Metabohub Gif Sur Yvette, France.
authors: François
authors: F
authors: Fenaille
authors: CEA, iBiTec-S, SPI, LEMM, Metabohub Gif Sur Yvette, France.
authors: Benoît
authors: B
authors: Colsch
authors: CEA, iBiTec-S, SPI, LEMM, Metabohub Gif Sur Yvette, France.
authors: Christophe
authors: C
authors: Junot
authors: CEA, iBiTec-S, SPI, LEMM, Metabohub Gif Sur Yvette, France; Université Paris VI (UPMC), CNRS UMR 7201, Paris Cedex 05, France.
authors: Jean-Claude
authors: JC
authors: Tabet
mesh_terms: D000641:Ammonia / Q000737:chemistry; D000838:Anions / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry*; D002245:Carbon Dioxide / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D011522:Protons; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013237:Stereoisomerism; D014867:Water / Q000737:chemistry
chemical_list: D000838:Anions; D011522:Protons; D014867:Water; D002245:Carbon Dioxide; D001224:Aspartic Acid; D018698:Glutamic Acid; D000641:Ammonia
keywords: Electrospray high-resolution mass spectrometry; Regioselective dissociation; Unexpected cleavage of aspartate anion