Characterisation of phosphorylated nucleotides by collisional and electron‐based tandem mass spectrometry

Abstract

RationaleTandem mass spectrometry of phosphorylated ions can often yield a limited number of product ions owing to the labile nature of phosphate groups. Developing techniques to improve dissociation for this type of ion has implications for the structural characterisation of many different phosphorylated ions, such as those from nucleotides, pharmaceutical compounds, peptides and polymers.MethodsSolutions of adenosine monophosphate, diphosphate and triphosphate (AMP, ADP and ATP) were studied in a hybrid linear ion trap–Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Precursor ions with an overall single positive charge, including protonated nucleotides or nucleotide cations containing one, two or three sodium atoms, were isolated for tandem mass spectrometry. Collision‐induced dissociation (CID) was performed in the linear ion trap, with electron‐induced dissociation (EID) being conducted in the FTICR cell.ResultsEID resulted in many product ions not seen in CID. EID product ion spectra were seen to vary for AMP, ADP and ATP when the nucleotide cation contained zero, one, two or three sodiums. Precursor cations that contain two or three sodiums mainly formed product ions derived from the phosphate group. Conversely, when a precursor ion containing no sodium underwent EID, product ions mainly relating to the non‐phosphate end of the ion were observed. The number of phosphate groups was not seen to greatly affect either CID or EID product ion spectra.ConclusionsThe presence of sodium in a precursor ion directs electron‐induced bond dissociation, thus enabling targeted, and therefore tuneable, fragmentation of groups within that precursor ion. For all precursor ions, the most useful product ion spectra were obtained by EID for a precursor ion containing one sodium, with bond dissociation occurring across the entire nucleotide cation. The findings of this study can be used to improve the structural elucidation of many phosphorylated molecules by broadening the range of product ions achievable. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

Comparison of Fragmentation Techniques for the Structural Characterization of Singly Charged Agrochemicals

“…13 Lioe and O'Hair also observed minimal fragmentation via CAD and more extensive fragmentation via EID. 19 In several studies, EID has produced cross-ring fragmentation of species 8,13,34,35 along with aiding in the localization of double bonds and acyl chains 36 leading to increased confidence of assignments. Lopez-Clavijo et al 37 and Mosely et al 7 found that EID and CAD can produce complementary fragments for various species.…”

Radiation‐Induced Transfer of Charge, Atoms, and Energy within Isolated Biomolecular Systems

“…The Bernstein group has used laser desorption to bring four different neutral nucleotides in the gas phase, and irradiated them with a VUV (10.49 eV) laser [44] . In all cases, only very weak peaks of m/z ratio larger than that of the nucleobase radical cation are observed: this is surprising, given the presence of many such peaks in the mass spectra of nucleosides after interaction with a variety of ionizing particles (see the previous section), but also in the mass spectra of protonated as well as sodiated adenosine mono‐, di‐ and triphosphate nucleotides ionized by electron impact at 26.5 eV [129] . Among these peaks, several are assigned to fragment ions that involve H and/or proton transfer: a major one corresponds to the protonated nucleobase, which has also been observed after UV photoabsorption of protonated adenosine monophosphate (AMP) in the 4–12.5 eV range [130] .…”

“…Both fragments are also formed after ionization of adenosine by several particles (the so‐called d and h fragment ions), and the same process (bond cleavage in the sugar group and H transfer) also occurs for other nucleosides (see the previous section and Scheme 2). This process seems to be quite general, since it has been observed after electron impact ionization of m/z ‐selected protonated as well as sodiated adenosine mono‐, di‐ and triphosphate nucleotides at a kinetic energy of 26.5 eV [129] . Interestingly, in the mass spectrum of adenosine triphosphate ions containing sodium, a peak is assigned to sodiated adenine, even though the triphosphate moiety is the most likely binding site for sodium, consistent with the fact that after collision‐induced dissociation, almost all sodium‐containing fragment ions include at least one phosphate group.…”

Liquid Extraction Surface Analysis (LESA) Electron-Induced Dissociation and Collision-Induced Dissociation Mass Spectrometry of Small Molecule Drug Compounds

“…EID is used here as a generic term to include methods which have been previously termed as electron-induced excitation of ions from organics (EIEIO) [3], electron excitation dissociation (EED) [3], and electron ionisation dissociation (also EID) [4]. EID has been applied to the structural characterisation of peptides [4][5][6][7][8], polyketides [9], metabolites [10], lipids [11], betaine dimers [12], amino acids [5], phosphorylated nucleotides [13], glycoconjugates (including glycopeptides, glycoalkaloids and glycolipids) [14], oxo-centered trinuclear carboxylate-bridged iron complexes [15] and octaethylporphoryin and its iron complex [16]. Mosely et al [17] compared the EID mass spectra of 33 small singly charged organic molecules of pharmaceutical interest with their corresponding collision-induced dissociation (CID) mass spectra.…”