The gas-phase structures of protonated thymidine, [dThd + H]+, and its modified form, protonated 5-methyluridine, [Thd + H]+, are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy combined with electronic structure calculations. IRMPD action spectra are measured over the ranges extending from ~600 to 1900 cm–1 and ~2800 to 3800 cm–1 using the FELIX free electron laser and an optical parametric oscillator/amplifier (OPO/OPA) laser system, respectively. Comparisons between the B3LYP/6-311+G(d,p) linear IR spectra calculated for the stable low-energy conformers and the measured IRMPD spectra are used to determine the most favorable tautomeric conformations of [dThd + H]+ and [Thd + H]+ and to identify those populated in the experiments. Both B3LYP and MP2 levels of theory predict a minor 2,4-dihydroxy tautomer as the ground-state conformer of [dThd + H]+ and [Thd + H]+ indicating that the 2'-hydroxyl substituent of Thd does not exert a significant impact on the structural features. [dThd + H]+ and [Thd + H]+ share parallel IRMPD spectral profiles and yields in both the FELIX and OPO regions. Comparisons between the measured IRMPD and calculated IR spectra suggest that minor 2,4-dihydroxy tautomers and O2 protonated conformers of [dThd + H]+ and [Thd + H]+ are populated in the experiments. Comparison of this work to our previous IRMPD spectroscopy study of protonated 2'-deoxyuridine and uridine suggests that the 5-methyl substituent alters the preferences of O2 versus O4 protonation.
The gas-phase conformations of transition metal cation-uracil complexes, [Ura+Cu]+ and [Ura+Ag]+, were examined via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical calculations. IRMPD action spectra were measured over the IR fingerprint and hydrogen-stretching regions. Structures and linear IR spectra of the stable tautomeric conformations of these complexes were initially determined at the B3LYP/6-31G(d) level. The four most stable structures computed were also examined at the B3LYP/def2-TZVPPD level to improve the accuracy of the predicted IR spectra. Two very favorable modes of binding are found for [Ura+Cu]+ and [Ura+Ag]+ that involve O2N3 bidentate binding to the 2-keto-4-hydroxy minor tautomer and O4 monodentate binding to the canonical 2,4-diketo tautomer of Ura. Comparisons between the measured IRMPD and calculated IR spectra enable elucidation of the conformers present in the experiments. These comparisons indicate that both favorable binding modes are represented in the experimental tautomeric conformations of [Ura+Cu]+ and [Ura+Ag]+. B3LYP suggests that Cu+ exhibits a slight preference for O4 binding, whereas Ag+ exhibits a slight preference for O2N3 binding. In contrast, MP2 suggests that both Cu+ and Ag+ exhibit a more significant preference for O2N3 binding. The relative band intensities suggest that O4 binding conformers comprise a larger portion of the population for [Ura+Ag]+ than [Ura+Cu]+. The dissociation behavior and relative stabilities of the [Ura+M]+ complexes, M+ = Cu+, Ag+, H+, and Na+) are examined via energy-resolved collision-induced dissociation experiments. The IRMPD spectra, dissociation behaviors, and binding preferences of Cu+ and Ag+ are compared with previous and present results for those of H+ and Na+.
Thymidine (dThd) is a fundamental building block of DNA nucleic acids, whereas 5-methyluridine (Thd) is a common modified nucleoside found in tRNA. In order to determine the conformations of the sodium cationized thymine nucleosides [dThd+Na]+ and [Thd+Na]+ produced by electrospray ionization, their infrared multiple photon dissociation (IRMPD) action spectra are measured. Complementary electronic structure calculations are performed to determine the stable low-energy conformations of these complexes. Geometry optimizations and frequency analyses are performed at the B3LYP/6-311+G(d,p) level of theory, whereas energies are calculated at the B3LYP/6-311+G(2d,2p) level of theory. As protonation preferentially stabilizes minor tautomers of dThd and Thd, tautomerization facilitated by Na+ binding is also considered. Comparisons of the measured IRMPD and computed IR spectra find that [dThd+Na]+ prefers tridentate (O2,O4',O5') coordination to the canonical 2,4-diketo form of dThd with thymine in a syn orientation. In contrast, [Thd+Na]+ prefers bidentate (O2,O2') coordination to the canonical 2,4-diketo tautomer of Thd with thymine in an anti orientation. Although 2,4-dihydroxy tautomers and O2 protonated thymine nucleosides coexist in the gas phase, no evidence for minor tautomers is observed for the sodium cationized species. Consistent with experimental observations, the computational results confirm that the sodium cationized thymine nucleosides exhibit a strong preference for the canonical form of the thymine nucleobase. Survival yield analyses based on energy-resolved collision-induced dissociation (ER-CID) experiments suggest that the relative stabilities of protonated and sodium cationized dThd and Thd follow the order [dThd+H]+ < [Thd+H]+ < [dThd+Na]+ < [Thd+Na]+.
A cationic degradation product, formed in solution from retinal Schiff base (RSB), is examined in the gas phase using ion mobility spectrometry, photoisomerization action spectroscopy, and collision induced dissociation (CID). The degradation product is found to be N-n-butyl-2-(β-ionylidene)-4-methylpyridinium (BIP) produced through 6π electrocyclization of RSB followed by protonation and loss of dihydrogen. Ion mobility measurements show that BIP exists as trans and cis isomers that can be interconverted through buffer gas collisions and by exposure to light, with a maximum response at λ = 420 nm.
Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) allows data-independent fragmentation of all ions in a sample and correlation of fragment ions to their precursors through the modulation of precursor ion cyclotron radii prior to fragmentation. Previous results show that implementation of 2D FT-ICR MS with infrared multi-photon dissociation (IRMPD) and electron capture dissociation (ECD) has turned this method into a useful analytical tool. In this work, IRMPD tandem mass spectrometry of calmodulin (CaM) has been performed both in one-dimensional and two-dimensional FT-ICR MS using a top-down and bottom-up approach. 2D IRMPD FT-ICR MS is used to achieve extensive inter-residue bond cleavage and assignment for CaM, using its unique features for fragment identification in a less time- and sample-consuming experiment than doing the same thing using sequential MS/MS experiments.
Ion mobility spectrometry (IMS) coupled with gas-phase hydrogen deuterium exchange (HDX)-mass spectrometry (MS) and molecular dynamic simulations (MDS) has been used for structural investigation of anions produced by electrospraying a sample containing a synthetic peptide having the sequence KKDDDDDIIKIIK. In these experiments the potential of the analytical method for locating charge sites on ions as well as for utilizing collision-induced dissociation (CID) to reveal the degree of deuterium uptake within specific amino acid residues has been assessed. For diffuse (i.e., more elongated) [M – 2H]2– ions, decreased deuterium content along with MDS data suggest that the D4 and D6 residues are charge sites, whereas for the more diffuse [M – 3H]3– ions, the data suggest that the D4, D7, and the C-terminus are deprotonated. Fragmentation of mobility-selected, diffuse [M – 2H]2– ions to determine deuterium uptake at individual amino acid residues reveals a degree of deuterium retention at incorporation sites. Although the diffuse [M – 3H]3– ions may show more HD scrambling, it is not possible to clearly distinguish HD scrambling from the expected deuterium uptake based on a hydrogen accessibility model. The capability of the IMS-HDX-MS/MS approach to provide relevant details about ion structure is discussed. Additionally, the ability to extend the approach for locating protonation sites on positively-charged ions is presented.
We report on the performance of a cryogenic 2D linear ion trap (cryoLIT) that is shown to be mass-selective in the temperature range of 17–295 K. As the cryoLIT is cooled, the ejection voltages during the mass instability scan decrease, which results in an effective mass shift to lower m/z relative to room temperature. This is attributed to a decrease in trap radius caused by thermal contraction. Additionally, the cryoLIT generates reproducible mass spectra from day-to-day, and is capable of performing stored waveform inverse Fourier transform (SWIFT) mass isolation of fragile N2-tagged ions for the purpose of background-free infrared dissociation spectroscopy.
The use of successive resonances for ion ejection is demonstrated here as a method of scanning quadrupole ion traps with improvement in both resolution and sensitivity compared with single frequency resonance ejection. The conventional single frequency resonance ejection waveform is replaced with a dual-frequency waveform. The two included frequencies are spaced very closely and their relative amplitudes are adjusted so that the first frequency that ions encounter excites them to higher amplitudes where space charge effects are less prominent, thereby giving faster and more efficient ejection when the ions come into resonance with the second frequency. The method is applicable at any arbitrary frequency, unlike double and triple resonance methods. However, like double and triple resonance ejection, ejection using successive resonances requires the rf and AC waveforms to be phase-locked in order to retain mass accuracy and mass precision. The improved performance is seen in mass spectra acquired by rf amplitude scans (resonance ejection) as well as by secular frequency scans.
A potassium-containing hexaazamacrocyclic dication, [M?H?K]2+, is able to add in the gas phase mono- and dicarboxylate anions as well as inorganic anions by forming the corresponding monocharged adducts, the structure of which markedly depends on the basicity of the anion. With anions, such as acetate or fluoride, the neutral hexaazamacrocycle M acts as an acceptor of monosolvated K+ ion. With less basic anions, such as trifluoroacetate or chloride, the protonated hexaazamacrocycle [M?H]+ performs the unusual functions of an acceptor of contact K+/anion pairs.
We have investigated the photoionization and photofragmentation yields of gas-phase multiply protonated melittin cations for photon energies at the K-shell absorption edges of carbon, nitrogen, and oxygen. Two similar experimental approaches were employed. In both experiments, mass selected [melittin+qH]q+ (q=2–4) ions were accumulated in radiofrequency ion traps. The trap content was exposed to intense beams of monochromatic soft X-ray photons from synchrotron beamlines and photoproducts were analyzed by means of time-of-flight mass spectrometry. Mass spectra were recorded for fixed photon energies, and partial ion yield spectra were recorded as a function of photon energy. The combination of mass spectrometry and soft X-ray spectroscopy allows for a direct correlation of protein electronic structure with various photoionization channels. Non-dissociative single and double ionization are used as a reference. The contribution of both channels to various backbone scission channels is quantified and related to activation energies and protonation sites. Soft X-ray absorption mass spectrometry combines fast energy deposition with single and double ionization and could complement established activation techniques.
Differential or field asymmetric waveform ion mobility spectrometry (FAIMS) operating at high electric fields fully resolves isotopic isomers for a peptide with labeled residues. The naturally present isotopes, alone and together with targeted labels, also cause spectral shifts that approximately add for multiple heavy atoms. Separation qualitatively depends on the gas composition. These findings may enable novel strategies in proteomic and metabolomic analyses using stable isotope labeling.
We present gas-phase structures of dimers of MnIII and FeIII meso-tetra(4-sulfonatophenyl)porphyrin multianions with various amounts of sodium and hydrogen counterions. The structural assignments are achieved by combining mass spectrometry, ion mobility measurements, quantum chemical calculations, and trajectory method collision cross section calculations. For a common charge state, we observe significant topological variations in the dimer structures of [(MTPPS)2+nX](6-n)- (M=MnIII, FeIII; X=H, Na; n?=?1–3) induced by replacing hydrogen counterions by sodium. For sodium, the dimer structures are much more compact, a finding that can be rationalized by the stronger interactions of the sodium cations with the anionic sulfonic acid groups of the porphyrins as compared to hydrogen.
The dominant gas-phase conformer of [M+3H]3+ ions of the model peptide acetyl-PSSSSKSSSSKSSSSKSSSSK has been examined with ion mobility spectrometry (IMS), gas-phase hydrogen deuterium exchange (HDX), and mass spectrometry (MS) techniques. The [M+3H]3+ peptide ions are observed predominantly as a relatively compact conformer type. Upon subjecting these ions to electron transfer dissociation (ETD), the level of protection for each amino acid residue in the peptide sequence is assessed. The overall per-residue deuterium uptake is observed to be relatively more efficient for the neutral residues than for the model peptide acetyl-PAAAAKAAAAKAAAAKAAAAK. In comparison, the N-terminal and C-terminal regions of the serine peptide show greater relative protection compared with interior residues. Molecular dynamics (MD) simulations have been used to generate candidate structures for collision cross section and HDX reactivity matching. Hydrogen accessibility scoring (HAS) for select structural candidates from MD simulations has been used to suggest conformer types that could contribute to the observed HDX patterns. The results are discussed with respect to recent studies employing extensive MD simulations of gas-phase structure establishment of a peptide system.
Ion mobility spectrometry provides information about molecular structures of ions. Hence, high resolving power allows separation of isomers which is of major interest in several applications. In this work, we couple our high-resolution ion mobility spectrometer (IMS) with a resolving power of Rp?=?100 to a time-of-flight mass spectrometer (TOF-MS). Besides, the benefit of an increased resolving power such an IMS-MS also helps analyzing and understanding the ionization processes in IMS. Usually, the coupling between IMS and TOF-MS is realized by synchronizing data acquisition of the IMS and MS resulting in two-dimensional data containing ion mobility and mass spectra. However, due to peak widths of less than 100 μs in our high-resolution IMS, this technique is not practicable due to significant peak broadening during the ion transfer into the MS and an insufficient data acquisition rate of the MS. Thus, a novel but simple interface between the IMS and MS has been designed which minimizes ion losses, allows recording of ion mobility at full IMS resolving power, and enables a shuttered transmission of ions into the MS. The interface is realized by replacing the Faraday plate used in IMS by a Faraday grid that is shielded by two additional aperture grids. For demonstration, positive product ions of benzene, toluene, and m-xylene in air are investigated. The IMS is equipped with a radioactive 3H source. Besides the well-known product ions M+ and M·NO+, a dimer ion is also observed for benzene and toluene, consisting of two molecules and three further hydrogen atoms.
The development of tandem ion mobility spectroscopy (IMS) known as IMS-IMS has led to extensive research into isomerizations of isolated molecules. Many recent works have focused on the retinal chromophore which is the optical switch used in animal vision. Here, we study a shortened derivative of the chromophore, which exhibits a rich IM spectrum allowing for a detailed analysis of its isomerization pathways, and show that the longer the chromophore is, the lower the barrier energies for isomerization are.
Gas phase infrared dissociation spectra of the radical cation, deprotonated and protonated forms of the hormone melatonin, and its complexes with alkali (Li+, Na+, and K+) and alkaline earth metal ions (Mg2+, Ca2+, and Sr2+) are measured in the spectral range 800–1800 cm?1. Minimum energy geometries calculated at the B3LYP/LACVP++** level are used to assign structural motifs to absorption bands in the experimental spectra. The melatonin anion is deprotonated at the indole-N. The indole-C linking the amide chain is the most favored protonation site. Comparisons between the experimental and calculated spectra for alkali and alkaline earth metal ion complexes reveal that the metal ions interact similarly with the amide and methoxy oxygen atoms. The amide I band undergoes a red shift with increasing charge density of the metal ion and the amide II band shows a concomitant blue shift. Another binding motif in which the metal ions interact with the amide-O and the π-electron cloud of the aromatic group is identified but is higher in energy by at least 18 kJ/mol. Melatonin is deprotonated at the amide-N with Mg2+ and the metal ion coordinates to the amide-N and an indole-C or the methoxy-O. These results provide information about the intrinsic binding of metal ions to melatonin and combined with future studies on solvated melatonin-metal ion complexes may help elucidate the solvent effects on metal ion binding in solution and the biochemistry of melatonin. These results also serve as benchmarks for future theoretical studies on melatonin-metal ion interactions.
Precise localization of post-translational modifications (PTMs) on proteins and peptides is an outstanding challenge in proteomics. While electron transfer dissociation (ETD) has dramatically advanced PTM analyses, mixtures of localization variants that commonly coexist in cells often require prior separation. Although differential or field asymmetric waveform ion mobility spectrometry (FAIMS) achieves broad variant resolution, the need for standards to identify the features has limited the utility of approach. Here we demonstrate full a priori characterization of variant mixtures by high-resolution FAIMS coupled to ETD and the procedures to systematically extract the FAIMS spectra for all variants from such data.
Extracted arrival time distributions of negative ion CID-derived fragments produced prior to traveling-wave ion mobility separation were evaluated for their ability to provide structural information on N-linked glycans. Fragmentation of high-mannose glycans released from several glycoproteins, including those from viral sources, provided over 50 fragments, many of which gave unique collisional cross-sections and provided additional information used to assign structural isomers. For example, cross-ring fragments arising from cleavage of the reducing terminal GlcNAc residue on Man8GlcNAc2 isomers have unique collision cross-sections enabling isomers to be differentiated in mixtures. Specific fragment collision cross-sections enabled identification of glycans, the antennae of which terminated in the antigenic α-galactose residue, and ions defining the composition of the 6-antenna of several of the glycans were also found to have different cross-sections from isomeric ions produced in the same spectra. Potential mechanisms for the formation of the various ions are discussed and the estimated collisional cross-sections are tabulated.
Ion isolation in a linear ion trap is demonstrated using dual resonance frequencies, which are applied simultaneously. One frequency is used to eject ions of a broad m/z range higher in m/z than the target ion, and the second frequency is set to eject a range of ions lower in m/z. The combination of the two thus results in ion isolation. Despite the simplicity of the method, even ions of low intensity may be isolated since signal attenuation is less than an order of magnitude in most cases. The performance of dual frequency isolation is demonstrated by isolating individual isotopes of brominated compounds.
Proof of concept evidence is presented for a new method for the determination of isoaspartate, an important post-translational modification. Chemical derivatization is performed using common reagents for the modification of carboxylic acids and shown to yield suitable diagnostic information with regard to isomerization at the aspartate residue. The diagnostic gas phase chemistry is probed by collision-induced dissociation mass spectrometry, on the timescale of the MS experiment and semi-quantitative calibration of the percentage of isoaspartate in a peptide sample is demonstrated.
