Energy-resolved Collision-induced Dissociation Research Articles

Differentiation of aminohydroxypropanesulfonic acid structural isomers using tandem mass spectrometry-based methods

d-Cysteinolic acid (D-CA) is an important metabolite within the biosulfur cycle. A structural isomer, (R)-3-amino-2-hydroxypropanesulfonate ((R)-AHPS), is less common in nature but potentially can be misidentified as D-CA due to their many shared physical properties. To support confident assignment of these two isomers by use of mass spectrometry alone, this study explores the fragmentation reactions of their [M+H]+ and [M−H]− ions using collision-induced dissociation (CID). Electrospray ionization mass spectrometry (ESI-MS) experiments were conducted on authentic standards using an ion trap mass spectrometer, while a triple-quadrupole (QqQ) mass spectrometer was used in the selective reaction monitoring (SRM) mode to record energy-resolved CID. Density-functional theory (DFT) calculations were carried out at the M06/6-31+G* level of theory to study gas-phase fragmentation mechanisms. The data generated revealed kinetically-controlled fragmentations involving participation of neighboring amino groups in the positive ion mode. Negative ion mode MS analysis could distinguish the structural isomers through different collision energy-resolved results for m/z 95 product ions, CH3SO3−. DFT calculations revealed an enthalpy (ΔH) (Gibbs energy (ΔG)) gap of 31.8 (31.4) kJ/mol between transition state barriers of a concerted mechanism for D-CA, and a more preferred stepwise mechanism for (R)-AHPS.

Experimental and Theoretical Structure Elucidation of the [2 : 1] Complex Ion of Carbo[n]helicene with n=6, 7 and 8 and Ag.

Gas-phase complexes of [n]helicenes with n=6, 7 and 8 and the silver(I) cation are generated utilizing electrospray ionization mass spectrometry (ESI-MS). Besides the well-established [1 : 1] helicene/Ag+ -complex in which the helicene provides a tweezer-like surrounding for the Ag+ , there is also a [2 : 1] complex formed. Density functional theory (DFT) calculations in conjunction with energy-resolved collision-induced dissociation (ER-CID) experiments reveal that the second helicene attaches via π-π stacking to the first helicene, which is part of the pre-formed [1 : 1] tweezer complex with Ag+ . For polycyclic aromatic hydrocarbons (PAHs) of planar structure, the [2 : 1] complex with silver(I) is typically structured as an Ag+ -bound dimer in which the Ag+ would bind to both PAHs as the central metal ion (PAH-Ag+ -PAH). For helicenes, the Ag+ -bound dimer is of similar thermochemical stability as the π-π stacked dimer, however, it is kinetically inaccessible. Coronene (Cor) is investigated in comparison to the helicenes as an essentially planar PAH. In analogy to the π-π stacked dimer of the helicenes, the Cor-Ag+ -Cor-Cor complex is also observed. Competition experiments using [n]helicene mixtures reveal that the tweezer complexes of Ag+ are preferably formed with the larger helicenes, with n=6 being entirely ignored as the host for Ag+ in the presence of n=7 or 8.

Two Rings Around One Ball: Stability and Charge Localization of [1 : 1] and [2 : 1] Complex Ions of [10]CPP and C60/70 [ *

We investigate the gas-phase chemistry of noncovalent complexes of [10]cycloparaphenylene ([10]CPP) with C60 and C70 by means of atmospheric pressure photoionization and electrospray ionization mass spectrometry. The literature-known [1 : 1] complexes, namely [10]CPP⊃C60 and [10]CPP⊃C70 , are observed as radical cations and anions. Their stability and charge distribution are studied using energy-resolved collision-induced dissociation (ER-CID). These measurements reveal that complexes with a C70 core exhibit a greater stability and, on the other hand, that the radical cations are more stable than the respective radical anions. Regarding the charge distribution, in anionic complexes charges are exclusively located on C60 or C70 , while the charges reside on [10]CPP in the case of cationic complexes. [2 : 1] complexes of the ([10]CPP2 ⊃C60/70 )+ ⋅/- ⋅ type are observed for the first time as isolated solitary gas-phase species. Here, C60 -based [2 : 1] complexes are less stable than the respective C70 analogues. By virtue of the high stability of cationic [1 : 1] complexes, [2 : 1] complexes show a strongly reduced stability of the radical cations. DFT analyses of the minimum geometries as well as molecular dynamics calculations support the experimental data. Furthermore, our novel gas-phase [2 : 1] complexes are also found in 1,2-dichlorobenzene. Insights into the thermodynamic parameters of the binding process as well as the species distribution are derived from isothermal titration calorimetry (ITC) measurements.

Hydrogen Bonding Effect on the Oxygen Binding and Activation in Cobalt(III)-Peroxo Complexes.

Cobalt(III)peroxo complexes serve as model metal complexes mediating oxygen activation. We report a systematic study of the effect of hydrogen bonding on the O2 binding energy and the O-O bond activation within the cobalt(III)peroxo complexes. To this end, we prepared a series of tris(pyridin-2-ylmethyl)amine-based cobalt(III)peroxo complexes having either none, one, two, or three amino groups in the secondary coordination sphere. The hydrogen bonding between the amino group(s) and the peroxo ligand was investigated within the isolated complexes in the gas phase using helium tagging infrared photodissociation spectroscopy, energy-resolved collision-induced dissociation experiments, and density functional theory. The results show that the hydrogen bonding stabilizes the cobalt(III)peroxo core, but the effect is only 10-20 kJ mol-1. Introducing the first amino group to the secondary coordination sphere has the largest stabilization effect; more amino groups do not change the results significantly. The amino group can transfer a hydrogen atom to the peroxo ligands, which results in the O-O bond cleavage. This process is thermodynamically favored over the O2 elimination but entropically disfavored.

5-Halogenation of Uridine Suppresses Protonation-Induced Tautomerization and Enhances Glycosidic Bond Stability of Protonated Uridine: Investigations via IRMPD Action Spectroscopy, ER-CID Experiments, and Theoretical Calculations.

Uridine (Urd), a canonical nucleoside of RNA, is the most commonly modified nucleoside among those that occur naturally. Uridine has also been an important target for the development of modified nucleoside analogues for pharmaceutical applications. In this work, the effects of 5-halogenation of uracil on the structures and glycosidic bond stabilities of protonated uridine nucleoside analogues are examined using tandem mass spectrometry and computational methods. Infrared multiple photon dissociation (IRMPD) action spectroscopy experiments and theoretical calculations are performed to probe the structural influences of these modifications. Energy-resolved collision-induced dissociation experiments along with survival yield analyses are performed to probe glycosidic bond stability. The measured IRMPD spectra are compared to linear IR spectra predicted for the stable low-energy conformations of these species computed at the B3LYP/6-311+G(d,p) level of theory to determine the conformations experimentally populated. Spectral signatures in the IR fingerprint and hydrogen-stretching regions allow the 2,4-dihydroxy protonated tautomers (T) and O4- and O2-protonated conformers to be readily differentiated. Comparisons between the measured and predicted spectra indicate that parallel to findings for uridine, both T and O4-protonated conformers of the 5-halouridine nucleoside analogues are populated, whereas O2-protonated conformers are not. Variations in yields of the spectral signatures characteristic of the T and O4-protonated conformers indicate that the extent of protonation-induced tautomerization is suppressed as the size of the halogen substituent increases. Trends in the energy-dependence of the survival yield curves find that 5-halogenation strengthens the glycosidic bond and that the enhancement in stability increases with the size of the halogen substituent.

Synthesis and Stability of Mixed-Diphosphine Ligated Gold Clusters

Sub-nanometer gold clusters are promising size- and composition-tunable materials that may be used for advanced technological applications such as catalysis, energy generation, and microelectronics. Synthesis and characterization of phosphine ligated gold clusters containing different ligands provide insight into how steric and electronic effects resulting from changes in chemical functionality influence cluster size, stability, and formation in solution. Herein, we demonstrate that synthesizing gold clusters using two different diphosphines in solution at the same time results in a broad distribution of novel mixed-ligand clusters. In comparison, adding a second diphosphine to a solution of gold clusters presynthesized with another diphosphine does not result in extensive formation of mixed-ligand species. Utilizing high-mass resolution electrospray ionization mass spectrometry, we determined novel cluster compositions and observed size-dependent trends in gold clusters that undergo ligand exchange forming mixed diphosphine species. Adjacent peaks in the mass spectra, separated by characteristic mass-to-charge ratios, provide evidence for multiple 1,3-bis(diphenylphosphino)propane (L3) and 1,5-bis(diphenylphosphino)pentane (L5) ligands on cationic clusters containing 8, 10, 11, and 22 gold atoms. Energy-resolved collision-induced dissociation experiments provide qualitative insight into how different diphosphine ligands affect the relative stability of specific size gold clusters. Our results indicate that mixed-ligand clusters containing both L3 and L5 are generally more stable than their single ligand counterparts containing either L3 or L5. These molecular-level insights will facilitate the rational and scalable synthesis of gold clusters for targeted applications.

Energy-Resolved Fragmentation Aiding the Structure Elucidation of Steroid Biomarkers.

The identification and confirmation of steroid sulfate metabolites in biological samples are essential to various fields, including anti-doping analysis and clinical sciences. Ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) is the leading method for the detection of intact steroid conjugates in biofluids, but because of the inherent complexity of biological samples and the low concentration of many targets of interest, metabolite identification based solely on mass spectrometry remains a major challenge. The confirmation of new metabolites typically depends on a comparison with synthetically derived reference materials that encompass a range of possible conjugation sites and stereochemistries. Herein, energy-resolved collision-induced dissociation (CID) is used as part of UHPLC-HRMS/MS analysis to distinguish between regio- and stereo-isomeric steroid sulfate compounds. This wholly MS-based approach was employed to guide the synthesis of reference materials to unambiguously confirm the identity of an equine steroid sulfate biomarker of testosterone propionate administration.

Insights into Cisplatin Binding to Uracil and Thiouracils from IRMPD Spectroscopy and Tandem Mass Spectrometry.

The monofunctional primary complexes cis-[PtCl(NH3)2(L)]+, formed by the reaction of cisplatin, a major chemotherapeutic agent, with four nucleobases L, i.e., uracil (U), 2-thiouracil (2SU), 4-thiouracil (4SU), and 2,4-dithiouracil (24dSU), have been studied by a combination of infrared multiple photon dissociation (IRMPD) action spectroscopy in both the fingerprint (900–1900 cm–1) and the N–H/O–H stretching (3000–3800 cm–1) ranges, energy-resolved collision-induced dissociation (CID) mass spectrometry, and density functional calculations at the B3LYP/LACVP/6-311G** level. On the basis of the comparison across the experimental features and the linear IR spectra of conceivable structures, the cisplatin residue is found to promote a monodentate interaction preferentially with the O4(S4) atoms of the canonical forms of U, 4SU, and 24dSU and to the S2 atom of 2SU, yielding the most stable structures. Additional absorptions reveal the presence of minor, alternative tautomers in the sampled ion populations of 2SU and 24dSU, underlying the ability of cisplatin to increase the prospect of (therapeutically beneficial) nucleic acid strand disorder. Implication of these evidence may provide insights into drug mechanism and design.

Parallel Determination of Polypeptide and Oligosaccharide Connectivities by Energy-Resolved Collison-Induced Dissociation of Protonated O-Glycopeptides Derived from Nonspecific Proteolysis.

Collision-induced dissociation (CID) is by far the most broadly applied dissociation method used for tandem mass spectrometry (MS/MS). This includes MS/MS-based structural interrogation of glycopeptides for applications in glycoproteomics. The end goal of such measurements is to determine the monosaccharide connectivity of the glycan, the amino acid sequence of the peptide, and the site of glycosylation for each glycopeptide of interest. In turn, this allows inferences with respect to the glycoprofile of the intact glycoprotein. For glycopeptide analysis, CID is best known for the ability to determine glycosidic topology of the oligosaccharide group; however, CID has also been shown to produce amide bond cleavage of the polypeptide group. Whether structural information is obtained for the glycan or the peptide has been found to depend on the applied collision energy. While these energy-resolved fragmentation pathways have been the subject of several studies on N-linked glycopeptides, there remains a dearth of similar work on O-linked glycopeptides. In this study, MS/MS via CID was shown to provide substantial peptide backbone fragmentation, in addition to glycosidic fragmentation, in an energy-dependent manner. While qualitatively similar to previous findings for N-glycopeptides, the energy-resolved CID (ER-CID) of O-glycopeptides was found to be substantially more sensitive to the collision energy setting. Thus, deliberately obtaining either glycan or peptide dissociation is a more delicate undertaking for O-glycopeptides. Establishing a more complete understanding of O-glycopeptide ER-CID is likely to have a substantive impact on how O-glycoproteomic analysis is approached in the future.

Structural and Energetic Effects of O2'-Ribose Methylation of Protonated Pyrimidine Nucleosides.

The 2'-substituents distinguish DNA from RNA nucleosides. 2'-O-methylation occurs naturally in RNA and plays important roles in biological processes. Such 2'-modifications may alter the hydrogen-bonding interactions of the nucleoside and thus may affect the conformations of the nucleoside in an RNA chain. Structures of the protonated 2'-O-methylated pyrimidine nucleosides were examined by infrared multiple photon dissociation (IRMPD) action spectroscopy, assisted by electronic structure calculations. The glycosidic bond stabilities of the protonated 2'-O-methylated pyrimidine nucleosides, [Nuom+H]+, were also examined and compared to their DNA and RNA nucleoside analogues via energy-resolved collision-induced dissociation (ER-CID). The preferred sites of protonation of the 2'-O-methylated pyrimidine nucleosides parallel their canonical DNA and RNA nucleoside analogues, [dNuo+H]+ and [Nuo+H]+, yet their nucleobase orientation and sugar puckering differ. The glycosidic bond stabilities of the protonated pyrimidine nucleosides follow the order: [dNuo+H]+ < [Nuo+H]+ < [Nuom+H]+. The slightly altered structures help explain the stabilization induced by 2'-O-methylation of the pyrimidine nucleosides.

Ion Mobility Spectrometry and Tandem Mass Spectrometry Analysis of Estradiol Glucuronide Isomers.

Estradiol is an estrogenic steroid that can undergo glucuronidation at two different sites, which results in two estradiol glucuronide regioisomers. These isomers can be produced by different enzymes and can have different biological activities before being eliminated from the body. Although there have been previous methods that can distinguish the two isomers, these methods often have long acquisition times or high cost per analysis. In this study, traveling wave ion mobility spectrometry (TWIMS) coupled with mass spectrometry (MS) was employed to separate estradiol glucuronides using alkali metal adduction in positive ion mode, where the sodiated dimer adduct provided adequate separation both in single-component standards and in two-component mixtures. Additionally, in negative ion mode, tandem mass spectrometry (MS/MS) was used to quantitatively determine the relative composition of the two isomers. This was possible due to differences in the energetic requirements for loss of the glucuronic acid, which was characterized by energy-resolved collision-induced dissociation (CID). This work demonstrated that the intensity of the glucuronic acid neutral loss product as compared with the intensity of the intact precursor ion can be used to determine the percentage of each isomer present in a mixture. Overall, TWIMS successfully separated estradiol glucuronide isomers in positive ion mode and MS/MS via CID enables relative quantitation of each isomer in negative ion mode.

Structures and Relative Glycosidic Bond Stabilities of Protonated 2'-Fluoro-Substituted Purine Nucleosides.

The 2'-substituent is the primary distinguishing feature between DNA and RNA nucleosides. Modifications to this critical position, both naturally occurring and synthetic, can produce biologically valuable nucleoside analogues. The unique properties of fluorine make it particularly interesting and medically useful as a synthetic nucleoside modification. In this work, the effects of 2'-fluoro modification on the protonated gas-phase purine nucleosides are examined using complementary tandem mass spectrometry and computational methods. Direct comparisons are made with previous studies on related nucleosides. Infrared multiple photon dissociation action spectroscopy performed in both the fingerprint and hydrogen-stretching regions allows for the determination of the experimentally populated conformations. The populated conformers of protonated 2'-fluoro-2'-deoxyadenosine, [Adofl+H]+, and 2'-fluoro-2'-deoxyguanosine, [Guofl+H]+, are highly parallel to their respective canonical DNA and RNA counterparts. Both N3 and N1 protonation sites are accessed by [Adofl+H]+, stabilizing syn and anti nucleobase orientations, respectively. N7 protonation and anti nucleobase orientation dominates in [Guofl+H]+. Spectroscopically observable intramolecular hydrogen-bonding interactions with fluorine allow more definitive sugar puckering determinations than possible for the canonical systems. [Adofl+H]+ adopts C2'-endo sugar puckering, whereas [Guofl+H]+ adopts both C2'-endo and C3'-endo sugar puckering. Energy-resolved collision-induced dissociation experiments with survival yield analyses provide relative glycosidic bond stabilities. The N-glycosidic bond stabilities of the protonated 2'-fluoro-substituted purine nucleosides are found to exceed those of their canonical analogues. Further, the N-glycosidic bond stability is found to increase with increasing electronegativity of the 2'-substituent, i.e., H < OH < F. The N-glycosidic bond stability is also greater for the adenine nucleoside analogues than the guanine nucleoside analogues.

Dissociation pathways of protic ionic liquid clusters: Alkylammonium nitrates.

Protic ionic liquids are promising candidates for many applications, including as spacecraft propellants. For both fundamental interest and understanding clustering and dissociation during electrospray-based propulsion, it is useful to explore the dissociation pathways of protic ionic liquid clusters, as well as the factors affecting the relative contributions of each pathway to the observed MS/MS spectra. With that said, most of the published reports on ionic liquid cluster dissociation have focused on aprotic ionic liquids. The purpose of the current work is to explore the dissociation pathways (eg, loss of amine, nitric acid, or ion pair) of alkylammonium nitrates using energy-resolved collision-induced dissociation. Here, it was found that, in general, protic ionic liquids have multiple dissociation pathways-namely, protic ionic liquids can lose their neutralized cation (here, an alkylamine) or neutralized anion (here, nitric acid)-in addition to the ion pair dissociation familiar to aprotic salt and aprotic ionic liquid clusters. In general, increasing the basicity of the cation (here, through increasing the degree of alkylation) decreases the propensity to follow these alternative pathways. Interestingly, increasing the cluster size has a similar effect: as cluster size increases, nitric acid loss decreases. These results will help better model and design protic ionic liquids for electrospray-based spacecraft propulsion and help provide a better understanding for the general behavior of protic ionic liquids versus aprotic ionic liquids within mass spectrometers.

IRMPD action spectroscopy, ER-CID experiments, and theoretical approaches investigate intrinsic L-thymidine properties compared to D-thymidine: Findings support robust methodology

L-Thymidine (L-dThd) is the enantiomer of D-thymidine (dThd), a naturally-occurring pyrimidine nucleoside found within DNA nucleic acids. L-dThd, also known as Telbivudine, does not occur naturally, but in the last decade has found successful application as an antiviral medication for hepatitis B virus infection. In this work, the gas-phase conformers of the protonated and sodium cationized forms of L-dThd, [L-dThd+H]+ and [L-dThd + Na]+, are investigated using infrared multiple photon dissociation (IRMPD) action spectroscopy complemented by electronic structure calculations performed at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory. Comparisons between the experimental IRMPD spectra and theoretical linear IR spectra elucidate the stable low-energy conformations adopted by these L-dThd complexes generated by electrospray ionization. Minor 2,4-dihydroxy tautomers (T) and O2 protonated conformers contribute to the experimental [L-dThd+H]+ population, whereas conformers involving tridentate binding of Na+ to the O2, O4′, and O5′ atoms primarily contribute to the experimental [L-dThd + Na]+ population. Theory predicts a tautomer as the protonated ground conformer of [L-dThd+H]+ with thymine in an anti orientation and a tridentate (O2O4′O5′) sodium cationized ground conformer with a syn thymine orientation, consistent with theoretical predictions for [dThd+H]+ and [dThd + Na]+, respectively. Both protonated and sodium cationized L-dThd and dThd illustrate highly parallel IRMPD spectral features as expected. Survival yield analyses of data from energy-resolved collision-induced dissociation experiments elucidate the relative stabilities of [L-dThd+H]+ and [L-dThd + Na]+ as compared to the corresponding enantiomeric systems. Identical results are exhibited in the survival yield analyses as anticipated for enantiomeric complexes to simple cations. This work employs the same robust methodology that has provided structural characterization and energetic insight for similar systems preceding it to validate the parallel theoretical and experimental behaviors expected for enantiomers.

Alternated Branching Ratios by Anomaly in Collision-Induced Dissociation of Proton-Bound Hoogsteen Base Pairs of 1-Methylcytosine with 1-Methylguanine and 9-Methylguanine

A comparative study on the proton-bound complexes of 1-methylcytosine (1-mC) with 1-methylguanine (1-mG) and 9-methylguanine (9-mG), [1-mC:1-mG:H]+ and [1-mC:9-mG:H]+, respectively, was carried out using energy-resolved collision-induced dissociation (ER-CID) experiments in combination with quantum chemical calculations. In ER-CID experiments, the measured survival yields indicated an essentially identical stability for the two proton-bound complexes. In comparison with the lowest-energy structures and base-pairing energetics predicted at the B3LYP/6-311+G(2d,2p) theory level, both complexes produced in this study were suggested to be proton-bound Hoogsteen base pairs. Curiously, despite the similarity in structures, binding energetics, and potential energy surfaces predicted by the B3LYP theory, the fragment branching ratios exhibited an intriguing alternation between the two proton-bound Hoogsteen base pairs. The CID of [1-mC:1-mG:H]+ produced protonated cytosines, [1-mC:H]+, more abundantly than [1-mG:H]+, whereas that of [1-mC:9-mG:H]+ gave rise to a more pronounced production of protonated guanines, [9-mG:H]+. However, using the proton affinities of moieties predicted by the high-accuracy methods, including CBS-QB3 and the Guassian-4 theory, the anomaly known for [Cytosine:Guanine:H]+ (J. Am. Soc. Mass Spectrom. 29, 2368-2379 (2018)) successfully accounted for the alternated branching ratios. Thereby, the anomaly, more specifically, the production of proton-transferred fragments of O-protonated cytosines in the CID of proton-bound Hoogsteen base pairs, is indeed real, which is disclosed as the alternated branching ratios in the CID spectra of [1-mC:1-mG:H]+ and [1-mC:9-mG:H]+ in this study. Graphical Abstract .

Influence of 2′-fluoro modification on glycosidic bond stabilities and gas-phase ion structures of protonated pyrimidine nucleosides

The effects of 2′-fluoro substitution on the protonated gas-phase ions of the pyrimidine nucleosides are examined and compared with their previously reported canonical RNA and DNA forms. N-Glycosidic bond cleavage is the only collision-induced dissociation (CID) fragmentation pathway of protonated 2′-fluoro-2′-deoxycytidine, [Cydfl+H]+, and the major pathway of protonated 2′-fluoro-2′-deoxyuridine, [Urdfl+H]+. Based on energy-resolved CID and survival yield analysis, the N-glycosidic bond of [Cydfl+H]+ is more stable than that of [Urdfl+H]+. Further, the N-glycosidic bond stability of protonated pyrimidine nucleosides increases with increasing 2′-substituent electronegativity and follows the order F > OH > H. Gas-phase conformations of [Cydfl+H]+ and [Urdfl+H]+ are studied via infrared multiple photon dissociation (IRMPD) action spectroscopy coupled with theoretical calculations. IRMPD action spectra are measured over the IR fingerprint and hydrogen-stretching regions. Comparisons of theoretical and experimental spectra indicate that the experimentally accessed [Cydfl+H]+ and [Urdfl+H]+ conformers are highly parallel to those populated for their canonical counterparts. Evidence for gas-phase intramolecular O3′H⋯F2′ hydrogen-bonding in the IRMPD spectra of [Cydfl+H]+ and [Urdfl+H]+ allows for more definitive sugar puckering determinations than possible in the analogous canonical nucleosides.

Origin and Prediction of Highly Specific Bond Cleavage Sites in the Thermal Activation of Intact Protein Ions.

Predicting the fragmentation patterns of proteins would be beneficial for the reliable identification of intact proteins by mass spectrometry. However, the ability to accurately make such predictions remains elusive. An approach to predict the specific cleavage sites in whole proteins resulting from collision-induced dissociation by use of an improved electrostatic model for calculating the proton configurations of highly-charged protein ions is reported. Using ubiquitin, cytochrome c, lysozyme and β-lactoglobulin as prototypical proteins, this approach can be used to predict the fragmentation patterns of intact proteins. For sufficiently highly charged proteins, specific cleavages occur near the first low-basicity amino acid residues that are protonated with increasing charge state. Hybrid QM/QM' (QM=quantum mechanics) and molecular dynamics (MD) simulations and energy-resolved collision-induced dissociation measurements indicated that the barrier to the specific dissociation of the protonated amide backbone bond is significantly lower than competitive charge remote fragmentation. Unlike highly charged peptides, the protons at low-basicity sites in highly charged protein ions can be confined to a limited sequence of low-basicity amino acid residues by electrostatic repulsion, which results in highly specific fragmentation near the site of protonation. This research suggests that the optimal charge states to form specific sequence ions of intact proteins in higher abundances than the use of less specific ion dissociation methods can be predicted a priori.

Dissociation of [b5 – H][rad]+ ions composed of one α-methyltryptophan and four alanine residues: The effect of the α-methyl group

Collision-induced dissociation (CID) of hexapeptide radical cations consisting of four alanine residues, one α-methyltryptophan and a glycine residue at the C-terminus results predominantly in bond cleavages around the sterically crowded α-methyltryptophan residue, but all generated [b5 – H]+ ions in moderate abundance. Unlike in the dissociations of the [b5 – H]+ ions of the analogous [A4Woxa]+ ions, where regardless of the initial location of the unmodified tryptophan residue the CID spectra were identical, the α-methyltryptophan-containing [b5 – H]+ ions all gave different CID spectra, indicating that there was no evidence of isomerization. Losses of imine-amides and the neutral of mass 141 Da provide evidence that the radical centre is located at the α-carbon of the N-terminal residue. This N-terminal captodative structure inhibits macrocyclization, thereby preventing isomerization and leading to different dissociation products. Density functional theory (DFT) calculations on [AAAWα-MeAoxa]+, the ion that gave the largest variety of dissociation products, established that the captodative structure at the N-terminus is at the global minimum, 27 kJ mol−1 lower in energy than the π-radical cation. Calculations also showed that losses of the dialanine radical from the captodative structure and the side chain of α-methyltryptophan from the π-radical are the two lowest-energy dissociation pathways, consistent with the onsets observed in the experimental energy-resolved CID curves. The overall conclusion is that [b5 – H]+ ions that contain an unmodified tryptophan have the radical centre located on the α-carbon of the tryptophan residue, and this leaves the N-terminal amino group free to initiate macrocyclization leading to isomerization prior to dissociation. When the α-carbon is blocked by a methyl group, as in α-methyltryptophan, then the captodative structure at the N-terminal residue is favoured and this prevents macrocyclization and isomerization.

Structural and Energetic Effects of O2'-Ribose Methylation of Protonated Purine Nucleosides.

The chemical difference between DNA and RNA nucleosides is their 2'-hydrogen versus 2'-hydroxyl substituents. Modification of the ribosyl moiety at the 2'-position and 2'-O-methylation in particular, is common among natural post-transcriptional modifications of RNA. 2'-Modification may alter the electronic properties and hydrogen-bonding characteristics of the nucleoside and thus may lead to enhanced stabilization or malfunction. The structures and relative glycosidic bond stabilities of the protonated forms of the 2'-O-methylated purine nucleosides, 2'-O-methyladenosine (Adom) and 2'-O-methylguanosine (Guom), were examined using two complementary tandem mass spectrometry approaches, infrared multiple photon dissociation action spectroscopy and energy-resolved collision-induced dissociation. Theoretical calculations were also performed to predict the structures and relative stabilities of stable low-energy conformations of the protonated forms of the 2'-O-methylated purine nucleosides and their infrared spectra in the gas phase. Low-energy conformations highly parallel to those found for the protonated forms of the canonical DNA and RNA purine nucleosides are also found for the protonated 2'-O-methylated purine nucleosides. Importantly, the preferred site of protonation, nucleobase orientation, and sugar puckering are preserved among the DNA, RNA, and 2'-O-methylated variants of the protonated purine nucleosides. The 2'-substituent does however influence hydrogen-bond stabilization as the 2'-O-methyl and 2'-hydroxyl substituents enable a hydrogen-bonding interaction between the 2'- and 3'-substituents, whereas a 2'-hydrogen atom does not. Further, 2'-O-methylation reduces the number of stable low-energy hydrogen-bonded conformations possible and importantly inverts the preferred polarity of this interaction versus that of the RNA analogues. Trends in the CID50% values extracted from survival yield analyses of the 2'-O-methylated and canonical DNA and RNA forms of the protonated purine nucleosides are employed to elucidate their relative glycosidic bond stabilities. The glycosidic bond stability of Adom is found to exceed that of its DNA and RNA analogues. The glycosidic bond stability of Guom is also found to exceed that of its DNA analogue; however, this modification weakens this bond relative to its RNA counterpart. The glycosidic bond stability of the protonated purine nucleosides appears to be correlated with the hydrogen-bond stabilization of the sugar moiety.

Relative glycosidic bond stabilities of naturally occurring methylguanosines: 7-methylation is intrinsically activating.

The frequency and diversity of posttranscriptional modifications add an additional layer of chemical complexity beyond canonical nucleic acid sequence. Methylations are particularly frequently occurring and often highly conserved throughout the kingdoms of life. However, the intricate functions of these modified nucleic acid constituents are often not fully understood. Systematic foundational research that reduces systems to their minimum constituents may aid in unraveling the complexities of nucleic acid biochemistry. Here, we examine the relative intrinsic N-glycosidic bond stabilities of guanosine and five naturally occurring methylguanosines (O2'-, 1-, 7-, N2,N2-di-, and N2,N2,O2'-trimethylguanosine) probed by energy-resolved collision-induced dissociation tandem mass spectrometry and complemented with quantum chemical calculations. Apparent glycosidic bond stability is generally found to increase with increasing methyl substitution (canonical < mono- < di- < trimethylated). Many biochemical transformations, including base excision repair mechanisms, involve protonation and/or noncovalent interactions to increase nucleobase leaving-group ability. The protonated gas-phase methylguanosines require less activation energy for glycosidic bond cleavage than their sodium cationized forms. However, methylation at the N7 position intrinsically weakens the glycosidic bond of 7-methylguanosine more significantly than subsequent cationization, and thus 7-methylguanosine is suggested to be under perpetually activated conditions. N7 methylation also alters the nucleoside geometric preferences relative to the other systems, including the nucleobase orientation in the neutral form, sugar puckering in the protonated form, and the preferred protonation and sodium cation binding sites. All of the methylated guanosines examined here are predicted to have proton affinities and gas-phase basicities that exceed that of canonical guanosine. Additionally, the proton affinity and gas-phase basicity trends exhibit a roughly inverse correlation with the apparent glycosidic bond stabilities.