Positive-ion mass spectra and collision-induced dissociation of some 2,3-didehydro amino acids

The positive-ion mass spectra of a number of didehydro amino acids, ionized by electron impact and/or thermospray, and collision-induced dissociation spectra taken at collision energies of a few electron volts and keV have been performed on multiple quadrupole and reversed geometry sector instruments. Observed differences in the mass spectra and in the fragmentation patterns are explained in terms of different isomeric structures, different internal excitation energies and different ion transit times between the ion source and the collision cell. Molecular ions of unhydrated amino acids are efficiently formed both by electron impact and thermospray, whilst molecular ions of the hydrated compounds are formed more efficiently by the latter technique. The present investigation demonstrates that the use of different ionization techniques combined with mass spectrometry/mass spectrometry measurements at different collision energies yields a wealth of information relevant to structural characterization of this important class of molecules.     

Collision-induced dissociation of ester enolate ions using fourier transform mass spectrometry

A series of ester enolates was investigated by the technique of collision-induced dissociation (CID) using a Fourier transform mass spectrometer. Two primary modes of fragmentation were observed which led to CID products characteristic of the acyl and alkoxyl moieties of the ester enolates. An investigation of the fragmentation mechanism revealed that the primary fragmentation mode appears to be a sensitive function of the structure and the proton affinities of the two possible product ions. In most cases the anion (ketenyl or alkoxide) with the lower proton affinity was observed as the threshold product, suggestive of a proton-bound dimer intermediate. Interesting secondary dissociations were observed to occur from the primary product ions, as alkoxide ions fragmented to enolate ions or other stabilized anions.     

a compact high pressure ion source for high and low energy collision-induced dissociation studies of cluster ions on a VG analytical ZAB-2FQ

A compact, field-free high pressure ion source designed to replace, with minimum disruption, the electron impact/chemical ionization ion source of a VG Analytical ZAB-2FQ hybrid BEqQ mass spectrometer is described. This ion source may be operated at temperatures from ≈40 to 250 °C and at pressures up to 4–5 torr and, thus, is capable of producing proton-bound cluster ions up to hexamers in good yields. Examples of high energy collision-induced dissociation, low energy collision-induced dissociation, and neutralization-reionization studies of proton-bound cluster ions produced in this source are presented.     

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.     

Energy dependence of the fragmentation of ester enolate ions

The energy dependence of the fragmentation of a selection of ester enolate ions has been studied by variable, low-energy collision-induced dissociation experiments in the quadrupole collision cell of a hybrid BEQQ mass spectrometer. The dominant fragmentation reactions observed are where ΔH₁ ? ΔH₂=PA([RCCO]^?) ? PA([?O]^?) (PA=proton affinity). The anion of lowest proton affinity is formed preferentially at low internal energies with the yield of the anion of higher proton affinity increasing with increasing internal energy. The [CH₃OCOCOCH₂]^? anion derived from methyl pyruvate forms [CH₃OCO]^? by reaction (2); this anion readily fragments to [CH₃G]^?+ CO consistent with a structure represented by a dipole-stabilized cluster of [CH₃O]^? and CO. Comparison of the 8-keV with the 50-eV collision-induced dissociation mass spectra indicated that the average internal energy of the fragmenting ions is considerably lower in the high-energy collisional experiments than it is in the low-energy collisional experiments.     

Implementation of dipolar direct current (DDC) collision-induced dissociation in storage and transmission modes on a quadrupole/time-of-flight tandem mass spectrometer

Means for effecting dipolar direct current collision-induced dissociation (DDC CID) on a quadrupole/time-of-flight in a mass spectrometer have been implemented for the broadband dissociation of a wide range of analyte ions. The DDC fragmentation method in electrodynamic storage and transmission devices provides a means for inducing fragmentation of ions over a large mass-to-charge range simultaneously. It can be effected within an ion storage step in a quadrupole collision cell that is operated as a linear ion trap or as ions are continuously transmitted through the collision cell. A DDC potential is applied across one pair of rods in the quadrupole collision cell of a QqTOF hybrid mass spectrometer to effect fragmentation. In this study, ions derived from a small drug molecule, a model peptide, a small protein, and an oligonucleotide were subjected to the DDC CID method in either an ion trapping or an ion transmission mode (or both). Several key experimental parameters that affect DDC CID results, such as time, voltage, low mass cutoff, and bath gas pressure, are illustrated with protonated leucine enkephalin. The DDC CID dissociation method gives a readily tunable, broadband tool for probing the primary structures of a wide range of analyte ions. The method provides an alternative to the narrow resonance conditions of conventional ion trap CID and it can access more extensive sequential fragmentation, depending upon conditions. The DDC CID approach constitutes a collision analog to infrared multiphoton dissociation (IRMPD).     

On the mature of the chemical noise in MALDI mass spectra

The so-called "chemical noise background" imposes a major limit on the practical sensitivity of MALDI mass spectrometry. Typically, as the amount of material of interest subjected to MALDI analysis is reduced, the signal decreases to the point where it can no longer be differentiated from the chemical noise. Using a newly designed MALDI-ion trap mass spectrometer, we describe experiments intended to throw light on the nature of the chemical noise background and to reduce its effects. Single-stage mass spectrometric signals from peptides were observed to disappear into the noise when the amount of sample applied to the MALDI sample stage was decreased to less than a femtomole. At these low levels, analysis of the collision-induced fragmentation spectra revealed the presence of ions originating from the peptide as well as cluster ions that originate from the chemical noise. The fragmentation pattern arising from dissociation of the cluster species suggests that they are composed largely of matrix molecules. A significant fraction of these cluster ions can be dissociated at activation energies lower than the threshold for peptide fragmentation. We used this finding to collisionally pre-activate MALDI ions to remove a significant portion of the chemical noise from the spectrum, allowing us to obtain readily discernible single stage MS signals from 100 attomols of peptide. The strategy also yielded high quality MS/MS spectra from 100 attomols of peptide. Different possibilities of collisional pre-activation for improving sensitivity are considered.     

Mass spectral studies of N,N-dialkylaminoethanols

Some dialkylaminoethanols, precursors of chemical warfare agents such as V-agents and nitrogen mustards, were analyzed by electron impact (EI) and electrospray ionization (ESI) mass spectrometry. The fragmentation pathways in EI and ESI-MS/MS methods are rationalized. The collision-induced dissociation (CID) spectra of [M+H](+) ions of aminoethanols in ESI mode are clearly distinguishable from one another, including those of isomeric normal and branched chain dialkylaminoethanols. Structures can be proposed based on the general fragmentation pathways of these molecules.     

Fluorinated methyl cation (CF(3)(+), CFH(2)(+))-alkyl nitrile cluster ions

Ion-molecule reactions of CF(3)(+) or CFH(2)(+) with an acetonitrile-butyronitrile mixture yield product cluster ions in which the two neutral molecules are associated with the cation. The structures of the ion-molecule product ions were investigated by collision-induced dissociation in a multi-quadrupole mass spectrometer. The cluster ions fragment by loss of one neutral alkyl nitrile. The abundance of the fragment ions shows an unexpected inverse correlation with the proton affinity of the alkyl nitrile. This result is inconsistent with the formation of a simple cation-bound dimeric cluster containing two alkyl nitrile molecules and a fluorinated methyl cation; instead, a pair of non-symmetrical dimeric complexes, separated by a large internal barrier, is indicated. This result is analogous to that observed for the putative methyl cation-bound heterodimer of acetonitrile and butyronitrile. Collision energy dependence studies and ab initio calculations suggest that the dimeric complexes are formed in potential energy wells located in an approximately symmetrical potential energy surface. Quasi-equilibrium theory calculations were used in order to obtain additional insights into the experimental data.     

Forensic identification of explosive oxidizers by electrospray ionization mass spectrometry

The mass spectrometry of a group of inorganic oxidizers was studied using the electrospray ionization technique. It was found that a series of cluster ions were predominant in both positive- and negative-ion mode, allowing for the characterization of the investigated oxidizers. The identity of the recorded cluster ions was further confirmed by using some isotopically labeled compounds and tandem mass spectrometry with collision-induced dissociation. The use of electrospray ionization mass spectrometry for positive identification of major oxidizer components in explosive formulations was demonstrated by three samples of forensic interest.     

Gas-phase dissociation of oligoribonucleotides and their analogs studied by electrospray ionization tandem mass spectrometry

Oligoribonucleotides (RNA) and modified oligonucleotides were subjected to low-energy collision-induced dissociation in a hybrid quadrupole time-of-flight mass spectrometer to investigate their fragmentation pathways. Only very restricted data are available on gas-phase dissociation of oligoribonucleotides and their analogs and the fundamental mechanistic aspects still need to be defined to develop mass spectrometry-based protocols for sequence identification. Such methods are needed, because chemically modified oligonucleotides can not be submitted to standard sequencing protocols. In contrast to the dissociation of DNA, dissociation of RNA was found to be independent of nucleobase loss and it is characterized by cleavage of the 5'-P-O bond, resulting in the formation of c- and their complementary y-type ions. To evaluate the influence of different 2'-substituents, several modified tetraribonucleotides were analyzed. Oligoribonucleotides incorporating a 2'-methoxy-ribose or a 2'-fluoro-ribose show fragmentation that does not exhibit any preferred dissociation pathway because all different types of fragment ions are generated with comparable abundance. To analyze the role of the nucleobases in the fragmentation of the phosphodiester backbone, an oligonucleotide lacking the nucleobase at one position has been studied. Experiments indicated that the dissociation mechanism of RNA is not influenced by the nucleobase, thus, supporting a mechanism where dissociation is initiated by formation of an intramolecular cyclic transition state with the 2'-hydroxyl proton bridged to the 5'-phosphate oxygen.     

精氨酸残基在质子化多肽RRMKWKK的气相碰撞诱导解离过程中的作用

用ESI/MS-MS方法研究了质子化多肽RRMKWKK 在低能气相碰撞诱导解离(CID)条件下的碰撞能和解离路径. 研究结果表明, [M+2H]2+和[M+3H]3+的CID断裂曲线和断裂位点相似. 但质子化多肽所含正电荷个数不同时, 产生同一碎片离子的初始碰撞能不同. 碱性氨基酸残基精氨酸(Arg)的支链是多肽RRMKWKK质子化时质子优先结合的位点, 导致含有Arg的多肽在气相碰撞诱导解离条件下解离时需要较高的碰撞能. 在用质谱方法研究含精氨酸残基的多肽时应选择质子个数比多肽中Arg个数多1个的母体离子. 质子化多肽RRMKWKK的结构AM1计算结果表明, 质子化RRMKWKK中两个相邻精氨酸在空间上相互分离, 库伦斥力的影响不足以改变质子的优先结合位点.     

Collision-induced dissociation of cytidine and its derivatives

The collision-induced dissociation of adenosine, uridine and guanosine, and their corresponding nucleobases has been published previously.1-3 Here we report the collision-induced dissociation of cytidine and the elucidation of its fragmentation pathways using stable isotope-labeled cytidines, through a quadrupole ion trap for tandem mass spectrometry up to MS(4). Furthermore, we investigated the collision-induced dissociation of five cytidine derivatives: 3-methylcytidine, N(4)-methyl-2'-deoxycytidine, 5-methylcytidine, 2-thiocytidine and N(4)-acetylcytidine. The primary fragmentation pathway was the neutral loss of ribose. MS(3) on the retained nucleobase generally resulted in an intense signal from the elimination of ammonia, but also in fragment ions characteristic of the different cytosine derivatives. On the basis of the MS(n) data, fragmentation pathways and plausible mechanisms are suggested.     

Methylating peptides to prevent adduct ion formation also directs cleavage in collision-induced dissociation mass spectrometry

We investigated the effect of N-terminal amino group and carboxyl group methylation on peptide analysis by electrospray mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Permethylation of the N-terminal amino group and the carboxyl groups can reduce metal ion adducts but does not enhance sensitivity in electrospray as previously observed for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. N-terminal trimethylated peptides exhibit collision-induced dissociation (CID) tandem mass spectra that differ from their unmodified analogs; the results support the mobile proton hypothesis of peptide fragmentation. A permanent positive charge at the N-terminus leads to competition between permanent-charge directed processes and loss of the N-terminal trimethyl amino group. Carboxyl methylation has no effect on fragmentation behavior other than to shift the mass of fragments containing methylated carboxyl groups. Comparison of regular and tandem mass spectra of different methylated peptides allowed probing the location of incomplete methylation, the proton displaced by alkali metal ions and the purity of a mass-selected methylated peptide ion.     

Structure analysis of branched oligosaccharides using post-source decay in matrix-assisted laser desorption ionization mass spectrometry

Post-source decay matrix-assisted laser desorption ionization (PSD-MALDI) of sodium ion-attached branched oligosaccharides derived from glycoproteins was demonstrated as a method of structure analysis by reflectron time-of-flight (TOF) mass spectrometry. Mono-, di- and triantennary structures were investigated. The fragmentation patterns of these (structurally related) substances as obtained in the positive-ion mode showed characteristic differences correlated with branching sites and linkage positions. Two-bond ring cleavages as known from fast atom bombardment/collision-induced dissociation and IR laser desorption mass spectrometry were also observed. Internal fragment ions formed by up to four consecutive cleavages were obtained with high intensity, allowing the branching structure of complex carbohydrates to be identified. PSD-MALDI of oligosaccharides is characterized by high sensitivity, very good signal-to-noise ratios and high reproducibility of fragmentation patterns and signal intensities.     

Application of the atoms in molecules theory and computational chemistry in mass spectrometry analysis of 1,4-naphthoquinone derivatives

Mass spectrometry analysis of 2-(acylamino)-1,4-naphthoquinone derivatives was carried out using electrospray ionization ion source in combination with tandem mass spectrometry. Protonated molecules were dissociated by application of the collision-induced dissociation (CID), and the protonation sites were suggested on the basis of the HOMO, molecular electrostatic potential map (MEP), proton affinity, and Fukui functions calculated by B3LYP/6-31+G(d,p). The main fragmentation mechanisms undergone by the protonated ions were elucidated on the basis of energy, geometry, and topology analysis of equilibrium geometries. Compounds exhibiting only aliphatic hydrogens at the lateral chain undergo interesting ketene elimination. On the other hand, only the benzoylium ion formation is detected for 2-benzoylamino-1,4-naphthoquinone. The bonds geometric and atoms in molecules parameters give evidence that acidic hydrogen atoms play an important role in the fragmentation pathways.     

Erratum to: Investigation of metal-oligonucleotide complexes by nanoelectrospray tandem mass spectrometry in the positive mode

The formation and fragmentation of multiply metal-coordinated oligonucleotides was studied by nanoelectrospray tandem mass spectrometry in the positive ion mode. Fundamental aspects of the gas-phase behavior of metal-oligonucleotide complexes are revealed. The addition of transition metal ions, such as iron(II), iron(III), and zinc(II), leads to very stable metal-oligonucleotide complexes which show heavily altered fragmentation patterns in contrast to uncomplexed oligonucleotides. The site of metal ion complexation was located by collision-induced dissociation (CID) experiments. It was found that all three metal ions investigated predominantly coordinate to the central phosphate groups of the oligonucleotides. Furthermore, it is demonstrated that the fragmentation of such complexes depends highly upon the metal ion complexed as well as on the sequence of the nucleobases in the oligonucleotide.     

Investigation of metal-oligonucleotide complexes by nanoelectrospray tandem mass spectrometry in the positive mode

The formation and fragmentation of multiply metal-coordinated oligonucleotides was studied by nanoelectrospray tandem mass spectrometry in the positive ion mode. Fundamental aspects of the gas-phase behavior of metal-oligonucleotide complexes are revealed. The addition of transition metal ions, such as iron(II), iron(III), and zinc(II), leads to very stable metal-oligonucleotide complexes which show heavily altered fragmentation patterns in contrast to uncomplexed oligonucleotides. The site of metal ion complexation was located by collision-induced dissociation (CID) experiments. It was found that all three metal ions investigated predominantly coordinate to the central phosphate groups of the oligonucleotides. Furthermore, it is demonstrated that the fragmentation of such complexes depends highly upon the metal ion complexed as well as on the sequence of the nucleobases in the oligonucleotide.     

Improving peptide fragmentation by N-terminal derivatization with high proton affinity

An improved method of de novo peptide sequencing based on mass spectrometry using novel N-terminal derivatization reagents with high proton affinity has been developed. The introduction of a positively charged group into the N-terminal amino group of a peptide is known to enhance the relative intensity of b-ions in product ion spectra, allowing the easy interpretation of the spectra. However, the physicochemical properties of charge derivatization reagents required for efficient fragmentation remain unclear. In this study, we prepared several derivatization reagents with high proton affinity, which are thought to be appropriate for peptide fragmentation under low-energy collision-induced dissociation (CID) conditions, and examined their usefulness in de novo peptide sequencing. Comparison of the effects on fragmentation among three derivatization reagents having a guanidino or an amidino moiety, which differ in proton affinity, clearly indicated that there was an optimal proton affinity for efficient fragmentation of peptides. Among reagents tested in this study, derivatization with 4-amidinobenzoic acid brought about the most effective fragmentation. This derivatization approach will offer a novel de novo peptide sequencing method under low-energy CID conditions.     

Mechanism of cross-ring cleavage reactions in dirhamnosyl lipids: effect of the alkali ion

Liquid secondary ion mass spectrometry and high-energy collision-induced dissociation were used to analyze a dirhamnosyl lipid mixture. The negative fast-atom bombardment spectrum reveals a mixture of four homologous dirhamnosyl lipids with the following general structure: Rha-Rha-Cn-Cm (where Cn and Cm denote 3-hydroxy fatty acid moieties). The mass region 450-600 u in the collision-induced dissociation spectra of the negative [M - H]- ions shows product ions that can be rationalized by terminal loss of a 3-hydroxyalkanoic acid residue; these ions can be used for the characterization of the fatty acid substituents. A unique effect of alkali-metal ions on the course of fragmentation of dirhamnosyl lipid attachment ions was observed. The strong chelation of sodium is revealed from the stability of the [M - H + 2Na]+ ion that does not lose a sodium ion with the eliminated neutrals, contrary to what is observed for the dilithium adduct. Cross-ring cleavages occur during high-energy collision-induced dissociation of both positively and negatively charged precursor ions. The results suggest a concerted decomposition pathway involving the six-membered rings of the monosaccharide residues. The formation of cross-ring cleavage products, which retain the C10-C10 moiety during high-energy collision-induced dissociation of all the precursor ions that contain sodium or lithium, strongly supports a retro [2 + 2 + 2] mechanism.