Mass spectrometric study of some protonated and lithiated 2,5-disubstituted-1,3,4-oxadiazoles

The mass spectrometric behavior of lithiated derivatives of 2,5-disubstituted-1,3,4-oxadiazoles has confirmed the skeletal rearrangement presented earlier for protonated derivatives. In the case of [M + H](+) ions the loss of isocyanic acid was observed and for [M + Li](+) ions the loss of lithium isocyanate occurred. On the other hand, benzoyl ions [RCO](+) were formed from [M + H](+) ions, but not from [M + Li](+) ions. Formation of benzoyl ions was in agreement with the differences between bond orders calculated for [M + H](+) ions and neutral molecules. From [M + Li](+) ions the [RCNLi](+) fragment ions were formed, but the formation of [RCNH](+) fragment ions from [M + H](+) ions was not observed. This result can be explained on the basis of theoretically calculated stabilities of these fragment ions, since the calculated heats of formation of [RCNLi](+) ions were found to be substantially lower than those of the respective [RCNH](+) ions.     

Near 100% selectivity in anion exchange reactions of layered zinc hydroxy nitrate

The anionic clay, zinc hydroxy nitrate was found to selectively intercalate fluoride ions from a mixture of halide ions and chloride ions from a mixture of chloride, bromide and iodide ions. In a binary mixture of chloride and bromide ions, the selectivity for chloride ions was very high (94%) even when the bromide concentration was eight times in excess. The selectivity was achieved in both concentrated and dilute salt solutions. The trend in selectivity observed here is different from what has been observed for layered double hydroxides.     

Collision-induced fragmentation of underivatized N-linked carbohydrates ionized by electrospray

The electrospray mass spectra and collision-induced fragmentation of neutral N-linked glycans obtained from glycoproteins were examined with a Q-TOF mass spectrometer. The glycans were ionized most effectively as adducts of alkali metals, with lithium providing the most abundant signal and caesium the least. Singly charged ions generally gave higher ion currents than doubly charged ions. Addition of formic acid could be used to produce [M + H]+ ions, but these ions were always accompanied by abundant cone-voltage fragments. The energy required for collision-induced fragmentation was found to increase in a linear manner as a function of mass with the [M + Na]+ ions requiring about four times as much energy as the [M + H]+ ions for complete fragmentation of the molecular ions. Fragmentation of the [M + H]+ ions gave predominantly B- and Y-type glycosidic fragments whereas the [M + Na]+ and [M + Li]+ ions produced a number of additional fragments including those derived from cross-ring cleavages. Little fragmentation was observed from the [M + K]+ and [M + Rb]+ ions and the only fragment to be observed from the [M + Cs]+ ion was Cs+. The [M + Na]+ and [M + Li]+ ions from all the N-linked glycans gave abundant fragments resulting from loss of the terminal GlcNAc moiety and prominent, though weaker, ions as the result of 0,2A and 2,4A cross-ring cleavages of this residue. Most other ions were the result of successive additional losses of residues from the non-reducing terminus. This pattern was particularly prominent with glycans containing several non-reducing GlcNAc residues where successive losses of 203 u were observed. Many of the ions in the low-mass range were products of several different fragmentation routes but still provided structural information. Possibly of most diagnostic importance was an ion formed by loss of 221 u (GlcNAc molecule) from an ion that had lost the 3-antenna and the chitobiose core. This latter ion, although coincident in mass with some other 'internal' fragments, often provided additional information on the composition of the antennae. Other ions defining antenna composition were weak cross-ring fragments produced from the core branching mannose residue. Glycans containing Gal-GlcNAc residues showed successive losses of this moiety, particularly from the B-type fragments resulting from loss of the reducing-terminal GlcNAc residue. The [M + Na]+ and [M + Li]+ ions from high-mannose and hybrid glycans gave a series of ions of composition (Man)nNa/Li+ where n = 1 to the total number of glycans in the molecule, allowing these sugars to be distinguished from the more highly processed complex glycans. Other ions in the spectra of the high-mannose glycans were diagnostic of chain branching but insufficient information was available to determine their mode of formation.     

Origin and removal of adducts (molecular mass = 98 u) attached to peptide and protein ions in electrospray ionization mass spectra

Electrospray ionization of peptides and proteins often produces intense adduct ions resulting from the attachment of a moeity with mass 98 u. The formation of these adduct ions results in a substantial reduction in the mass spectrometric sensitivity and an undesirable increase in the complexity of the mass spectra. In the present study it was shown that the removal of the attached adducts from peptide and protein ions can be affected by collisional activation and that the adducts arise from the attachment of sulfuric acid or phosphoric acid to peptide and protein ions. When sulfate and phosphate ions are removed from the samples by chemical means, adduct free ions are obtained from proteins yielding spectra with improved quality and sensitivity.     

N-(2-diethylamino)ethyl-4-aminobenzamide derivative for high sensitivity mass spectrometric detection and structure determination of N-linked carbohydrates

N-Linked glycans were derivatised by reductive amination using N-(2-diethylamino)ethyl-4-aminobenzamide (DEAEAB, procainamide) and examined by electrospray mass spectrometry. This derivative ionised primarily by protonation of the tertiary amine group and attachment of an alkali metal to give [M + H + X](2+) ions which were much more abundant that doubly charged ions from glycans derivatised with other aromatic amines. Fragmentation of these ions depended on the nature of the alkali metal (X). Lithium and sodium adducts fragmented to give prominent ions produced by cleavages within the DEAEAB derivative whereas the other adducts produced more abundant ions from fragmentation of the carbohydrate. Elimination of a sugar fragment, usually by cleavage adjacent to GlcNAc or sialic acid, together with a hydrogen atom, produced the most abundant singly charged fragment ions. These ions then formally fragmented by glycosidic cleavages. Potassium, rubidium and caesium adducts produced abundant losses of the alkali metal, but the resulting ions appeared not to undergo extensive fragmentation. Most fragment ions from all of the adducts were singly charged, the remainder being doubly charged. Although the spectra of the [M + X + H](2+) ions were not as informative as those from the singly charged ions from other derivatives, they, nevertheless, provided much valuable information on the structure of these glycans.     

Study of the dissociation of a charge-reduced phosphopeptide formed by electron transfer from an alkali metal target

Doubly protonated phosphopeptide (YGGMHRQET(p)VDC) ions obtained by electrospray ionization were collided with Xe and Cs targets to give singly and doubly charged positive ions via collision-induced dissociation (CID). The resulting ions were analyzed and detected by using an electrostatic analyzer (ESA). Whereas doubly charged fragment ions resulting from collisionally activated dissociation (CAD) were dominant in the CID spectrum with the Xe target, singly charged fragment ions resulting from electron transfer dissociation (ETD) were dominant in the CID spectrum with the Cs target. The most intense peak resulting from ETD was estimated to be associated with the charge-reduced ion with H2 lost from the precursor. Five c-type fragment ions with amino acid residues detached consecutively from the C-terminal were clearly observed without a loss of the phosphate group. These ions must be formed by N--Calpha bond cleavage, in a manner similar to the cases of electron capture dissociation (ECD) and ETD from negative ions. Although the accuracy in m/z of the CID spectra was about +/-1 Th because of the mass analysis using the ESA, it is supposed from the m/z values of the c-type ions that these ions were accompanied by the loss of a hydrogen atom. Four z-type (or y--NH3, or y--H2O) ions analogously detached consecutively from the N-terminal were also observed. The fragmentation processes took place within the time scale of 4.5 micros in the high-energy collision. The present results demonstrated that high-energy ETD with the alkali metal target allowed determination of the position of phosphorylation and the amino acid sequence of post-translational peptides.     

Coulomb effects in binding of heme in gas-phase ions of myoglobin

Coulomb effects in binding of heme in gas-phase holomyoglobin ions are studied. Positive and negative ions are formed from solution myoglobin with Fe(2+) (ferromyoglobin) and Fe(3+) (ferrimyoglobin). The energy that must be added to the resulting holomyoglobin ions to cause heme loss has been measured by triple-quadrupole tandem mass spectrometry. With negative ions, neutral heme is lost regardless of the charge state of Fe in solution. It is likely that the Fe(3+) is reduced to Fe(2+) in the negative electrospray process. With positive ions, predominantly neutral heme loss is observed with ions formed from ferromyoglobin in solution, and positive heme loss with ions formed from ferrimyoglobin in solution. The energies required to induce neutral heme loss are similar for positive and negative ions. The energies required to induce charged heme loss from positive holomyoglobin ions are significantly less. Coulomb repulsion between the charged heme and charged protein appears to lower the barrier for heme loss. These results are consistent with a simple model potential with a long-range Coulomb repulsion and short-range attraction between the heme and protein.     

Evidence for thermal decomposition contributions to the mass spectra of chlorinated phenoxyacid herbicides obtained by particle beam liquid chromatography mass spectrometry

The spectral quality of a group of chlorinated phenoxyacid herbicides has been shown to degrade under certain conditions upon introduction into the mass spectrometer by a particle beam interface. Experiments were performed to investigate these changes in spectra. Normalized ion chromatograms were generated for the herbicides, and the results showed a broadening of the profiles of some ions, indicating a longer residence time in the ion source. These ions were postulated as coming from the ionization of thermal degradation products from the herbicides. The generation of these ions was dependent on ion source temperature, analyte concentration, and, by implication, ion source cleanliness. Tandem mass spectrometry experiments were performed on these ions from the herbicides and ions from the corresonding phenols. The tandem mass spectra of the ions from the herbicides were similar to the tandem mass spectra of the ions from the phenols. Therefore, it appears that the particle beam mass spectra of the chlorinated phenoxyacid herbicides are composite spectra with contributions from the gas phase ionization of the parent herbidides and thermal decomposition products.     

Halogeno-substituted 2-aminobenzoic acid derivatives for negative ion fragmentation studies of N-linked carbohydrates

Negative ion electrospray mass spectra of high-mannose N-linked glycans derivatised with 2-aminobenzoic acids and ionised from solutions containing ammonium hydroxide gave prominent [M-H](-) ions accompanied by weaker [M-2H](2-) ions. Fragmentation of both types of ions gave prominent singly charged glycosidic cleavage ions containing the derivatised reducing terminus and ions from the non-reducing terminus that appeared to be products of cross-ring cleavages. Differentiation of these two groups of ions was conveniently achieved in a single spectrum by use of chloro- or bromo-substituted benzoic acids in order to label ions containing the derivative with an atom with a distinctive isotope pattern. Fragmentation of the doubly charged ions gave more abundant fragments, both singly and doubly charged, than did fragmentation of the singly charged ions, but information of chain branching was masked by the appearance of prominent ions produced by internal cleavages.     

Collision-induced fragmentation of negative ions from N-linked glycans derivatized with 2-aminobenzoic acid

N-Linked glycans from bovine ribonuclease B, chicken ovalbumin, bovine fetuin, porcine thyroglobulin and human alpha(1)-acid glycoprotein were derivatized with 2-aminobenzoic acid by reductive amination and their tandem mass spectra were recorded by negative ion electrospray ionization with a quadrupole time-of-flight mass spectrometer. Derivatives were also prepared from 2-amino-5-methyl- and 2-amino-4,5-dimethoxybenzoic acid in order to confirm the identity of fragment ions containing the reducing terminus. Major fragments from the [M - H](-) ions from the neutral glycans retained the derivative (Y-type cleavages) and provided information on sequence and branching. Other major fragments were products of A-type cross-ring cleavages giving information on antenna structure. Singly doubly and triply charged ions were formed from sialylated glycans. They produced major fragments by loss of sialic acid and a series of singly charged ions that were similar to those from the neutral analogues. Doubly charge ions were also produced by the neutral glycans and were fragmented to form product ions with one and two charges. Again, the fragment ions with a single charge were similar to those from the singly charged parents, but branching information was less obvious because of the occurrence of more abundant ions produced by multiple cleavages. Detection limits were around 200 fmol (3 : 1 signal-to-noise ratio).     

Multiply charged carbon ions produced from C2H2 and CH4 molecules in energetic,heavy ion impact

Multiply charged carbon ions up to C⁵⁺ ions have been observed from C₂H₂ and CH₄ molecules in 1.05 MeV/amu Ar¹²⁺ ion impact. These ions are believed to be produced through processes where multiply charged molecular ions are produced first by direct ionization and, then, dissociate via Coulomb explosion into atomic ions. The peak positions of ions from C₂H₂ molecules are found to be shifted with respect to those of ions from CH₄ molecules and this shift is understood to be due to the initial kinetic energy provided through the Coulomb potential between the dissociating ions.     

Decationization and dealumination of clinoptilolite tuff and ammonium exchange on acid-modified tuff

The paper presents results of investigation of exchange of the clinoptilolite tuff cations with hydrogen ions from HCl solution of concentration 0.1 mmol cm(-3) and ammonium ions solutions of concentrations 0.0071 to 2.6 mmol cm(-3). Molal concentrations, x (mmol g(-1)) of cations exchanged in acid solution and in ammonium ions solutions were compared with molal concentrations of cations obtained by determination of the cation-exchange capacity of clinoptilolite tuff. The obtained results show that at ammonium ion concentrations lower than 0.1 mmol cm(-3), with regard to exchange capacity for particular ions, best exchanged are Na+ ions, followed by Mg2+ and Ca2+ ions, while exchange of K+ ions is the poorest (Na+ > Mg2+ > Ca2+ > K+). At ammonium concentrations from 0.2 to 1 mmol cm(-3) the order is Na+ > Ca2+ > Mg2+ > K+. At concentrations higher than 1 mmol cm(-3) the order is Na+ > Ca2+ > K+ > Mg2+. The results are a consequence of the uptake of hydrogen ions by zeolite samples in ammonium ions solutions at concentrations lower than 1 mmol cm(-3) and indicate the importance of Mg2+ (besides Na+ ions) for the exchange between clinoptilolite cations and H+ ions, in contrast to K+ ions, whose participation in the reaction with H+ ions is the lowest. During decationization of the clinoptilolite in acid solution, best exchanged are Na+, Mg2+, and Ca2+ ions, while exchange of K+ ions is the poorest. Due to poor exchange of K+ and H+ ions and good exchange of Na+, Mg2+, and Ca2+ ions, it is to be assumed that preservation of stability of the clinoptilolite structure is caused by K+ ions present in the channel C. Clinoptilolite is dissolved in the clinoptilolite A and B channels where Na+, Mg2+, and Ca2+ ions are present. On the acid-modified clinoptilolite samples, exchange of ammonium ions is poorer than on natural zeolite. The longer the contact time of the zeolite and acid solution, the worse ammonium ions exchange. It can be assumed that H+ ions exchanged with zeolite cations are consumed for solution of aluminum in the clinoptilolite structure; therefore the concentration of H+ ions as exchangeable cations decreases. In the ammonium ion solution at a concentration of 0.0065 mmol cm(-3), from the acid-modified zeolite samples, Al3+ ions are exchanged best, followed by Na+, Mg2+, Ca2+, and K+ ions. Further to the results, it is to be assumed that exchangeable Al3+ ions available from clinoptilolite dissolution are best exchanged with H+ ions in acid solution.     

Evidence for Sequence Scrambling in Collision-Induced Dissociation of y-Type Fragment Ions

Sequence scrambling from y-type fragment ions has not been previously reported. In a study designed to probe structural variations among b-type fragment ions, it was noted that y fragment ions might also yield sequence-scrambled ions. In this study, we examined the possibility and extent of sequence-scrambled fragment ion generation from collision-induced dissociation (CID) of y-type ions from four peptides (all containing basic residues near the C-terminus) including: AAAAHAA-NH₂ (where “A” denotes carbon thirteen (¹³C₁) isotope on the alanine carbonyl group), des-acetylated-α-melanocyte (SYSMEHFRWGKPV-NH₂), angiotensin II antipeptide (EGVYVHPV), and glu-fibrinopeptide b (EGVNDNEEGFFSAR). We investigated fragmentation patterns of 32 y-type fragment ions, including y fragment ions with different charge states (+1 to +3) and sizes (3 to 12 amino acids). Sequence-scrambled fragment ions were observed from ~50 % (16 out of 32) of the studied y-type ions. However, observed sequence-scrambled ions had low relative intensities from ~0.1 % to a maximum of ~12 %. We present and discuss potential mechanisms for generation of sequence-scrambled fragment ions. To the best of our knowledge, results on y fragment dissociation presented here provide the first experimental evidence for generation of sequence-scrambled fragments from CID of y ions through intermediate cyclic “b-type” ions.

ViscosityB-coefficients,structural entropies and heat capacities,and the effects of ions on the structure of water

The water-structural contributions to the entropies and heat capacities of hydration of over 120 ions and the viscosity B-coefficients of nearly 80 aqueous ions are tabulated and correlated. B-coefficients for many more ions are predicted from this relationship and from their dependence on ionic size and charge. The structural entropies determine a unique scale of water structure making and breaking by the ions.     

Surface induced reactions of cluster ions

First results are presented from a new apparatus, consisting of a supersonic beam for generating neutral clusters, a variable energy electron gun for ionizing the clusters, and a tandem mass spectrometer set-up for studying surface induced reactions of mass and energy selected cluster ions. Rare gas cluster ions, fragment ions from SF₆, benzene ions and benzene cluster ions have been investigated so far. Cluster ion dissociation, intracluster ion molecule reactions and surface reactions with adsorbed hydrocarbons have been shown to be important reaction channels for these ion-surface collision at energies ranging from a few eV to 500 eV. The surface induced fragmentation spectrum is demonstrated to be a useful tool for probing binding energy and structure of cluster ions.     

Two-dimensional analysis of phospholipids by capillary liquid chromatography/electrospray ionization mass spectrometry

A phospholipid mixture extracted from cultured cells was directly analyzed by capillary (Cap) liquid chromatography (LC)/electrospray ionization (ESI) mass spectrometry (MS). Using a quadrupole mass spectrometer, we analyzed positive molecular ions, negative molecular ions, positive fragment ions and negative fragment ions under four different functions. In the analysis of the elution patterns of the phospholipids, a two-dimensional map, in which the first dimension is elution time and the second dimension is mass, proved useful. Consequently, four different maps can be obtained by each of four different functions. Among them, from negative fragment ions at high cone voltage in the negative ion mode, ions that originated from acyl fatty acid and phosphorylcholine, phosphorylethanolamine and cyclic inositol phosphate can be detected at specific elution times. The map from positive fragment ions at high cone voltage in the positive ion mode indicated ions such as diradylglycerol and derivatives of 1-alkyl or 1-alkenyl cyclic phosphatidic acid from phosphatidylethanolamine (PE), and phosphorylcholine from choline-containing phospholipids. The map produced from positive molecular ions indicated choline-containing phospholipids such as phosphatidylcholine, sphingomyelin, lysophosphatidylcholine and PE. The map of negative molecular ions effectively indicated acidic phospholipids such as phosphatidylinositol. We were able to obtain more than 500 molecular species of phospholipids by this method within a few hours immediately after extraction from culture cells using a mixture of chloroform and methanol (2:1). In this context, we concluded that the combination of Cap-LC and ESIMS seems to be very effective in the analysis of phospholipid classes and their molecular species.     

Time-resolved observation of product ions generated by 157 nm photodissociation of singly protonated phosphopeptides

Vacuum UV photodissociation tandem mass spectra of singly charged arginine-terminated phosphopeptides were recorded at times ranging from 300 ns to ms after photoexcitation, to investigate when the phosphate group falls off from the precursor and product ions and whether loss of phosphate can be eliminated in tandem mass spectra. For peptide ions containing phosphoserine and phosphothreonine, little loss of 98 Da from the product ions was observed up to 1 μs after photoexcitation. However, neutral losses from the precursor ions were considerable just 300 ns after photoactivation. Loss of 98 Da from product ions first appears about 1 μs after laser irradiation and becomes more common 13 μs after photoexcitation. Consistent with previous reports, phosphotyrosine was more stable than either phosphoserine or phosphothreonine.     

Determination of calcium in complex samples using functional magnetic beads combined with electrodeless/sheathless electrospray ionization mass spectrometry

Conventionally, electrospray ionization ion trap mass spectrometry (ESI-ITMS) is not used for the analysis of metal ions because metal ions may be of too low mass to be trapped by the mass analyzer. Furthermore, metal ions can easily precipitate during ESI processes because of their poor volatilities. We present an approach for solving these two problems using milk as the sample to demonstrate the feasibility of using ESI-ITMS as the detection method for metal ions. Iron oxide magnetic beads with nitrilotriacetic acid (NTA) immobilized on their surface were used as affinity probes for calcium ions from dairy drinks. Ethylenediaminetetraacetic acid (EDTA) was used for the elution of the metal ions chelated by NTA on the beads. The EDTA eluent was analyzed by electrodeless/sheathless ESI-ITMS. The use of functional magnetic beads to trap metal ions from complex samples can effectively reduce the interference from sample matrix. Using the EDTA eluent as the sample avoids the problem of the low volatility of metal ions during ESI-ITMS analysis. This method gives measurements of calcium ions in dairy drinks quantitatively very close to the true values (<4% error).     

Dissociation of multiply charged negative ions for hirudin (54–65), fibrinopeptide B, and insulin A (oxidized)

Collision-induced dissociation (CID) was performed on multiply deprotonated ions from three commercial peptides: hirudin (54-65), fibrinopeptide B, and oxidized insulin chain A. Ions were produced by electrospray ionization in a Fourier transform ion cyclotron resonance mass spectrometer. Each of these peptides contains multiple acidic residues, which makes them very difficult to ionize in the positive mode. However, the peptides deprotonate readily making negative ion studies a viable alternative. The CID spectra indicated that the likely deprotonation sites are acidic residues (aspartic, glutamic, and cysteic acids) and the C-terminus. The spectra are rife with c, y, and internal ions, although some a, b, x, and z ions form. Many of the fragment ions were formed from cleavage adjacent to acidic residues, both N- and C-terminal to the acidic site. In addition, neutral loss (e.g., NH3, CH3, H2O, and CO2) was prevalent from both the parent ions and from fragment ions. These neutral eliminations were often indicative of specific amino acid residues. The fragmentation patterns from several charge states of the parent ions, when combined, provide significant primary sequence information. These results suggest that negative mode CID of multiply deprotonated ions provides useful structural information and can be worthwhile for highly acidic peptides that do not form positive ions in abundance.     

Secondary-ion and electron production from surfaces bombarded by large polyatomic ions.

Heavy molecular ions with energies in the range 10-20 keV and masses from 276 u to 132,000 u, produced by matrix-assisted laser desorption, were used as primary projectiles to produce secondary-ion spectra from a variety of surfaces in a tandem time-of-flight mass spectrometer. In the negative mode the ratio of electron emission to secondary-ion emission was found to decrease rapidly with increasing projectile mass. Ion emission was found to dominate for primary ions larger than approximately 10,000 u. Positive or negative molecular ions and cations were observed from several organic targets of masses up to 1140 u (gramicidin S) for incident projectiles up to mass 132,000 u, i.e., for projectile speeds down to approximately 7000 m/s. Other ions characteristic of the target were also observed for these projectiles. Thus, large polyatomic ions can cause secondary-ion desorption even at very low velocity. The background ions of both polarities are similar to those found in keV particle bombardment by monatomic projectiles. The same ions are observed for all the projectiles; most can be identified with hydrocarbon background. The relative intensities of the background positive ions are largely independent of projectile, and for both polarities the ratio of the ions characterizing the target to those forming the background is approximately constant for all the projectiles. These results strongly suggest that the background ions come from the usual layer of organic impurities attached to the target surface. No direct evidence for surface-induced dissociation was observed in this mass and energy range.