Fast atom bombardment tandem mass spectrometry employing ion-molecule reactions for the differentiation of phospholipid classes

Fast atom bombardment tandem mass spectrometry, employing ion-molecule reactions with ethyl vinyl ether in a triple-quadrupole mass spectrometer, is used to differentiate classes of phospholipids. The phospholipids are desorbed and ionized by fast atom bombardment, mass-selected by the first quadrupole, and reacted with ethyl vinyl ether in the second quadrupole; the resulting product ions are analyzed by the third quadrupole. The protonated molecules and reaction product ions observed permit the differentiation of various phospholipid classes. The pattern of addition reaction products formed is shown to depend solely on the functionality of the lipid polar head group and not on the fatty acyl constituents. Neutral gain scans that are specific for each phospholipid class are performed. Ion dissociation products are observed in the same scan as the ion reaction products to provide data on the fatty acyl composition and position on the glycerophosphate core along with the phospholipid class. Although this method is less sensitive than neutral loss scanning for most phospholipid classes, it can (1) identify phospholipids that do not readily lose their head group as a neutral fragment and (2) detect phospholipids in mixtures containing species that give interfering neutral losses.     

The reaction intermediate spectrum. A new type of experiment in tandem mass spectrometry

A new scan is described which responds to ions that are intermediates in the dissociation of a mass-selected parent ion (m_p) to give a mass-selected daughter ion (m_d). The scan gives a simple mass v. abundance output for ions which satisfy this condition. It is implemented here on a BEQQ hybrid mass spectrometer using, in sequence, collision-induced dissociation occurring at high energy in the first reaction region, and low-energy collisional activation in the collision quadrupole. The experiment provides information on reaction sequences not available from single scans of other types. In the several cases examined, it is demonstrated that, among many conceivable fragmentation routes connecting a parent ion with a particular fragment ion, only a few are significant. Examination of reaction intermediate spectra also appears to be a fruitful new approach to mechanistic questions, as illustrated by consideration of the behavior of several isomeric octanones. These new spectra also have analytical value: they show good signal-to-noise ratios and allow ready distinction between isobaric and isomeric ions. A comparison of the reaction intermediate spectrum with a daughter spectrum obtained by the B/E linked-scanning technique reveals the contributions of artifact peaks which result from poor parent ion mass resolution in the latter. Reaction intermediate spectra combine information from the daughter spectra of m_p and the parent spectra of m_d and, as a specified portion of this data domain, have unique characteristics.     

Ion structure determinations and ion—molecule reactions by double quadrupole mass spectrometry

The QQ mass spectrometer is shown to be applicable to ion structure determination via collision-induced dissociations of mass-selected ions. The instrument can be scanned so as to record the products of dissociation as well as those of ion—molecule association reactions. The dissociations correspond to those observed at high kinetic energy in mass-analyzed ion kinetic energy spectrometers and the association reactions show parallels with reactions seen in ion cyclotron resonance spectroscopy and in high-pressure mass spectrometry     

Nature of methyl cation bonding to dihydroxybenzenes elucidated using an ion trap mass spectrometer

Methylation of the isomeric dihydroxybenzeres using the dimethylfluoronium ion ([CH₃FCH₃]⁺) was studied in a quadrupole ion trap mass spectrometer. The products were characterized by tandem mass spectrometry using collision-activated dissociation. A comparison of the daughter ion spectra of the methylated products with those of model ions, generated by protonation of substituent- and ring-methylated analogs, demonstrates that a mixture of methylated products is generated. Included are structures in which the methyl is σ-bonded to the ring and others with σ-bonds to the heteroatom, the latter being favored in catechol and hydroquinone. The energy-resolved daughter ion spectra for the methylated isomers, acquired by varying the amplitude of the a.c. voltage used to excite resonantly the mass-selected ions, support these conclusions regarding the site of methylation.     

Quantitative selected ion flow tube mass spectrometry: The influence of ionic diffusion and mass discrimination

Selected ion flow tube mass spectrometry, (SIFT-MS), involves the partial conversion of mass-selected precursor ions to product ions in their reactions with the trace gases in an air sample that is introduced into helium carrier gas in a flow tube. The precursor and product ions are then detected and counted by a downstream quadrupole mass spectrometer. Quantification of particular trace gases is thus achieved from the ratio of the total count rate of the product ions to that for the precursor ions. However, it is important to appreciate that in this ion chemistry the light precursor ions (usually H3O+ ions) are invariably converted to heavier product ions. Hence, the product ions diffuse to the flow tube walls more slowly and thus they are more efficiently transported to the downstream mass spectrometer sampling orifice. This phenomenon we refer to as diffusion enhancement. Further, it is a well-known fact that discrimination can occur against ions of large mass-to-charge ratio, (m/z), in quadrupole mass spectrometers. If not accounted for, diffusion enhancement usually results in erroneously high trace gas concentrations and mass discrimination results in erroneously low concentrations. In this experimental investigation, we show how both these counteracting effects can be accounted for to increase the accuracy of SIFT-MS quantification. This is achieved by relating the currents of ions of various m/z that arrive at the downstream mass spectrometer sampling orifice disc to their count rates at the ion detector after mass analysis. Thus, both diffusion enhancement and mass discrimination are parameterized as a function of m/z and these are combined to provide an overall discrimination factor for the particular analytical instrument.     

Analysis of natural products by tandem mass spectrometry employing reactive collisions with ethyl vinyl ether

Representative natural products from the diterpene dilactone, psorospermin and quabalactone classes were protonated, mass-selected and reacted with ethyl vinyl ether in a triple-quadrupole mass spectrometer. Minor differences in the structures of the compounds led to different reactivities toward the reagent, as indicated by the relative abundances of products such as the ethylated and vinylated compounds. Additional information is obtained from the dissociation products formed upon non-reactive, inelastic collisions with the neutral reagent. The daughter spectra have excellent signal: noise ratios and good reproducibility. The results demonstrate that the use of reactive collisions may supplement collision-activated dissociation in chemical analysis of large organic molecules by tandem mass spectrometry.     

Estimation of mutagenic/carcinogenic potential of environmental contaminants by ion-molecule reactions and tandem mass spectrometry

The ability to produce and detect products of model DNA/carcinogen ion-molecule reactions is demonstrated in the ion source and the collision cell of a triple quadrupole tandem mass spectrometer. Reaction between adenine and benzoyl chloride in the ion source is shown to produce the DNA adduct benzoyl adenine. The daughter ion mass spectrum of the reaction product is compared to that of the synthesized standard. Mass chromatograms of the reaction between mass-selected pyridine ions and various analytes eluting from a GC column into the collision cell are demonstrated and illustrate the ability to detect only the GC eluates that react with pyridine. This technique could provide a rapid and sensitive method for screening complex environmental samples for carcinogens, as well as for estimating the relative mutagenic/carcinogenic potential of environmental contaminants.     

Matrix-assisted laser desorption/ionization tandem reflectron time-of-flight mass spectrometry of fullerenes

Atandem reflectron time-of-flight mass spectrometer developed in our laboratory provides a unique opportunity to investigate the collision-induced dissociation of fullerene ions formed by matrix-assisted laser desorption/ionization (MALDI). Specifically, this opportunity arises from the ability to utilize high energy collisional activation (normally available only on tandem sector instruments by using continuous ionization techniques) for ions formed by pulsed laser desorption, whereas most MALDI time-of-flight instruments record product ion mass spectra of ions formed by metastable or postsource decay. In this study we investigate the products of mass-selected and collisionally activated C ₆₀ ⁺ and C ₇₀ ⁺ ions by using different target gases over a range of target gas pressures. In general, heavier target gases produce more extensive fragmentation and improve the mass resolution of lower mass ionic products because a greater portion of these ions are formed by single collisions. Additionally, the tandem time-of-flight instrument utilizes a nonlinear (curved-field) reflectron in the second mass analyzer that enables high energy collision-induced dissociation spectra to be recorded without scanning or stepping the reflectron voltage.     

Selective isotopic exchange of polyfimctional ions in tandem mass spectrometry: Methodology,applications and mechanism

Isotopic exchange of mass-selected odd- and even-electron molecular ions of aromatic compounds upon collision with deuterated gases was investigated as a function of reagent gas, interaction time and collision energy. Use of ND₃ as reagent allows exchange of all active hydrogens for the compound types studied, providing a count of the total number of active hydrogens present in the analyte. CH₃OD exchanges specific types of active hydrogens, such as phenolic and carboxylic hydrogens, without exchanging amino hydrogens. This selectivity assists in the identification and enumeration of different types of active hydrogens present in polyfunctional compounds. The H–D exchange patterns serve to differentiate isomeric aromatic compounds containing methoxy, amino, hydroxy and carboxylic acid substituents. Trapping of mass-selected ions in the collision region of a triple quadrupole mass spectrometer greatly enhances the degree of H–D exchange, thereby facilitating determination of the number of active hydrogens in the analyte. Triple stage mass spectrometric experiments, performed in a pentaquadrupole mass spectrometer, help elucidate the exchange process. Isotopic exchange in the collision region of a tandem mass spectrometer also provides insights into the site of protonation in molecules containing several functional groups. The proximity of the functional groups and the proton affinity difference between the analyte and the reagent gas are important factors in site-specific H–D exchange in polyfunctional compounds. An investigation of the effects of collision energy reveals that cluster ion formation plays a major role in the exchange mechanism operating in the triple quadrupole and that H–D exchange, ion-molecule adduct formation and endothermic fragmentation are competitive reaction channels.     

Collision-induced dissociation of glycero phospholipids using electrospray ion-trap mass spectrometry.

Characterisation of phospholipids was achieved using collision-induced dissociation (CID) with an ion-trap mass spectrometer. The product ions were compared with those obtained with a triple quadrupole mass spectrometer. In the negative ion mode the product ions were mainly sn-1 and sn-2 lyso-phospholipids with neutral loss of ketene in combination with neutral loss of the polar head group. Less abundant product ions were sn-1 and sn-2 carboxylate anions. CID using a triple quadrupole mass spectrometer, however, gave primarily the sn-1 and sn-2 carboxylate anions together with lyso-phosphatidic acid with neutral loss of water. For the ion trap a charge-remote-type mechanism is proposed for formation of the lyso-phospholipid product ions by loss of alpha-hydrogen on the fatty acid moiety, electron rearrangement and neutral loss of ketene. A second mechanism involves nucleophilic attack of the phosphate oxygen on the sn-1 and sn-2 glycerol backbone to form carboxylate anions with neutral loss of cyclo lyso-phospholipids. CID (MS(3) and MS(4)) of the lyso-phospholipids using the ion-trap gave the same carboxylate anions as those obtained with a triple quadrupole instrument where multiple collisions in the collision cell are expected to occur. The data demonstrate that phospholipid species determination can be performed by using LC/MS(n) with an ion-trap mass spectrometer with detection of the lyso-phospholipid anions. The ion-trap showed no loss in sensitivity in full scan MS(n) compared to multiple reaction monitoring data acquisition. In combination with on-line liquid chromatography this feature makes the ion-trap useful in the scanning modes for rapid screening of low concentrations of phospholipid species in biological samples as recently described (Uran S, Larsen A, Jacobsen PB, Skotland T. J. Chromatogr. B 2001; 758: 265).     

Solvation in electrospray mass spectrometry: effects on the reaction kinetics of fragmentation mediated by ion-neutral complexes

In electrospray ionization (ESI) on a triple quadrupole mass spectrometer, benzydamine, a molecule with an N,N-dimethylaminopropoxyl side chain, showed a fragmentation pattern in Q1 scans that is dramatically different from the mass-selected collision-induced dissociation (CID) of its MH(+) ion. The N,N-dimethylimmonium ion, which dominates in Q1 scans at higher energies, is only a minor product in all CID spectra. By using a smaller model molecule, N,N,N',N'-tetramethyl-1,3-propanediamine, with the kinetic energy release measured for the corresponding reaction, we have demonstrated that an ion-neutral complex composed of the N,N-dimethylazetidine cation and a neutral counterpart is involved. When the ion-neutral complex intermediate evolves toward elimination to form the immonium ion, the transition state is stabilized by the neutral species. Solvation of the ion-neutral complex, which obstructs the separation of the two partners by the resulting tighter enclosure, facilitates the elimination by enhancing the stabilization of the transition state. Therefore, the prevalence of the immonium ion in Q1 scans was a result of solvation in the ESI source. In CID reactions, where the decomposing ions are mass-selected and thus solvation does not exist, the immonium ion was a minor product, and the separation of the ion-neutral complex became dominant.     

Application of the statistical test of equivalent pathways (STEP) method to the triple quadrupole mass spectrometer

Recently, we demonstrated a new method, STEP (Statistical Test of Equivalent Pathways) analysis, which differentiates first-generation product ions (primary product ions) from second-generation product ions (secondary product ions) obtained in tandem mass spectrometric (MS/MS) experiments on a quadrupole ion trap mass spectrometer. The study presented here defines how to adapt the STEP method to a more routinely used mass analyzer, the triple quadrupole. New ion activation conditions were developed to adapt the STEP method to the triple quadrupole mass spectrometer using peptides and carbohydrates. The application of this method to the triple quadrupole is useful because it provides an efficient approach to differentiate primary and secondary ions on this instrument. Out of the total number of ions that were subjected to the STEP analysis, this method correctly identified 96% of ions as primary or secondary, indicating that this analysis is effective for carbohydrates and peptides undergoing collision-induced dissociation (CID) on a triple quadrupole mass spectrometer.     

The determination of glycopeptides by liquid chromatography/mass spectrometry with collision-induced dissociation

Glycopeptides derived from ribonuclease B and ovomucoid have been subjected to collision-induced dissociation (CID) in the second quadrupole of a triple quadrupole mass spectrometer. Doubly charged parent ions gave predictable fragmentation that yielded partial sequence information of the attached oligosaccharide as Hex and HexNAc units. Common oxonium ions are observed in the product ion mass spectra of the glycopeptides that correspond to HexNAc⁺ (m/z 204) and HexHexNAc⁺ (m/z 366). A strategy for locating the glycopeptides in the proteolytic digest mixtures of glycoproteins by ions spray liquid chromatography mass spectrometry (LC/MS) is described by utilizing CID in the declustering region of the atmospheric pressure ionization mass spectrometer to produce these characteristic oxonium ions. This LC/CID/MS approach is used to identify glycopeptides in proteolytic digest mixtures of ovomucoid, asialofetuin, and fetuin. LC/CID/MS in the selected ion monitoring mode may be used to identify putative glycopeptides from the proteolytic digest of fetuin.     

Applications of collision dynamics in quadrupole mass spectrometry

Some applications of collision dynamics in the field of quadrupole mass spectrometry are presented. Previous data on the collision induced dissociation of ions in triple quadrupole mass spectrometers is reviewed. A new method to calculate the internal energy distribution of activated ions directly from the increase in the cross section for dissociation with center of mass energy is presented. This method, although approximate, demonstrates explicitly the high efficiency of transfer of translational to internal energy of organic ions. It is argued that at eV center of mass energies, collisions between protein ions and neutrals such as Ar are expected to be highly inelastic. The discovery and application of collisional cooling in radio frequency quadrupoles is reviewed. Some previously unpresented data on fragment ion energies in triple quadrupole tandem mass spectrometry are shown that demonstrate directly the loss of kinetic energy of fragment ions in the cooling process. The development of the energy loss method to measure collision cross sections of protein ions in triple quadrupole instruments is reviewed along with a new discussion of the effects of inelastic collisions in these experiments and related ion mobility experiments.     

The fragmentation of the ions of nonan-4-one: A study by triple quadrupole mass spectrometry

The fragmentation pathways for the ions generated by electron impact from nonan-4-one have been studied using low energy collision induced dissociation in a triple quadrupole mass spectrometer. Over 400 fragmentation pathways have been identified. These results are compared with data from earlier ion kinetic energy spectrometry studies of nonan-4-one which employed metastable decompositions.     

High amplitude short time excitation: A method to form and detect low mass product ions in a quadrupole ion trap mass spectrometer

Collision induced dissociation (CID) in a quadrupole ion trap mass spectrometer using the conventional 30 ms activation time is compared with high amplitude short time excitation (HASTE) CID using 2 ms and 1 ms activation times. As a result of the shorter activation times, dissociation of the parent ions using the HASTE CID technique requires resonance excitation voltages greater than conventional CID. After activation, the rf trapping voltage is lowered to allow product ions below the low mass cut-off to be trapped. The HASTE CID spectra are notably different from those obtained using conventional CID and can include product ions below the low mass cut-off for the parent ions of interest. The MS/MS efficiencies of HASTE CID are not significantly different when compared with the conventional 30 ms CID. Similar results were obtained with a two-dimensional (linear) ion trap and a three-dimensional ion trap.     

Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins

The use of a new electrospray qQq Fourier transform ion cyclotron mass spectrometer (qQq-FTICR MS) instrument for biologic applications is described. This qQq-FTICR mass spectrometer was designed for the study of post-translationally modified proteins and for top-down analysis of biologically relevant protein samples. The utility of the instrument for the analysis of phosphorylation, a common and important post-translational modification, was investigated. Phosphorylation was chosen as an example because it is ubiquitous and challenging to analyze. In addition, the use of the instrument for top-down sequencing of proteins was explored since this instrument offers particular advantages to this approach. Top-down sequencing was performed on different proteins, including commercially available proteins and biologically derived samples such as the human E2 ubiquitin conjugating enzyme, UbCH10. A good sequence tag was obtained for the human UbCH10, allowing the unambiguous identification of the protein. The instrument was built with a commercially produced front end: a focusing rf-only quadrupole (Q0), followed by a resolving quadrupole (Q1), and a LINAC quadrupole collision cell (Q2), in combination with an FTICR mass analyzer. It has utility in the analysis of samples found in substoichiometric concentrations, as ions can be isolated in the mass resolving Q1 and accumulated in Q2 before analysis in the ICR cell. The speed and efficacy of the Q2 cooling and fragmentation was demonstrated on an LCMS-compatible time scale, and detection limits for phosphopeptides in the 10 amol/muL range (pM) were demonstrated. The instrument was designed to make several fragmentation methods available, including nozzle-skimmer fragmentation, Q2 collisionally activated dissociation (Q2 CAD), multipole storage assisted dissociation (MSAD), electron capture dissociation (ECD), infrared multiphoton induced dissociation (IRMPD), and sustained off resonance irradiation (SORI) CAD, thus allowing a variety of MS(n) experiments. A particularly useful aspect of the system was the use of Q1 to isolate ions from complex mixtures with narrow windows of isolation less than 1 m/z. These features enable top-down protein analysis experiments as well structural characterization of minor components of complex mixtures.     

A novel structural analysis of glycerophosphocholines as TFA/K(+) adducts by electrospray ionization ion trap tandem mass spectrometry

When collisionally activated dissociation (CAD) of glycerophosphocholine (GPC) species is examined using quadrupole ion trap mass spectrometry (QITMS), the spectral patterns differ from those obtained using sector or quadrupole mass spectrometry. Methods employed in the structural analysis of GPCs using a sector or quadrupole mass spectrometers are not necessarily useful for an ion trap mass spectrometer. A novel method is presented for structurally analyzing GPCs that involves the CAD of trifluoroacetic acid (TFA) adducts of kaliated GPCs. Solutions of GPCs in 0.1% TFA/methanol were electrosprayed to produce precursor ions by attaching a trifluoroacetic acid (TFA) molecule to a kaliated GPC molecule. The CAD-MS/MS spectra obtained by QITMS revealed a dramatic increase in the abundance of fragment ions, corresponding to the losses of sn-1 and sn-2 fatty acyl substituents. A preferential loss of the sn-1 fatty acyl group over the loss of the sn-2 fatty acyl group was observed among the GPC standards examined. A GPC extract from egg yolk was directly analyzed by this method without prior separation. The identities and positions of fatty acyl substituents of over 20 GPC species were identified. Some isomers present in very low relative abundance, which could not be analyzed by QITMS/MS using other ions as precursors, were identified by the TFA attachment method.     

Energy deposition in iron pentacarbonyl ions undergoing surface-induced dissociation in a Fourier transform mass spectrometer

Internal energy deposition into iron pentacarbonyl positive ions undergoing surface-induced dissociation (SID) in a Fourier transform mass spectrometer is estimated from the abundances and known critical energies of the product fragment ions. A narrow energy distribution, comparable to that reported in earlier BQ and tandem quadrupole SID studies of the same compound, is observed. As judged by the ratio of fragment ions to incident parent ions observed, SID of iron pentacarbonyl in the 3 T Fourier transform mass spectrometer is more efficient, but results in lower conversion of laboratory to internal energy. This may be a result of the more shallow collision incidence angle employed in the Fourier transform mass spectrometer measurements (a few degrees), which contrasts with the 32–60° collision angles used in the earlier BQ and tandem quadrupole mass spectrometry studies. Collision-induced dissociation with He under single collision conditions is also reported, Not unexpectedly, conversion of kinetic to internal energy was lower than found in a previous Fourier transform mass spectrometer study of the iron pentacarbonyl cation employing argon as collision gas under multiple collision conditions.     

Organization of nucleobase‐functionalized β‐peptides investigated by soft electrospray ionization mass spectrometry

The development and validation of analytical methods is a key to succeed in investigating noncovalent interactions between biomolecules or between small molecules and biomolecules. Electrospray ionization mass spectrometry (ESI‐MS) was applied with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR‐MS) as well as a quadrupole/time‐of‐flight tandem mass spectrometer (QqToF‐MS) for a systematic investigation of noncovalent complexes based on nucleobase pairing in an artificial and noncharged backbone topology. Synthetical β‐peptide helices covalently modified with nucleobases were organized by recognition of a sequence of four nucleobases. Specific duplexes of β‐peptide helices were obtained on the basis of hydrogen bonding base pair complementarity. Oligomer interactions were detected with defined stoichiometry and sensitivity for the respective duplex stability. FTICR‐MS and QqToF‐MS were used equally well to indicate double strand stabilities in agreement with the dissociation data determined by UV spectroscopy. Furthermore, the dissociation energies of gas phase ions of the noncovalent complexes were analyzed with collision induced dissociation (CID)‐MS/MS and infrared multiphoton dissociation (IRMPD)‐MS/MS. The CID conditions turned out to be too harsh for a differentiation of the duplex stabilities, whereas IRMPD might be developed as a technique to detect even small interaction energy differences. Copyright © 2009 John Wiley & Sons, Ltd.