Thermally assisted infrared multiphoton photodissociation in a quadrupole ion trap.

Thermally assisted infrared multiphoton photodissociation (TA-IRMPD) provides an effective means to dissociate ions in the quadrupole ion trap mass spectrometer (QITMS) without detrimentally affecting the performance of the instrument. IRMPD can offer advantages over collision-induced dissociation (CID). However, collisions with the QITMS bath gas at the standard pressure and ambient temperature cause IR-irradiated ions to lose energy faster than photons can be absorbed to induce dissociation. The low pressure required for IRMPD (< or = 10(-5) Torr) is not that required for optimal performance of the QITMS (10(-3) Torr), and sensitivity and resolution suffer. TA-IRMPD is performed with the bath gas at an elevated temperature. The higher temperature of the bath gas results in less energy lost in collisions of the IR-excited ions with the bath gas. Thermal assistance allows IRMPD to be used at or near optimal pressures, which results in an approximately 1 order of magnitude increase in signal intensity. Unlike CID, IRMPD allows small product ions, those less than about one-third the m/z of the parent ion, to be observed. IRMPD should also be more easily paired with fluctuating ion sources, as the corresponding fluctuations in resonant frequencies do not affect IRMPD. Finally, while IR irradiation nonselectively causes dissociation of all ions, TA-IRMPD can be made selective by using axial expansion to move ions away from the path of the laser beam.

Dissociation pathways of alkali-cationized peptides: opportunities for C-terminal peptide sequencing.

Dissociation pathways of alkali-cationized peptides have been studied using multiple stages of mass spectrometry (MSx) with a quadrupole ion trap mass spectrometer. Over 100 peptide ions ranging from 2 to 10 residues in length and containing each of the 20 common amino acids have been examined. The formation of the [b(n-1) + Na + OH]+ product ion is the predominant dissociation pathway for the majority of the common amino acids. This product corresponds to a sodium-cationized peptide one residue shorter in length than the original peptide. In a few cases, product ions such as [b(n-1) + Na - H]+ and those formed by loss, or partial loss, of a sidechain are observed. Both [b(n-1) + Na + OH]+ and [b(n-1) + Na - H]+ product ions can be selected as parent ions for a subsequent stage of tandem mass spectrometry (MS/MS). It is shown that these dissociation patterns provide opportunities for peptide sequencing by successive dissociation from the C-terminus of alkali-cationized peptides. Up to seven stages of MS/MS have been performed on a series of [b + Na + OH]+ ions to provide sequence information from the C-terminus. This method is analogous to Edman degradation except that the cleavage occurs from the C-terminus instead of the N-terminus, making it more attractive for N-terminal blocked peptides. The isomers leucine and isoleucine cannot be differentiated by this method but the isobars lysine and glutamine can.

Evidence for ionization-related conformational differences of peptide ions in a quadrupole ion trap.

The differences in boundary-activated dissociation (BAD) onsets have been investigated for peptide ions that were generated by two different ionization techniques, nanoflow electrospray ionization (nanoESI) and liquid secondary-ion mass spectrometry (LSIMS). BAD onsets of these ions were determined to compare the relative internal energies of the ions. Protonated peptide ions formed by nanoESI had lower BAD onsets than ions formed by LSIMS. The BAD onsets of peptides derivatized to have a fixed charge on the N-terminus also were lower for those generated by nanoESI than those generated by LSIMS. The BAD onsets of ions formed by nanoESI did not change with the variation of collisional cooling periods after gating ions into the ion trap and after isolating them prior to dissociation, indicating that the ions formed by the two ionization techniques would not adopt the same energy distributions. It is proposed that the ions formed by the two techniques differ in secondary structure, and the LSIMS ions are collisionally cooled to a lower local minimum along the potential energy surface than the nan

C-terminal peptide sequencing using acetylated peptides with MSn in a quadrupole ion trap.

MS/MS has been used to sequence peptides and small proteins for a number of years. This method allows one to isolate the peptide of interest, which makes it possible to analyze impure samples and unseparated mixtures, such as protein digests. Collision-induced dissociation (CID) of the selected peptide ion generates the product ions that provide sequence information. However, often the MS/MS spectrum does not provide adequate information for complete sequence determination. The quadrupole ion trap has the capability to do multiple stages of mass spectrometry, MSn, which can increase the information available to determine the peptide sequence. A regular and predictable dissociation pattern for peptides further simplifies this analysis. By forming predominantly one type of ion, ambiguity is removed as to whether the ion is N- or C-terminal. This pattern can also be advantageous in that ion intensity remains concentrated for the next stage of MS/MS. In this work, a method to take advantage of the MSn capabilities of the quadrupole ion trap by controlling the dissociation pathways is explored. Dissociation is altered by acetylating the N-terminus of the peptide. MSn of a variety of acetylated peptides is used to determine the effects of the identity of the C-terminal residue and the length of the peptide on the dissociation pathways observed.

A new approach for effecting surface-induced dissociation in an ion cyclotron resonance mass spectrometer: a modeling study.

With the increasing use of ion cyclotron resonance (ICR) for tandem mass spectrometry (MS/MS) analysis of biomolecules, surface-induced dissociation (SID) should be given serious consideration as an ion activation technique. There are at least two compelling reasons to consider SID: it can deposit significant amounts of internal energy into large ions, and no collision gas is required. These potential advantages have led us to undertake a modeling study of the SID process in an ICR using the ion optics program SIMION. The various methods previously used to obtain SID spectra are compared to a new approach for effecting SID in an ICR. Through simulations, many different parameters present in the experiment are correlated to the kinetic energy of the parent ion upon impact and the overall product ion collection efficiency (and hence the signal intensity) expected. The modeling results suggest this new approach allows larger, more precise, and controllable impact energies to be used, as well as providing higher collection efficiencies. The validity of the modeling results is supported by good qualitative agreement with previously reported experimental results.

Determination of the dissociation kinetics of a transient intermediate.

Tandem mass spectrometry provides information on the dissociation pathways of gas-phase ions by providing a link between product ions and parent ions. However, there exists a distinct possibility that a parent ion does not dissociate directly to the observed product ion, but that the reaction proceeds through unobserved reaction intermediates. This work describes the discovery and kinetic analysis of an unobserved reaction intermediate with a quadrupole ion trap. [a4 - NH3] ions formed from [YG beta FL + H] ions dissociate to [(F*YG - NH3) - CO] ions. It is expected, however, from previous results, that [F*YG - NH3] ions should form prior to [(F*YG - NH3) - CO] ions. Double-resonance experiments are used to demonstrate the existence of intermediate [F*YG - NH3] ions. Various kinetic analyses are then performed using traditional collision-induced dissociation kinetics and double-resonance experiments. The phenomenological rates of formation and decay of peptide rearrangement ion dissociation products are determined by curve fitting decay and formation data generated with the kinetics experiments. The data generated predict an observable level of the intermediate in a time frame accessible but previously not monitored. By examining early product-ion formation, the intermediate ions, [F*YG - NH3]+, are observed.

C-terminal peptide sequencing via multistage mass spectrometry.

Results are presented showing the ability to obtain C-terminal sequence information from peptides by multiple stages of mass spectrometry. Under typical low-energy collision-induced dissociation conditions of quadrupole ion trap and ion cyclotron resonance mass spectrometers, lithium- and sodium-cationized peptides dissociate predominantly by reaction at the C-terminal peptide bond or an adjacent bond. For the majority of cases studied, the dominant reaction is a rearrangement process that results in the loss of the C-terminal residue and formation of a product ion that is one amino acid shorter than the original peptide ion. Using the multistage MS/MS capabilities of quadrupole ion trap and ion cyclotron resonance mass spectrometers, a subsequent stage of MS/MS can be performed to determine the identity of the new C-terminal residue. Up to eight stage of MS/MS have been performed with both quadrupole ion trap and ion cyclotron resonance mass spectrometers. In general, the same dissociation pathways are observed with both instruments, although occasionally there are significant differences in the branching ratios of competing pathways.

New method to study the effects of peptide sequence on the dissociation energetics of peptide ions.

A new method has been developed to study the dissociation patterns of singly protonated peptides by using a quadrupole ion trap mass spectrometer. The new approach involves using boundary-activated dissociation to characterize the ease of dissociation of peptide ions. Insight can be gained into the effect of specific peptide sequences on the dissociation energetics of protonated peptides. Increased knowledge of the effects of specific sequences on the dissociation patterns of peptide ions should improve the ability to interpret complex spectra from tandem mass spectrometry (MS/MS) experiments. This method has confirmed the previously observed increase in the energy needed for the dissociation of peptide ions containing basic residues. In addition, this technique has revealed the effect of the location of proline residues on the dissociation energetics of peptides with this amino acid.

Collision-induced signal enhancement: a method to increase product ion intensities in MS/MS and MSn experiments.

Collision-induced signal enhancement (CISE), a new technique to enhance the MSn capabilities of the quadrupole ion trap, is demonstrated. CISE is based on the chemistry, i.e., the dissociation pathways, of the analyte examined. Polysaccharides up to hexamers are used to demonstrate the capabilities of CISE to enhance signal in two distinct functional modes. Mode 1 CISE is designed to enhance the signal of an ion desired for MSn analysis. Mode 2 CISE is designed to enhance structurally significant product ions in an MS/MS spectrum. Two different approaches can be utilized to effect the two functional modes of CISE. Both approaches use conventional resonant excitation techniques to effect dissociation, which is performed nonanalytically, i.e., without isolation of the ions to be dissociated. The two approaches are (1) single-frequency resonance excitation, and (2) broad-band wave form resonant excitation. Experimental results for Mode 1 CISE analysis demonstrate up to a 17.3-fold signal increase for the single-frequency approach and 5.3-fold using broad-band excitation. Mode 2 CISE analysis shows up to a 16.3-fold increase in signal strength with single-frequency excitation and 3.3-fold using broad-band excitation.

Boundary-activated dissociation of peptide ions in a quadrupole ion trap.

Boundary-activated dissociation (BAD) of peptides has been investigated as an alternative to the use of resonant excitation to effect collision-induced dissociation in the quadrupole ion trap. BAD's nonresonant excitation mechanism overcomes a major drawback in resonant excitation, namely, the variation of the resonant excitation frequency as a function of ion space charging. As with resonant excitation, the pulsed introduction of heavy gases (argon, xenon) extends the applicability of BAD when tandem mass spectrometry is performed on peptide ions. The presence of heavy gases during ion activation allows greater internal energy deposition and also enables BAD to be performed at much lower trapping field strengths (lower q values) than previously reported for this technique. This extends the mass range over which product ions can be collected.

Origin of product ions in the MS/MS spectra of peptides in a quadrupole ion trap.

Stored waveform inverse Fourier transform and double resonance techniques have been used in conjunction with a quadrupole ion trap to study the dissociation patterns of peptide ions. These experiments provide insight into the origin of individual product ions in an MS/MS spectrum. Results show for a series of leucine enkephalin analogues with five amino acid residues that the b4 ion is the main product ion through which many other product ions arise. It was also observed that the percentage of the a4 product ions that are formed directly from the protonated molecule (M + H)+ depends on the nature of the fourth amino acid residue. In addition, it was determined that in the peptides studies here lower series b ions (e.g., b3) arise from direct dissociation of higher series b ions (e.g., b4) only about 50% of the time.

A MALDI probe for mass spectrometers.

A new MALDI probe has been designed that uses transmission geometry. This geometry allows the probe to be fashioned after typical EI/CI solid probes which enables it to be introduced into spatially constrained ion source regions such as encountered in quadrupole ion trap mass spectrometers. In the probe design demonstrated here, light from a fiber optic irradiates the backside of a sample through a small piece of quartz on which the sample has been directly deposited. The performance characteristics exhibited by utilizing this probe for MALDI on a quadrupole ion trap mass spectrometer are similar to those which can be obtained through the traditional methods of implementing MALDI. Spectra have been obtained from 50 fmol of total loading of bombesin, MS/MS has been performed on 5 pmol of des-Arg9-bradykinin, and the analyte ion signal is shown to last for over 2500 laser shots for 2 pmol of bombesin. Optical micrographs showing the crystal distribution of a sample containing 2 pmol of bombesin have been obtained as a function of the number of laser shots for a single sample loading. Although this probe was designed for use with the quadrupole ion trap, it can be adapted for use with all types of mass spectrometers. Thus, with only one laser, one fiber optic, and this probe, MALDI can be performed on multiple instruments in a lab.

Correlation of kinetic energy losses in high-energy collision-induced dissociation with observed peptide product ions.

In collision-induced dissociation, some of an incident parent ion's kinetic energy is converted into internal energy upon collision with a neutral target. The kinetic energy lost is related to the amount of internal energy deposited into any individual ion. To see dissociations of different critical energies on the same time scale, different amounts of internal energy need to be deposited. This should be reflected in the kinetic energy lost by the parent ion in the formation of different product ions. Variable amounts of energy loss in the formation of different peptide product ions are reported here. It is seen that different product ion types (b, y, a) show ordered patterns of energy losses. A greater energy loss is observed for the formation of b-type product ions than for y-type, and even greater energy losses are observed for the formation of a-type product ions. A very good correlation between ion type energy loss and ion mass is observed. Thus, measuring the energy losses in the formation of product ions may provide a means for classifying the product ion type.

Strategy for pulsed ionization methods on a sector mass spectrometer.

A method to help facilitate efficient implementation of pulsed ionization methods on a double-focusing sector mass spectrometer is described here. This method involves the addition of an inductive detector between the electric and magnetic sectors. The inductive detector will allow a crude, but complete time-of-flight mass spectrum to be acquired with as little as a single laser shot, thereby avoiding the necessity of sequentially compiling limited mass ranges as has been done previously. Mass analysis by the complete sector instrument can be performed simultaneously with the time-of-flight analysis.

Effects of heavy gases on the tandem mass spectra of peptide ions in the quadrupole ion trap.

Heavy gases (xenon, argon, krypton, methane) have been used to improve the performance of the quadrupole ion trap when performing collision-induced dissociation on peptides. MS/MS spectra reveal that increased amounts of internal energy can be deposited into peptide ions and more structural information can be obtained. Specifically, the pulsed introduction of the heavy gases (as reported previously by Doroshenko, V. M.; Cotter, R. J. Anal. Chem. 1996, 68, 463) provides greater energy deposition without the deleterious effects that static pressures of heavy gas have on spectra. Internal energy deposition as indicated by a qualitative evaluation of MS/MS spectra shows pulsed introduction of heavy gases enables ions to obtain more internal energy than possible by using static pressures of the same heavy gases. A linear correlation is observed between the percentage of heavy gas added and the ratio of product ions used to reflect internal energy deposition. Results here also show that upon pulsed introduction of heavy gases, empirical optimization of a single frequency resonant excitation signal is no longer needed to obtain good MS/MS spectrometry efficiency. The presence of many low mass-to-charge ratio ions and the absence of side chain cleavages in the MS/MS spectra of peptides suggests that the propensity for consecutive fragmentations is increased with the pulsed introduction of heavy gases. In addition, by varying the delay time between introduction of the gas and application of the resonant excitation signal, the amount of fragmentation observed in MS/MS spectra can be changed.

Reactions of the phenylium cation with small oxygen- and nitrogen-containing molecules.

The reactions of phenylium with water and ammonia and their methyl homologs have been investigated using a quadrupole ion trap and semiempirical molecular orbital calculations. The results indicate that both types of molecules react with phenylium through lone pair electrons even though, for methyl-containing compounds, insertion into a C-H bond would lead to more stable products. For the excited adducts formed by reaction with methyl-containing reactant neutrals, the only dissociation observed is loss of a methyl radical. Neutral losses of H2 or CH4, which are more thermodynamically stable, are not observed, which indicates that these reactions are either not kinetically competitive or have high energy transition states due to the fact that the reactions would need to occur via orbital symmetry forbidden 1,2 eliminations.

Reaction from isomeric parent ions in the dissociation of dimethylpyrroles.

The major dissociation pathways of the [M-H](+) (loss of NH3 or CH4) and the [M+H](+) (loss of NH3 or CH3) ions from dimethylpyrroles have been determined to occur from isomeric parent ions. For the [M-H](+) ion (formed by loss of a methyl hydrogen), loss of NH3 leads to the formation of the phenylium ion and is preceded by consecutive carbon ring expansions followed by a ring contraction to form protonated aniline. Loss of CH4 occurs after the first carbon ring expansion, which forms protonated picoline. The relative partitioning between the two dissociation paths depends upon the internal energy content of the parent ion; the highest point on the potential energy surface is the second ring expansion step. The [M+H](+) ion reacts through a similar pathway via dihydro analogs of picoline and aniline. The proposed reaction pathways are supported by results of semiempirical molecular orbital calculations.

Ion trap mass spectrometry. Using high-pressure ionization.

The quadrupole ion trap, operated with a relatively high-pressure bath gas, occupies a unique place among mass analyzers. As an ion storage device, it shares many characteristics with the ICR instrument, but the use of a light bath gas and mass-selective instability for mass analysis contrasts with normal ICR operation. The bath gas facilitates the capture of ions injected into the oscillating quadrupole field and cools the ions to the center of the ion trap before mass-selective ejection. The bath gas also provides a conduit for heating ions. Mild collisional activation conditions can dissociate clusters, such as solvated analyte ions, formed by ES. Dissociation of covalently bound ions can be used to obtain structural information as part of a tandem MS experiment or to destroy polyatomic species in elemental analyses. The moderate background pressure requirements of the ion trap using mass-selective instability make it a natural mass analyzer to be coupled with high-pressure ionization methods. The ion trap can provide a significant advantage in efficiency over beam-type mass spectrometers, which translates into lower detection limits. Lower detection limits can be realized from superior duty cycle and MS/MS efficiency, although transmission for single-stage MS is comparable. The greatest advantage in efficiency can be obtained with weak ion beams that require long ion accumulation times and therefore high duty cycles. This, in part, makes the ion trap particularly attractive for ES. Bright ion sources can provide similar advantages if major ion beam components can be ejected during ion accumulation to minimize the deleterious effects caused by ion-ion interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Competition between resonance ejection and ion dissociation during resonant excitation in a quadrupole ion trap.

The competition between ion dissociation and ion ejection during resonant excitation in a quadrupole ion trap is investigated. Ions of similar mass but with a range of critical energies for the onset of dissociation have been examined. The effects of the amplitude and duration of the resonant excitation, the well depth in which the ions are trapped, and the pressure and nature of the collision gas are explored. Once the onset of ion ejection is reached, the rate of ion ejection increases with increased amplitude of the resonant excitation signal. The rate of ejection decreases or stays constant as a function of the duration of the resonant excitation, depending upon the ion species being excited. Increasing the trapping well depth increases the relative amount of dissociation versus ejection as does increasing the pressure of the bath gas. Adding heavier bath gases lowers the onset of ion dissociation and raises the onset of ion ejection.

Collisional activation with random noise in ion trap mass spectrometry.

Random noise applied to the end caps of a quadrupole ion trap is shown to be an effective means for the collisional activation of trapped ions independent of mass/charge ratio and number of ions. This technique is compared and contrasted with conventional single-frequency collisional activation for the molecular ion of N,N-dimethylaniline, protonated cocaine, the molecular anion of 2,4,6-trinitrotoluene, and doubly pronated neuromedin U-8. Collisional activation with noise tends to produce more extensive fragmentation than the conventional approach due to the fact that product ions are also kinetically excited in the noise experiment. The efficiency of the noise experiment in producing detectable product ions relative to the conventional approach ranges from being equivalent to being a factor of 3 less efficient. Furthermore, discrimination against low mass/charge product ions is apparent in the data from multiply charged biomolecules. Nevertheless, collisional activation with random noise provides a very simple means for overcoming problems associated with the dependence of single-frequency collisional activation on mass/charge ratio and the number of ions in the ion trap.