Positive ion transmission mode ion/ion reactions in a hybrid linear ion trap

A triple quadrupole mass spectrometer capable of ion trapping experiments has been adapted for ion/ion reaction studies. The instrument is based on a commercially available linear ion trap (LIT) tandem mass spectrometer (i.e., an MDS SCIEX 2000 Q TRAP) that has been modified by mounting an atmospheric sampling glow discharge ionization (ASGDI) source to the side of the vacuum manifold for production of singly charged anions. The ASGDI source is located line of sight to the side of the third quadrupole of the triple quadrupole assembly (Q3). Anions are focused into the side of the rod array (i.e., anion injection occurs orthogonal to the normal ion flight path). A transmission mode method to perform ion/ion reactions has been developed whereby positive ions are transmitted through the pressurized collision quadrupole (Q2) while anions are stored in Q2. The Q2 LIT is used to trap negative ions whereas the Q3 LIT is used to accumulate positive ions transmitted from Q2. Anions are injected to Q3 and transferred to Q2, where they are stored and collisionally cooled. Multiply charged protein/peptide ions, formed by electrospray, are then mass selected by the first quadrupole assembly (Q1) operated in the rf/dc mode and injected into Q2. The positive ions, including the residual precursor ions and the product ions arising from ion/ion proton-transfer reactions, are accumulated in Q3 until they are analyzed via mass-selective axial ejection for mass analysis. The parameters that affect ion/ion reactions are discussed, including pressure, nature of the gas in Q2, and operation of Q2 as a linear accelerator. Ion/ion reactions in this mode can be readily utilized to separate ions with the same m/z but largely different mass and charge, e.g., +1 bradykinin and +16 myoglobin, in the gas phase.     

Selective ion filtering by digital thresholding: a method to unwind complex ESI-mass spectra and eliminate signals from low molecular weight chemical noise

In this work, we present a simple method by which to preferentially detect either high molecular weight or low molecular weight ions generated by electrospray ionization. This approach, termed selective ion filtering by digital thresholding (SIFdT) is demonstrated on a commercial ESI-TOF instrument that employs a fast digitizer coupled to a microchannel plate detector. The digital representation of each individual scan is digitally filtered prior to spectral coaddition. As larger, more highly charged ions induce a more intense response than low molecular weight singly charged species, a digital threshold can be set that precludes the detection of singly charged species yet permits the efficient detection of larger, more highly charged species. In this work, we demonstrate the applicability of this approach to eliminate low molecular weight chemical noise in ESI-TOF spectra of oligonucleotide and protein ions, demonstrate improved dynamic range for analyte solutions containing high levels of low MW constituents, and show that spectra acquired at different digital thresholds can be subtracted to yield spectra of low molecular weight constituents with improved mass measurement accuracies. A notional scheme is presented in which an alternative digitization approach is employed using multiple differentially thresholded data streams to allow improved internal mass calibration and higher resolution ion partitioning.     

Increasing the negative charge of a macroanion in the gas phase via sequential charge inversion reactions

Protonated and deprotonated biological molecules in the gas phase play an important role in life sciences research. The structural information accessible from the ions is highly dependent upon their charge states. Therefore, it is desirable to develop means for increasing absolute charge states, particularly for ionization methods, such as MALDI, that yield relatively low charge ions. The work presented here demonstrates the formation of a doubly deprotonated polypeptide or oligonucleotide ion (dianion) from a singly deprotonated analogue via two sequential ion/ion proton-transfer reactions involving charge inversion. The high exoergicity and the large cross section arising from the long-range attractive Coulomb potential of ion/ion reactions make this process plausible. In this example, an overall efficiency of conversion of singly charged ions to doubly charged ions of roughly 8% for polypeptide was noted while lower efficiency (roughly 2%) observed with an oligonucleotide is likely due to a greater degree of neutralization. No other approach to increasing the net negative charge of an anion in the gas phase has as yet been reported.     

Fragmentation of singly protonated peptides via a combination of infrared and collisional activation

The coupling of matrix-assisted laser desorption/ionization (MALDI) to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) provides an exceptionally capable platform for peptide analysis, but an important limitation of this approach is the difficulty in obtaining informative tandem mass spectra (MS/MS) of singly protonated peptides. This difficulty is especially pronounced with peptide ions containing basic amino acid residues (for example, tryptic peptides). While such ions can be fragmented in some instrument configurations, most FTICR instruments have comparatively little facility for high-energy fragmentation. Here, a novel MS/MS approach implemented with MALDI-FTICR-MS and specifically intended for enhanced fragmentation of singly protonated peptides is described. The method involves infrared irradiation in concert with the simultaneous application of sustained off-resonance irradiation collision-induced dissociation (SORI-CID). This form of MS/MS, described as a combination of infrared and collisional activation (CIRCA), is shown to provide a greater capacity for dissociation of singly charged model peptide ions as compared to infrared multiphoton dissociation (IRMPD) or SORI-CID alone. Overall, the CIRCA approach is demonstrated to be a feasible technique for accessing useful fragmentation pathways of singly charged peptides, including those harboring basic amino acid residues--a crucial feature in the context of proteomics.     

Fragmentation of sialylated carbohydrates using infrared atmospheric pressure MALDI ion trap mass spectrometry from cation-doped liquid matrixes

Infrared atmospheric pressure matrix-assisted laser desorption/ionization on an ion trap mass spectrometer is used to study sialylated oligosaccharides desorbed from the liquid phase. Glycerol doped with various cations provides the opportunity to produce cation-adducted intact molecular ions of sugars. Distinct combinations of cations allow for sialic acid stabilization, as well as differential cleavage, resulting in more complete fragmentation coverage of the oligosaccharide. Alkali and transition metal cations are utilized to create three distinct molecular ion species, involving the adduction of a singly charged cation, two singly charged cations, or a doubly charged cation. From these different molecular ion types, complementary sequence, branching, and linkage information for sialylated oligosaccharides can be deduced.     

Analysis of megadalton ions using cryodetection MALDI time-of-flight mass spectrometry

Presented are initial results from the first commercially available matrix-assisted laser desorption/ionization time-of-flight mass spectrometer specifically designed for the sensitive detection of very high mass ions (macromizer, Comet AG). This new instrument utilizes a 16-element superconducting tunnel junction detector coupled with a fully adjustable gimbal-mounted ion source/focusing region that allows unparalleled sensitivity for detection of singly charged high molecular weight ions. Using this new technology, the singly charged ions in the megadalton region are detected from immunoglobulin M and von Willebrand factor proteins. This detector technology also measures the kinetic energy of the particles impacting the detector, which can be correlated to the charge of the particles. Immunoglobulin G and streptavidin were used to demonstrate the ability of the macromizer instrument to detect high-mass ions and to discern the charge state of the ions.     

Alternately pulsed nanoelectrospray ionization/atmospheric pressure chemical ionization for ion/ion reactions in an electrodynamic ion trap

The alternate operation of nanoelectrospray ionization and atmospheric pressure chemical ionization, using a common atmosphere/vacuum interface and ion path, has been implemented to facilitate ion/ion reaction experiments in a linear ion trap-based tandem mass spectrometer. The ion sources are operated in opposite polarity modes whereby one of the ion sources is used to form analyte ions while the other is used to form reagent ions of opposite polarity. This combination of ion sources is well-suited to implementation of experiments involving multiply charged ions in reaction with singly charged ions of opposite polarity. Three analytically useful ion/ion reaction types are illustrated: the partial deprotonation of a multiply protonated protein, the partial protonation of a multiply deprotonated oligonucleotide, and electron transfer to a multiply protonated peptide. The approach described herein is attractive in that it enables both single proton-transfer and single electron-transfer ion/ion reaction experiments to be implemented without requiring major modifications to the tandem mass spectrometer hardware. Furthermore, a wide range of reactant ions can be formed with these ionization methods and the pulsed nature of operation appears to lead to no significant compromise in the performance of either ion source.     

High resolution time-of-flight mass analysis of the entire range of intact singly-charged proteins

The proof of principle for high-resolution analysis of intact singly charged proteins of any size is presented. Singly charged protein ions were produced by electrospray ionization followed by surface-induced charge reduction at atmospheric pressure. The inlet and trapping system "stops" the forward momentum of the protein ions over a very broad range to be captured by the digitally produced electric fields of a large radius linear ion trap whereupon they are moved into a smaller radius linear ion trap and collected and concentrated in front of its exit end-cap electrode using digital waveform manipulation. The protein ions are then ejected on demand from the end of the small radius linear quadrupole in a tightly collimated ion beam with an instrumentally defined kinetic energy into the acceleration region of an orthogonal acceleration reflectron time-of-flight mass analyzer where their flight times were measured and detected with a Photonis BiPolar TOF detector. We present results that clearly prove that massive singly charged ions can yield high-resolution mass spectra with very low chemical noise and without loss of sensitivity with increasing mass across the entire spectrum. Analysis of noncovalently bound protein complexes was demonstrated with streptavidin-Cy5 bound with a biotinylated peptide mimic. Our results suggest proteins across the entire range can be directly quantified using our mass analysis technique. We present evidence that solvent molecules noncovalently adduct onto the proteins while yielding consistent flight time distributions. Finally, we provide a look into future that will result from the ability to rapidly measure and quantify protein distributions.     

Charge reduction electrospray mass spectrometry

A new mass spectrometric technique, charge reduction electrospray mass spectrometry (CREMS), allowing the analysis of complex mixtures of biological molecules is described. The charge state of ions produced by electrospray ionization may be reduced in a controlled manner to yield predominantly singly charged ions through reactions with bipolar (i.e., both positively and negatively charged) ions generated using a 210Po alpha particle source. The electrospray-generated multiply charged ions undergo charge reduction in a "neutralization chamber" positioned before the entrance nozzle to the mass spectrometer. The ions are detected using a commercial orthogonal electrospray time-of-flight mass spectrometer, although the neutralization chamber can be adapted to virtually any mass analyzer. The CREMS results obtained exhibit a signal intensity drop-off with increasing oligonucleotide size similar to that observed with matrix-assisted laser desorption/ionization mass spectrometry. Proton-transfer reactions were found to be responsible for reducing charge on proteins and oligonucleotides in both positive and negative ion mode.     

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry combined with mass spectrometry for peptide analysis

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (ultra-FAIMS) combined with mass spectrometry (MS) has been applied to the analysis of standard and tryptic peptides, derived from α-1-acid glycoprotein, using electrospray and nanoelectrospray ion sources. Singly and multiply charged peptide ions were separated in the gas phase using ultra-FAIMS and detected by ion trap and time-of-flight MS. The small compensation voltage (CV) window for the transmission of singly charged ions demonstrates the ability of ultra-FAIMS-MS to generate pseudo-peptide mass fingerprints that may be used to simplify spectra and identify proteins by database searching. Multiply charged ions required a higher CV for transmission, and ions with different amino acid sequences may be separated on the basis of their differential ion mobility. A partial separation of conformers was also observed for the doubly charged ion of bradykinin. Selection on the basis of charge state and differential mobility prior to tandem mass spectrometry facilitates peptide and protein identification by allowing precursor ions to be identified with greater selectivity, thus reducing spectral complexity and enhancing MS detection.     

Frequency-scanning MALDI linear ion trap mass spectrometer for large biomolecular ion detection

This study presents the first report on the development of a matrix-assisted laser desorption ionization (MALDI) linear ion trap mass spectrometer for large biomolecular ion detection by frequency scan. We designed, installed, and tested this radio frequency (RF) scan linear ion trap mass spectrometer and its associated electronics to dramatically extend the mass region to be detected. The RF circuit can be adjusted from 300 to 10 kHz with a set of operation amplifiers. To trap the ions produced by MALDI, a high pressure of helium buffer gas was employed to quench extra kinetic energy of the heavy ions produced by MALDI. The successful detection of the singly charged secretory immunoglobulin A ions indicates that the detectable mass-to-charge ratio (m/z) of this system can reach ~385 000 or beyond.     

Electron-induced dissociation of singly charged organic cations as a tool for structural characterization of pharmaceutical type molecules

Collision-induced dissociation (CID) and electron-induced dissociation (EID) have been investigated for a selection of small, singly charged organic molecules of pharmaceutical interest. Comparison of these techniques has shown that EID carried out on an FTICR MS and CID performed on a linear ion trap MS produce complementary data. In a study of 33 molecule-cations, EID generated over 300 product ions compared to 190 product ions by CID with an average of only 3 product ions per precursor ion common to both tandem MS techniques. Even multiple stages of CID failed to generate many of the product ions observed following EID. The charge carrying species is also shown to have a very significant effect on the degree of fragmentation and types of product ion resulting from EID. Protonated species behave much like the ammonium adduct with suggestion of a hydrogen atom from the charge carrying species strongly affecting the fragmentation mechanism. Sodium and potassium are retained by nearly every product ion formed from [M + Na](+) or [M + K](+) and provide information to complement the EID of [M + H](+) or [M + NH(4)](+). In summary, EID is proven to be a fitting partner to CID in the structural elucidation of small singly charged ions and by studying EID of a molecule-ion holding different charge carrying species, an even greater depth of detail can be obtained for functional groups commonly used in synthetic chemistry.     

Detecting singly charged ions during field evaporation of tantalum at high temperatures

The field evaporation of tantalum from point emitters in a broad range of temperatures from T = 300 to 2500 K was studied using a static magnetic mass spectrometer equipped with a special field ion source. The room-temperature mass spectrum of field-evaporated particles displayed only the peaks of triply charged ions (Ta³⁺). As the temperature was increased, the charge of field-evaporated ions exhibited a decrease: at T ～ 1000 K, the peaks of doubly charged ions (Ta²⁺) prevailed. The peaks of singly charged ions (Ta⁺) were detected for the first time at temperatures in the interval 1900 K < T < 2500 K. The rate of evaporation of singly charged tantalum ions was several orders of magnitude lower than that of doubly charged ions.     

Electron transfer ion/ion reactions in a three-dimensional quadrupole ion trap: reactions of doubly and triply protonated peptides with SO2*-

Ion-ion reactions between a variety of peptide cations (doubly and triply charged) and SO2 anions have been studied in a 3-D quadrupole ion trap, resulting in proton and electron transfer. Electron transfer dissociation (ETD) gives many c- and z-type fragments, resulting in extensive sequence coverage in the case of triply protonated peptides with SO2*-. For triply charged neurotensin, in which a direct comparison can be made between 3-D and linear ion trap results, abundances of ETD fragments relative to one another appear to be similar. Reactions of doubly protonated peptides with SO2*- give much less structural information from ETD than triply protonated peptides. Collision-induced dissociation (CID) of singly charged ions formed in reactions with SO2*- shows a combination of proton and electron transfer products. CID of the singly charged species gives more structural information than ETD of the doubly protonated peptide, but not as much information as ETD of the triply protonated peptide.     

De novo sequencing of neuropeptides using reductive isotopic methylation and investigation of ESI QTOF MS/MS fragmentation pattern of neuropeptides with N-terminal dimethylation

A stable-isotope dimethyl labeling strategy was previously shown to be a useful tool for quantitative proteomics. More recently, N-terminal dimethyl labeling was also reported for peptide sequencing in combination with database searching. Here, we extend these previous studies by incorporating N-terminal isotopic dimethylation for de novo sequencing of neuropeptides directly from tissue extract without any genomic information. We demonstrated several new sequencing applications of this method in addition to the identification of the N-terminal residue using the enhanced a(1) ion. The isotopic labeling also provides easier and more confident de novo sequencing of peptides by comparing similar MS/MS fragmentation patterns of the isotopically labeled peptide pairs. The current study on neuropeptides shows several distinct fragmentation patterns after N-terminal dimethylation which have not been reported previously. The y((n-1)) ion is enhanced in multiply charged peptides and is weak or missing in singly charged peptides. The MS/MS spectra of singly charged peptides are simplified due to the enhanced N-terminal fragments and suppressed internal fragments. The neutral loss of dimethylamine is also observed. The mechanisms for the above fragmentations are proposed. Finally, the structures of the immonium ion and related ions of N(alpha), N(epsilon)-tetramethylated lysine and N(epsilon)-dimethylated lysine are explored.     

MS/MS simplification by 355 nm ultraviolet photodissociation of chromophore-derivatized peptides in a quadrupole ion trap

Ultraviolet photodissociation (UVPD) of chromophore-modified peptides enhances the capabilities for de novo sequencing in a quadrupole ion trap mass spectrometer. Attachment of UV chromophores allows efficient photoactivation of not only the precursor ions but also any fragments that retain the chromophore functionality. For doubly protonated peptides, UVPD leads to a vast reduction in MS/MS complexity. The array of b and y ions typically seen upon collisionally activated dissociation is reduced to a single series of either y or b ions by UVPD depending on the location of the chromophore (i.e., N- or C-terminus). The sulfonation reagent Alexa Fluor 350 (AF350) provided the best overall results for the singly and doubly charged peptides by UVPD. The nonsulfonated analogue of AF350, 7-amino-4-methylcoumarin-3-acetic acid, also led to simplified spectra for doubly charged, but not singly charged, peptides by UVPD. Dinitrophenyl-peptides also yielded simplified spectra by UVPD albeit with a small amount of internal fragments accompanying the series of diagnostic y ions. The success of this MS/MS simplification process stems from extensive secondary fragmentation of any chromophore-containing fragments upon exposure to subsequent laser pulses. Energy-variable UVPD reveals that the abundances of non-chromophore-containing y fragment ions increase linearly with laser pulse energy, suggesting secondary dissociation of these species is insignificant. The abundances of chromophore-containing a/b fragment ions follow a quadratic trend due to the extensive secondary fragmentation at higher laser energies or multiple pulses.     

Generation of highly charged peptide and protein ions by atmospheric pressure matrix-assisted infrared laser desorption/ionization ion trap mass spectrometry

We show that highly charged ions can be generated if a pulsed infrared laser and a glycerol matrix are employed for atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry with a quadrupole ion trap. Already for small peptides like bradykinin, doubly protonated ions form the most abundant analyte signal in the mass spectra. The center of the charge-state distribution increases with the size of the analyte. For example, insulin is detected with a most abundant ion signal corresponding to a charge state of four, whereas for cytochrome c, the 10 times protonated ion species produces the most intense signal. Myoglobin is observed with up to 13 charges. The high m/z ratios allow us to use the Paul trap for the detection of MALDI-generated protein ions that are, owing to their high molecular weight, not amenable in their singly protonated charge state. Formation of multiple charges critically depends on the addition of diluted acid to the analyte-matrix solution. Tandem mass spectra generated by collision-induced dissociation of doubly charged peptides are also presented. The findings allow speculations about the involvement of electrospray ionization processes in these MALDI experiments.     

Chemical characterization of individual, airborne sub-10-nm particles and molecules

A nanoaerosol mass spectrometer (NAMS) is described for real-time characterization of individual airborne nanoparticles. The NAMS includes an aerodynamic inlet, quadrupole ion guide, quadrupole ion trap, and time-of-flight mass analyzer. Charged particles in the aerosol are drawn through the aerodynamic inlet, focused through the ion guide, and captured in the ion trap. Trapped particles are irradiated with a high-energy laser pulse to reach the "complete ionization limit" where each particle is thought to be completely disintegrated into atomic ions. In this limit, the relative signal intensities of the atomic ions give the atomic composition. The method is first demonstrated with sucrose particles produced with an electrospray generator. Under the conditions used, the particle detection efficiency (fraction of charged particles entering the inlet that are subsequently analyzed) reaches a maximum of 10(-4) at 9.5 nm in diameter and the size distribution of trapped particles has a geometric standard deviation of 1.1 based on a log-normal distribution. A method to deconvolute overlapping multiply charged ions (e.g. C3+ and O4+) is presented. When applied to sucrose spectra, the measured C/O atomic ratio is 1.1, which matches the expected ratio from the molecular formula. The spectra of singly charged bovine serum albumin (BSA) molecules are also presented, and the measured and expected C/N/O atomic ratios are within 15% of the each other. Also observed in the BSA spectra are signals from 13C and 32S which arise from 40 and approximately 34 atoms per molecule (particle), respectively. Potential applications of NAMS to atmospheric chemistry and biotechnology are briefly discussed.     

Electrospray ionization high-resolution ion mobility spectrometry-mass spectrometry

A hybrid atmospheric pressure ion mobility spectrometer is described which exhibits resolving power approaching the diffusion limit for singly and multiply charged ions (over 200 for the most favorable case). Using an electrospray ionization source and a downstream quadrupole mass spectrometer with electron multiplier as detector, this ESI-IMS-MS instrument demonstrates the potential of IMS for rapid analytical separations with a resolving power similar to liquid chromatography. The first measurements of gas-phase mobility spectra of mass-identified multiply charged ions migrating at atmospheric pressure are reported. These spectra confirm that collision cross sections are strongly affected by charge state. Baseline separations of multiply charged states of cytochrome c and ubiquitin demonstrate the improved resolving power of this instrument compared with previous atmospheric pressure ion mobility spectrometers. The effects of electric potential, initial pulse duration, ion-molecule reactions, ion desolvation, Coulombic repulsion, electric field homogeneity, ion collection, and charge on the resolving power of this ion mobility spectrometer are discussed.     

Fast multiple electron capture dissociation in a linear radio frequency quadrupole ion trap

We developed a fast electron capture dissociation (ECD) device using a linear radio frequency-quadrupole (RFQ) ion trap. The device dissociated peptides and proteins using a focused electron beam with an intensity of 0.5 microA and a diameter of 1 mm. The electron capture rate was 13%/ms for doubly charged peptides, and the total amount of ECD products was identical to the theoretical limit, i.e., 50% of incident precursor ions were observed as maximum ECD products by electron irradiation of 7 ms in a pulse counting detection scheme. Coupling this ECD device to a time-of-flight mass spectrometer, we applied multiple ECD. Protonated ubiquitin precursor ions with a charge state of 10 were repeatedly cleaved by ECD, i.e., charge-reduced species and their highly charged fragments were cleaved again and again, creating lower charged products, leaving only singly to triply charged states among the final products. Meanwhile with the amount of electron irradiated, lower charged products increased. Applying an electron beam for 8 ms, we obtained 96% of the total sequence coverage using a 40 fmol sample except at three proline sites. This fast ECD device should be widely applicable to proteomics including post-translational modification analysis and top-down analysis.