Implementation of ion/ion reactions in a quadrupole/time-of-flight tandem mass spectrometer

A commercial quadrupole/time-of-flight (QqTOF) tandem mass spectrometer has been adapted for ion/ion reaction studies. To enable mutual storage of oppositely charged ions in a linear ion trap, the oscillating quadrupole field of the second quadrupole of the system (Q2) serves to store ions in the radial dimension while auxiliary radio frequency is superposed on the end lenses of Q2 during the reaction period to create barriers in the axial dimension. A pulsed dual electrospray (ESI) source is directly coupled to the instrument interface for the purpose of proton transfer reactions. Singly and doubly charged protein ions as high in mass as 66 kDa are readily formed and observed after proton-transfer reactions. For the modified instrument, the mass resolving power is approximately 8000 for a wide m/z range, and the mass accuracy is approximately 20 ppm for external calibration and approximately 5 ppm for internal calibration after ion/ion reactions. Parallel ion parking is demonstrated with a six-component protein mixture, which shows the potential application of reducing spectral complexity and concentrating certain charge states. The current system has high flexibility with respect to defining MS(n) experiments involving collision-induced dissociation (CID) and ion/ion reactions. Protein precursor and CID product masses can be determined with good accuracy, providing an attractive platform for top-down proteomics. Electron transfer dissociation ion/ion reactions are implemented by using a pulsed nano-ESI/atmospheric pressure chemical ionization dual source for ionization. The reaction between protonated peptide ions and radical anions of 1,3-dinitrobenzene formed exclusively c- and z-type fragment ions.     

A quadrupole ion trap mass spectrometer with three independent ion sources for the study of gas-phase ion/ion reactions

An instrument for the study of gas-phase ion/ion reactions in which three independent sources of ions, namely, two electrospray ionization sources and one atmospheric sampling glow discharge ionization source, are interfaced to a quadrupole ion trap mass analyzer is described. This instrument expands the scope of gas-phase ion/ion reaction studies by allowing for manipulation of the charge states of multiply charged reactant and product ions. Examples are provided involving the formation of protein-protein complexes in the gas phase. Complexes with charge states that cannot be formed from reactant ion charge states present in the normal electrospray charge state distributions can be formed in the new apparatus. Strategies that rely on both reactant ion charge state manipulation and product ion charge state manipulation are demonstrated. In addition, simplification of product ion spectra generated from dissociation of complexes formed via ion/ion reactions can be effected by using the discharge source to reduce the charge state of the product ions to primarily 1+.     

Parallel ion parking of protein mixtures

The multiple charging phenomenon resulting from electrospray ionization of proteins, while useful for the ability to make several mass measurements on a single component, can lead to highly complex spectra when mixtures are analyzed, as each component can generate multiple ions of distinct mass-to-charge ratio. Ion/ion proton-transfer reactions can overcome this problem by reduction of all components to the +1 charge state, but this typically requires the ability to extend the mass range of the instrument well beyond that available in most commercial instruments. Furthermore, reduction of protein charge to +1 also results in a reduction in detector response. Here it is shown that application of a relatively high amplitude, low-frequency auxiliary ac signal to the end cap electrodes of a 3-D ion trap during an ion/ion reaction can slow the ion/ion reaction rates of ions over a broad m/z range, in a process termed HALF parallel ion parking. Adjustment of the frequency and amplitude of the applied voltage allows the mass range into which the initial ion signal is moved to be controlled, allowing for the simplification of multicomponent mixtures within a mass range that is more commonly available on commercial systems. In addition to decreasing spectral complexity, this is advantageous for mixtures with low-abundance components, as there is less compromise with detector response than in reduction to the +1 charge state. Preliminary evidence also suggests that the ion collision cross section may play an important role in determining which charge states are most significantly inhibited from further ion/ion reactions under a given set of ion parking conditions.     

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.     

Phosphorylation site identification via ion trap tandem mass spectrometry of whole protein and peptide ions: bovine alpha-crystallin A chain

Tandem mass spectrometry was applied both to ions of a tryptic fragment and intact protein of bovine alpha-crystallin A chain to localize the single site of phosphorylation. The [M + 19H](19+) to [M + 11H](11+) charge states of both phosphorylated and unphosphorylated bovine alpha-crystallin A chain whole protein ions were subjected to collisional activation in a quadrupole ion trap. Ion parking was used to increase the number of parent ions over that yielded by electrospray. Ion-ion proton-transfer reactions were used to reduce the product ion charge states largely to +1 to simplify spectral interpretation. In agreement with previous studies on whole protein ion fragmentation, both protein forms showed backbone cleavages C-terminal to aspartic acid residues at lower charge states. The phosphorylated protein showed competitive fragmentation between backbone cleavage and the neutral loss of phosphoric acid. Analysis of which backbone cleavage products did or did not contain the phosphate was used to localize the site of phosphorylation to one of two possible serine residues. A tryptic digest of the bovine alpha-crystallin A chain yielded a phosphopeptide containing one missed cleavage site. The peptide provided information complementary to that obtained from the intact protein and localized the modified serine to residue 122. Fragmentation of the triply charged phosphopeptide yielded five possible serine phosphorylation sites. Fragmentation of the doubly charged phosphopeptide, formed by ion/ion proton-transfer reactions, positively identified the phosphorylation site as serine-122.     

Sonic spray as a dual polarity ion source for ion/ion reactions

A single sonic spray source has been used to generate both positive and negative ions for subsequent ion/ion reaction experiments. Ion/ion reactions took place after ions of each polarity were sequentially injected into a linear ion trap, where axial trapping was effected by applying an auxiliary radio frequency voltage to one end lens. Absolute charge reductions via proton transfer were demonstrated for multiply charged protein/peptide cations and multiply charged oligonucleotide anions. Deprotonation of polypeptide cations occurs with anions derived from fluorinated compounds such as nonadecafluoro-1-decanol and perfluoro-1-octanol, while multiply charged oligonucleotide anions are efficiently protonated via reaction with proton sponge (N,N,N',N'-tetramethyl-1,8-naphthalenediamine) cations. No evidence for signal suppression of the biopolymer ions was noted to result from the presence of these reagents in the solution subjected to sonic spray. Several of the analytically useful applications of ion/ion proton-transfer reactions are demonstrated using a single sonic spray ion source. These include an ion parking experiment for the purpose of gas-phase ion concentration and charge-state reduction of product ions formed via beam-type and in-trap collision-induced dissociation of multiply charged oligonucleotide parent anions. Examples of complex formation are also given to illustrate the flexibility of the sonic spray-induced ion/ion reaction method.     

Activation of intact electron-transfer products of polypeptides and proteins in cation transmission mode ion/ion reactions

Cationic peptide electron-transfer products that do not fragment spontaneously are exposed to ion trap collisional activation immediately upon formation while they pass through a high-pressure collision cell (Q2), where the electron-transfer reagent anions are stored. Radial ion acceleration, which is normal to the ion flow, is implemented by applying an auxiliary dipolar alternating current to a pair of opposing rods of the Q2 quadrupole array at a frequency in resonance with the surviving electron-transfer products. Collisional cooling of cations in the pressurized Q2 ensures efficient overlap of the positive and negative ions for ion/ion reactions and also gives rise to relatively long residence times (milliseconds) for ions in Q2, making it possible to fragment ions via radial excitation during their axial transmission. The radial activation for transmission mode electron-transfer ion/ion reactions has been demonstrated with a doubly protonated tryptic peptide, a triply protonated phosphopeptide, and [M + 7H]7+ ions of ubiquitin. In all cases, significant increases in fragment ion yields and structural information from electron-transfer dissociation (ETD) were observed, suggesting the utility of this method for improving transmission mode ETD performance for relatively low charge states of peptides and proteins.     

Oligonucleotide mixture analysis via electrospray and ion/ion reactions in a quadrupole ion trap

Electrospray ionization combined with ion/ion reactions in a quadrupole ion trap can be used for the direct analysis of oligonucleotide mixtures. Elements to the success of this approach include factors related to ionization, ion/ion reactions, and mass analysis. This paper deals with issues regarding the ion polarity combination, viz., positive oligonucleotides/negative charge-transfer agent versus negative oligonucleotides/positive charge-transfer agent. Anions derived from perfluorocarbons appear to be directly applicable to mixtures of positive ions derived from electrospray of oligonucleotides, in direct analogy with positive protein ions. Conditions for forming positive oligonucleotide ions devoid of adducts were more difficult to establish than for forming relatively clean negative oligonucleotide ions. A new approach for manipulating negative ion charge states in the ion trap is described and is based on use of the electric field of the positive charge-transfer agent for storage of high-mass negative ions formed during the ion/ion reaction period. Oxygen cations are shown to be acceptable for charge-state manipulation of mixed-base oligomers but induce fragmentation in polyadenylate homopolymers. Protonated isobutylene (C4H9+), on the other hand, is shown to induce significantly less fragmentation of polyadenylate homopolymers.     

Identification of bacteriophage MS2 coat protein from E. coli lysates via ion trap collisional activation of intact protein ions

Collisional activation of the intact MS2 viral capsid protein with subsequent ion/ion reactions has been used to identify the presence of this virus in E. coli lysates. Tandem ion trap mass spectrometry experiments on the +7, +8, and +9 charge states, followed by ion/ion reactions, provided the necessary sequence tag information (and molecular weight data) needed for protein identification via database searching. The most directly informative structural information is obtained from those charge states that produce a series of product ions arising from fragmentation at adjacent residues. The formation of these product ions via dissociation at adjacent amino acid residues depends greatly on the charge state of the parent ion. Database searching of the charge-state-specific sequence tags was performed by two different search engines: the ProteinInfo program from the Protein information Retrieval On-line World Wide Web Lab or PROWL and the TagIdent program from the ExPASy molecular biology server. These search engines were used in conjunction with the sequence tag information generated via collisional activation of the intact viral coat protein. These programs were used to evaluate the feasibility of generating sequence tags from collisional activation of intact multiply charged protein ions in a quadrupole ion trap.     

An interface with a linear quadrupole ion guide for an electrospray-ion trap mass spectrometer system

A new ion sampling interface for an electrospray ionization 3D ion trap mass spectrometer system is described. The interface uses linear rf quadrupoles as ion guides and ion traps to enhance the performance of the 3D trap. Trapping ions in the linear quadrupoles is demonstrated to improve the duty cycle of the system. Dipolar excitation of ions trapped in a linear quadrupole is used to eject unwanted ions. A resolution of ejection of up to 254 is demonstrated for protonated reserpine ions (m/z 609.3). A composite waveform with a notch in frequency space is used to eject a wide range of matrix ions and to isolate trace analyte ions in a linear quadrupole before ions are injected into the 3D trap. This is useful to overcome space charge problems in the 3D trap caused by excess matrix ions. For trace reserpine in a 500-fold molar excess of poly(propylene glycol) (PPG), it is demonstrated that the resolution and sensitivity of the 3D trap can be increased dramatically with ejection of the excess PPG matrix ions. In comparison to ejection of matrix ions in the 3D trap with a similar broad-band waveform, a 5-fold increase in sensitivity with a 7 times shorter acquisition time was achieved.     

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.     

Dissociation of multiple protein ion charge states following a single gas-phase purification and concentration procedure

The formation of a range of precursor ion charge states from a single concentrated and purified charge state, followed by activation of each charge state, is introduced as a means to obtain more protein structural information than is available from dissociation of a single charge state alone. This approach is illustrated using off-resonance collisional activation of the [M + 8H]8+ to [M + 6H]6+ precursor ions of the bacteriophage MS2 viral coat protein following concentration and purification of the [M + 8H]8+ charge state. This range of charge states was selected on the basis of an ion trap collisional activation study of the effects of precursor ion charge state on the dissociation of the [M + 12H]12+ to [M + 5H]5+ ions. Gas-phase ion/ion proton-transfer reactions and the ion parking technique were applied to purify and concentrate selected precursor ion charge states as well as to simplify the product ion spectra. The high-charge-state ions fragment preferentially at the N-terminal side of proline residues while the product ion spectra of the lowest charge states investigated are dominated by C-terminal aspartic acid cleavages. Maximum structural information is obtained by fragmentation of the intermediate-charge states.     

Charge ratio analysis method: approach for the deconvolution of electrospray mass spectra

A new method to interpret electrospray mass spectral data based on calculating the ratio of mass-to-charge (m/z) values of multiply charged ions is described. The mass-to-charge ratios of any two multiply charged ions corresponding to a single compound are unique numbers that enable the charge states for each ion to be unequivocally identified. The multiply charged ions in electrospray mass spectra originate from the addition or abstraction of protons, cations, or anions to and from a compound under analysis. In contrast to existing deconvolution processes, the charge ratio analysis method (CRAM), identifies the charge states of multiply charged ions without any prior knowledge of the nature of the charge-carrying species. In the case of high-resolution electrospray mass spectral data, in which multiply charged ions are resolved to their isotopic components, the CRAM is capable of correlating the isotope peaks of different multiply charged ions that share the same isotopic composition. This relative ratio method is illustrated here for electrospray mass spectral data of lysozyme and oxidized ubiquitin recorded at low- to high-mass resolution on quadrupole ion trap and Fourier transform ion cyclotron mass spectrometers, and theoretical data for the protein calmodulin based upon a reported spectrum recorded on the latter.     

Charge-state-dependent sequence analysis of protonated ubiquitin ions via ion trap tandem mass spectrometry

One of the major factors governing the "top-down" sequence analysis of intact multiply protonated proteins by tandem mass spectrometry is the effect of the precursor ion charge state on the formation of product ions. To more fully understand this effect, electrospray ionization coupled to a quadrupole ion trap mass spectrometer, collision-induced dissociation, and gas-phase ion/ion reactions have been employed to examine the fragmentation of the [M + 12H]12+ to [M + H]+ ions of bovine ubiquitin. At low charge states (+1 to +6), loss of NH3 or H2O from the protonated precursor and directed cleavage at aspartic acid residues was observed. At intermediate charge states, (+7, +8, and +9), extensive nonspecific fragmentation of the protein backbone was observed, with 50% sequence coverage obtained from the [M + 8H]8+ ion alone. At high charge states, (+10, +11, +12), the single dominant channel that was observed was the preferential fragmentation of a single proline residue. These data can be readily explained in terms of the current model for intramolecular proton mobilization, that is, the "mobile proton model", the mechanisms for amide bond dissociation developed for protonated peptides, as well as the structures of the multiply charged ions of ubiquitin in the gas phase, examined by ion mobility and hydrogen/deuterium exchange measurements.     

Transmission mode ion/ion electron-transfer dissociation in a linear ion trap

Two related methods for effecting electron-transfer dissociation (ETD) are described that involve either the storage of analyte cations in a linear ion trap while reagent anions are transmitted through the cations or storage of the reagent anions with transmission of the analyte cations. In the former approach, the ETD products are captured and stored in the linear ion trap for subsequent mass analysis. In the latter approach, the ETD products pass through the linear ion trap and must be collected or directly mass-analyzed by an external device. In the present study, another linear ion trap is placed in series with the ion trap where the ion/ion reaction was employed. A pulsed dual ion source approach coupled with a hybrid triple quadrupole/linear ion trap instrument was used to illustrate these methods. The two approaches give similar results in terms of the identities and relative abundances of the ETD products. Under optimum conditions, the two approaches also give comparable extents of ion/ion reactions for the same reaction time. Also, conversions of precursor ions to product ions over the same reaction time are similar to those noted for experiments in which ions of both polarities are stored simultaneously. These approaches, therefore, provide expanded experimental options for the use of ETD. An advantage of transmission mode experiments that they hold over mutual storage mode experiments is that they do not require that any specialized measures be taken to enable the simultaneous storage of oppositely charged ions.     

Whole protein dissociation in a quadrupole ion trap: identification of an a priori unknown modified protein

A protein mixture derived from a whole cell lysate fraction of Saccharomyces cerevisiae, which contains roughly 19 proteins, has been analyzed to identify an a priori unknown modified protein using a quadrupole ion trap tandem mass spectrometer. Collection of the experimental data was facilitated by collision-induced dissociation and ion/ion proton-transfer reactions in multistage mass spectrometry procedures. Ion/ion reactions were used to manipulate charge states of both parent ions and product ions for the purpose of concentrating charge into the parent ion of interest and to reduce the product ion charge states for determination of product ion mass and abundance. The identification of the protein was achieved by matching the uninterpreted product ion spectrum against protein sequence databases with varying degrees of annotation, coupled with a scoring scheme weighted for the relative abundances of the experimentally observed product ions and the frequency of fragmentations occurring at preferential sites. The protein was identified to be an acetylated yeast heat shock protein, HS12_Yeast (11.6 kDa), with the initiating methionine residue removed. This constitutes the first example of the identification of an a priori unknown protein that is not present in an annotated protein database using a "top-down" approach with a quadrupole ion trap. This example illustrates the utility of relatively low cost instrumentation with modest mass analysis characteristics for the identification of modified proteins without recourse to enzymatic digestion. It also illustrates how experimental data can be used interactively with protein databases when the modified protein of interest is not initially present in the database.     

Development of a proton-transfer reaction-linear ion trap mass spectrometer for quantitative determination of volatile organic compounds

Currently, proton-transfer reaction mass spectrometry (PTR-MS) allows for quantitative determination of volatile organic compounds in real time at concentrations in the low ppt range, but cannot differentiate isomers or isobaric molecules, using the conventional quadrupole mass filter. Here we pursue the application of linear quadrupole ion trap (LIT) mass spectrometry in combination with proton-transfer reaction chemical ionization to provide the advantages of specificity from MS/MS. A commercial PTR-MS platform composed of a quadrupole mass filter with the addition of end cap electrodes enabled the mass filter to operate as a linear ion trap. The rf drive electronics were adapted to enable the application of dipolar excitation to opposing rods, for collision-induced dissociation (CID) of trapped ions. This adaptation enabled ion isolation, ion activation, and mass analysis. The utility of the PTR-LIT was demonstrated by distinguishing between the isomeric isoprene oxidation pair, methyl vinyl ketone (MVK) and methacrolein (MACR). The CID voltage was adjusted to maximize the m/ z 41 to 43 fragment ratio of MACR while still maintaining adequate sensitivity. Linear calibration curves for MVK and MACR fragments at m/ z 41 and 43 were obtained with limits of detection of approximately 100 ppt, which should enable ambient measurements. Finally, the PTR-LIT method was compared to an established GC/MS method by quantifying MVK and MACR production during a smog chamber isoprene-NO x irradiation experiment.     

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.     

Mass analysis of mobility-selected ion populations using dual gate, ion mobility, quadrupole ion trap mass spectrometry

An electrospray ionization, dual gate, ion mobility, quadrupole ion trap mass spectrometer (ESI-DG-IM-QIT-MS) was constructed and evaluated for its ability to select mobility-filtered ions prior to mass analysis. While modification of the common signal-averaged ion mobility experiment was required, no modifications to the QIT were necessary. The dual gate scanning mode of operation was used to acquire mobility spectra, whereas the single mobility monitoring experiment selectively filtered ions for concentration and subsequent fragmentation within the QIT. Ion mobility separation of positively charged peptides and negatively charged carbohydrates, followed by MS fragmentation, was demonstrated. For a 1-min acquisition time, it was possible to obtain complete de novo sequence information for the examined peptides. Fragmentation of the negative carbohydrate chlorine adducts yielded ions characteristic of cross-ring and glycosidic bond cleavage. Previous unions of atmospheric pressure ion mobility and mass spectrometry have been limited in their ability to reproducibly obtain MSn data for mobility separation ions. The union of high-pressure ion mobility with quadrupole ion trap mass spectrometry presents the unique opportunity to obtain more detailed information regarding the chemistries of gas-phase ions.     

Bidirectional ion transfer between quadrupole arrays: MSn ion/ion reaction experiments on a quadrupole/time-of-flight tandem mass spectrometer

Methods for bidirectional ion transmission between distinct quadrupole arrays were developed on a quadrupole/time-of-flight tandem mass spectrometer (QqTOF) containing three quadrupoles (ion guide Q0, mass filter Q1, and collision cell Q2) and a reflectron TOF analyzer, for the purpose of implementing multistage ion/ion reaction experiments. The transfer efficiency, defined as the percentage of ions detected after two transfer steps relative to the initial ion abundance, was found to be about 60% between Q2 and Q0 (with passage through the intermediate array (Q1)) and almost 100% between Q2 and Q1. Efficient ion transfer enabled new means for executing MSn experiments on an instrument of this type by operating Q1 in rf/dc mode for performing multiple steps of precursor/product ion isolation while passing ions through Q1 or trapping ions in Q1. In the latter case, the Q1 functioned as a linear ion trap. Either collision induced dissociation (CID) or ion/ion reactions can be conducted in between each stage of mass analysis. MS3 or MS4 experiments were developed to illustrate the charge increase of peptide ions via two steps of charge inversion ion/ion reactions, CID of electron-transfer dissociation (ETD) products and CID of a metal-peptide complex formed from ion/ion reactions.