Ion trap/ion mobility/quadrupole/time-of-flight mass spectrometry for peptide mixture analysis

An ion trap/ion mobility/quadrupole/time-of-flight mass spectrometer has been developed for the analysis of peptide mixtures. In this approach, a mixture of peptides is electrosprayed into the gas phase. The mixture of ions that is created is accumulated in an ion trap and periodically injected into a drift tube where ions separate according to differences in gas-phase ion mobilities. Upon exiting the drift tube, ions enter a quadrupole mass filter where a specific mass-to-charge (m/z) ratio can be selected prior to collisional activation in an octopole collision cell. Parent and fragment ions that exit the collision cell are analyzed using a reflectron geometry time-of-flight mass spectrometer. The overall configuration allows different species to be selected according to their mobilities and m/z ratios prior to collision-induced dissociation and final MS analysis. A key parameter in these studies is the pressure of the target gas in the collision cell. Above a critical pressure, the well-defined mobility separation degrades. The approach is demonstrated by examining a mixture of tryptic digest peptides of ubiquitin.     

Calibration of ion effective temperatures achieved by resonant activation in a quadrupole ion trap

The present paper describes a calibration of the ion effective temperatures as a function of the resonant activation amplitude in a quadrupole ion trap mass spectrometer. MS/MS experiments are performed on leucine enkephalin (M + H)+, bradykinin (M + H)+, (M + 2H)2+, and (M + 3H)3+, and ubiquitin (M + 11H)11+. For each amplitude, the effective temperature is calculated as the temperature that would give the same dissociation rate constant as the one observed and is calculated using published Arrhenius parameters. The effective temperature is found to be linearly dependent on the activation amplitude on the range investigated. The dependence of the slope and of the intercept of the T(eff) = f (amplitude) functions on the parent ion m/z is examined and an equation is derived to calibrate the ion effective temperature between 365 and 600 K. Below 365 K, a deviation from linearity is expected. Above 600 K, the validity of the equation will depend on whether the rapid energy exchange limit is still reached. Calculating backward, the Arrhenius parameters from the measured dissociation rates using this calibration show excellent agreement with the published values. The calibration can therefore be used to determine Arrhenius activation parameters from dissociation kinetics under resonant activation in quadrupole ion trap mass spectrometers.     

Consecutive ion activation for top down mass spectrometry: improved protein sequencing by nozzle-skimmer dissociation

Mass spectra produced by nozzle-skimmer dissociation (NSD) have been little used in the past for structural characterization. NSD cannot be used on mass-separated ions (MS/MS), and for electrosprayed protein ions, previous NSD spectra showed backbone cleavages similar to those from energetic methods such as collisionally activated dissociation (CAD) or infrared multiphoton dissociation (IRMPD). However, our experimental configuration with Fourier transform (FT) MS makes possible three consecutive steps of NSD ion activation: thermal in the entrance capillary and collisional in both the nozzle-skimmer (N-S) region and the region after the skimmer before the quadrupole entrance lens (S-Q). In the high-pressure N-S region of adjustable path length, ions undergo high-frequency, low-energy collisions to rupture weak noncovalent or covalent bonds, with these "denatured" products then subjected to high-energy collisions in the low-pressure S-Q region to cleave strong backbone bonds. These NSD spectra, plus those from variable capillary thermal activation, of 8+ to 11+ ubiquitin ions electrosprayed from denatured solution show backbone cleavages between 74 of 75 amino acid pairs, vs 66 for CAD and 50 for IRMPD in the FTMS cell. Thermal activation by the inlet capillary of the newly desolvated 6+, 7+ ubiquitin ions from electrospraying the native conformer increases the NSD yield from 8% at 56 degrees C to 96% at 76 degrees C, but with little change in product branching ratios; this capillary heating has no effect on CAD or IRMPD of these ions collected in the FTMS cell. Ion desolvation with its concomitant H-bond strengthening appears to produce a transiently stable conformer whose formation can be prevented by capillary heating. The far more complex and stable noncovalent tertiary structures of large protein ions in the gas phase have made MS/MS difficult; initial inhibition of tertiary structure formation with immediate NSD ("prefolding dissociation") appears promising for the top down characterization of a 200-kDa protein.     

Distortion of ion structures by field asymmetric waveform ion mobility spectrometry

Field asymmetric waveform ion mobility spectrometry (FAIMS) is emerging as a major analytical tool, especially in conjunction with mass spectrometry (MS), conventional ion mobility spectrometry (IMS), or both. In particular, FAIMS is used to separate protein or peptide conformers prior to characterization by IMS, MS/MS, or H/D exchange. High electric fields in FAIMS induce ion heating, previously estimated at <10 degrees C on average and believed too weak to affect ion geometries. Here we use a FAIMS/IMS/MS system to compare the IMS spectra for ESI-generated ubiquitin ions that have and have not passed FAIMS and find that some unfolding occurs for most charge states. These data and their comparison with the reported protein unfolding in a Paul trap imply that at least some structural transitions observed in FAIMS, or previously in an ion trap, are not spontaneous. The observed unfolding is similar to that produced by heating of approximately 50 degrees C above room temperature, consistent with the calculated heating of ions at FAIMS waveform peaks. Hence, the ion isomerization in FAIMS likely proceeds in steps during the "hot" periods, especially right after entering the device. The process distorts ion geometries and causes ion losses by a "self-cleaning" mechanism and thus should be suppressed as much as possible. We propose achieving that via cooling FAIMS by the amount of ion heating; in most cases, cooling by approximately 75 degrees C should suffice.     

Identification of sites of ubiquitination in proteins: a fourier transform ion cyclotron resonance mass spectrometry approach

Structural elucidation of posttranslationally modified peptides and proteins is of key importance in the understanding of an array of biological processes. Ubiquitination is a reversible modification that regulates many cellular functions. Consequences of ubiquitination depend on whether a single ubiquitin or polyubiquitin chain is added to the tagged protein. The lysine residue through which the polyubiquitin chain is formed is also critical for biological activity. Robust methods are therefore required to identify sites of ubiquitination modification, both in the target protein and in ubiquitin. Here, we demonstrate the suitability of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, in conjunction with activated ion electron capture dissociation (AI ECD) or infrared multiphoton dissociation (IRMPD), for the analysis of ubiquitinated proteins. Polyubiquitinated substrate protein GST-Ubc5 was generated in vitro. Tryptic digests of polyubiquitinated species contain modified peptides in which the ubiquitin C-terminal Gly-Gly residues are retained on the modified lysine residues. Direct infusion microelectrospray FT-ICR of the digest and comparison with an in silico digest enables identification of modified peptides and therefore sites of ubiquitination. Fifteen sites of ubiquitination were identified in GST-Ubc5 and four sites in ubiquitin. Assignments were confirmed by AI ECD or IRMPD. The Gly-Gly modification is stable and both tandem mass spectrometric techniques are suitable, providing extensive sequence coverage and retention of the modification on backbone fragments.     

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.     

Observation of hydrogen-deuterium exchange of ubiquitin by direct analysis of electrospray capillary-skimmer dissociation with Fourier transform ion cyclotron resonance mass spectrometry

The structure of ubiquitin, a small cytoplasmic protein with an extended beta-sheet and an alpha-helix surrounding a hydrophobic core, has been characterized by hydrogen-deuterium (H/D) exchange labeling in conjunction with successive analysis by capillary-skimmer dissociation with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium content of each fragment ion was investigated at different times, and the results indicate that the deuterium incorporation rate into the backbone amides of ubiquitin varied depending on the environment of the amide hydrogens. Amide hydrogens of the N-terminal beta-strand showed quite slow exchange while those of the 35-39 loop were exchanged within a short exposure time to deuterium oxide. It was also possible to evaluate the difference in hydrogen-bond stability. The present data are consistent with the structural features obtained by X-Ray and NMR analyses. Although some of the labeling information might be lost by the scrambling of amide protons during capillary-skimmer dissociation, the results demonstrate that the present method provides useful higher-order structural information for proteins.     

Control of ion distortion in field asymmetric waveform ion mobility spectrometry via variation of dispersion field and gas temperature

Field asymmetric waveform ion mobility spectrometry (FAIMS) has emerged as an analytical tool of broad utility, especially in conjunction with mass spectrometry. Of particular promise is the use of FAIMS and 2-D ion mobility methods that combine FAIMS with conventional IMS to resolve and characterize protein and other macromolecular conformers. However, FAIMS operation requires a strong electric field, and ions are inevitably heated by energetic collisions with buffer gas molecules. This may induce ion isomerization or dissociation, which distort the separation properties of FAIMS (and subsequent stages) or reduce instrumental sensitivity. As FAIMS employs a periodic waveform, whether those processes are controlled by ion temperature at maximum or average field intensity has been debated. Here we address this issue by measuring the unfolding of compact ubiquitin ion geometries as a function of waveform amplitude (dispersion field, E(D)) and gas temperature, T. The field heating is quantified by matching the dependences of structural transitions on E(D) and T: increasing E(D) from 12 to 16 or from 16 to 20 kV/cm is equivalent to heating the (N2) gas by approximately 15-25 degrees C. The magnitude of field heating for any E(D) can be estimated using the two-temperature theory, and raising E(D) by 4 kV/cm augments heating by approximately 15-30 degrees C for maximum and approximately 4-8 degrees C for average field in the FAIMS cycle. Hence, isomerization of ions in FAIMS appears to be determined by the excitation at waveform peaks.     

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.     

Macromolecular ion accelerator

Presented herein are the development of macromolecular ion accelerator (MIA) and the results obtained by MIA. This new instrument utilizes a consecutive series of planar electrodes for the purpose of facilitating stepwise acceleration. Matrix-assisted laser desorption/ionization (MALDI) is employed to generate singly charged macromolecular ions. A regular Z-gap microchannel plate (MCP) detector is mounted at the end of the accelerator to record the ion signals. In this work, we demonstrated the detection of ions with the mass-to-charge (m/z) ratio reaching 30,000,000. Moreover, we showed that singly charged biomolecular ions can be accelerated with the voltage approaching 1 MV, offering the evidence that macromolecular ions can possess much higher kinetic energy than ever before.     

Rectilinear ion trap mass spectrometer with atmospheric pressure interface and electrospray ionization source

A rectilinear ion trap (RIT) mass analyzer was incorporated into a mass spectrometer fitted with an electrospray ionization source and an atmospheric pressure interface. The RIT mass spectrometer, which was assembled in two different configurations, was used for the study of biological compounds, for which performance data are given. A variety of techniques, including the use of a balanced rf, elevated background gas pressure, automatic gain control, and resonance ejection waveforms with dynamically adjusted amplitude, were applied to enhance performance. The capabilities of the instrument were characterized using proteins, peptides, and pharmaceutical drugs. Unit resolution and an accuracy of better than m/z 0.2 was achieved for mass-to-charge (m/z) ratios up to 2000 Th at a scan rate of approximately 3000 amu/(charge.s) while reduced scan rates gave greater resolution and peak widths of less than m/z 0.5 over the same range. The mass discrimination in trapping externally generated ions was characterized over the range m/z 190-2000 and an optimized low mass cutoff value of m/z 120-140 was found to give equal trapping efficiencies over the entire range. The radial detection efficiency was measured as a function of m/z ratio and found to rise from 35% at low m/z values to more than 90% for ions of m/z 1800. The way in which the ion trapping capacity depends on the dc trapping potential was investigated by measuring the mass shift due to space charge effects, and it was shown that low trapping potentials minimize space charge effects by increasing the useful volume of the device. The collision-induced dissociation (CID) capabilities of the RIT instrument were evaluated by measuring isolation efficiency as a function of mass resolution as well as measuring peptide CID efficiencies. Overall CID efficiencies of more than 60% were easily reached, while isolation of an ion with unit resolution at m/z 524 was achieved with high rejection (>95%) of the adjacent ions. The overall analytical capabilities of the ESI-RIT instrument were demonstrated with the analysis of a mixture of pharmaceutical compounds using multiple-stage mass spectrometry.     

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.     

High-resolution field asymmetric waveform ion mobility spectrometry using new planar geometry analyzers

Field asymmetric waveform ion mobility spectrometry (FAIMS) has emerged as a powerful tool of broad utility for separation and characterization of gas-phase ions, especially in conjunction with mass spectrometry (MS). In FAIMS, ions are filtered by the dependence of mobility on electric field while being carried by gas flow through the analytical gap between two electrodes of either planar (p-) or cylindrical (c-) geometry. Most FAIMS/MS systems employ c-FAIMS because of its ease of coupling to MS, yet the merits of the two geometries have not been compared in detail. Here, a priori simulations reveal that reducing the FAIMS curvature always improves resolution at equal sensitivity. In particular, the resolving power of p-FAIMS exceeds that of c-FAIMS, typically by a factor of 2-4 depending on the ion species and carrier gas. We have constructed a new planar FAIMS incorporating a curtain plate interface for effective operation with an ESI ion source and joined to an MS using an ion funnel interface with a novel slit aperture. The resolution increases up to 4-fold over existing c-FAIMS, even though the analysis is approximately 2 times faster. This allows separation of species not feasible in previous FAIMS studies, e.g., protonated leucine and isoleucine or new bradykinin isomers. The improvement for protein conformers (of ubiquitin) is less significant, possibly because of multiple unresolved geometries.     

High-throughput miniature cylindrical ion trap array mass spectrometer

A fully multiplexed cylindrical ion trap (CIT) array mass spectrometer with four parallel ion source/mass analyzer/detector channels has been built to allow simultaneous high-throughput analysis of multiple samples. A multielement external chemical ionization/electron ionization source was coupled to a parallel array of CITs each of equal size (internal radius 2.5 mm), and the signal was recorded using an array of four miniature (2-mm inner diameter) electron multipliers. Using external electron ionization, the spectra of four separate samples were recorded simultaneously in real time using a four-channel preamplifier system and a data acquisition program written using LabVIEW software. These experiments mark the first demonstration of externally generated ions being successfully trapped in a miniature CIT mass analyzer. The instrument currently provides mass/charge range of approximately m/z 50-500. Average peak width is m/z 0.3, corresponding to a resolution of 1000 at m/z 300. The four-channel mass spectrometer is housed in a single vacuum manifold and operated with a single set of control electronics. The modular design of this instrument allows scale-up to many more channels of analysis for future applications in the areas of industrial process monitoring and combinatorial analysis and in the fields of proteomics and metabolomics.     

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.     

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.     

C60 secondary ion Fourier transform ion cyclotron resonance mass spectrometry

Secondary ion mass spectrometry (SIMS) has seen increased application for high spatial resolution chemical imaging of complex biological surfaces. The advent and commercial availability of cluster and polyatomic primary ion sources (e.g., Au and Bi cluster and buckminsterfullerene (C(60))) provide improved secondary ion yield and decreased fragmentation of surface species, thus improving accessibility of intact molecular ions for SIMS analysis. However, full exploitation of the advantages of these new primary ion sources has been limited, due to the use of low mass resolution mass spectrometers without tandem MS to enable enhanced structural identification capabilities. Similarly, high mass resolution and high mass measurement accuracy would greatly improve the chemical specificity of SIMS. Here we combine, for the first time, the advantages of a C(60) primary ion source with the ultrahigh mass resolving power and high mass measurement accuracy of Fourier transform ion cyclotron resonance mass spectrometry. Mass resolving power in excess of 100?000 (m/Δm(50%)) is demonstrated, with a root-mean-square mass measurement accuracy below 1 part-per-million. Imaging of mouse brain tissue at 40 μm pixel size is shown. Tandem mass spectrometry of ions from biological tissue is demonstrated and molecular formulas were assigned for fragment ion identification.     

Focused ion beam fabrication of novel core-shell nanowire structures

A novel method of indirect deposition by means of a focused ion beam (FIB) is utilized to develop metal/insulator/semiconductor nanowire core-shell structures. This method is based upon depositing an annular pattern centered on a nanowire, with secondary deposition then coating the wire. Typical cross-sectional deposition area increments as a function of ion doses are 1.3 × 10(-2)?μm(2)?nC(-1) for Pt and 3.5 × 10(-2)?μm(2)?nC(-1) for SiO(2). The structures are examined with a transmission electron microscope (TEM) using a new nanowire TEM sample preparation method that allows direct examinations of individually selected core-shell nanowires fabricated under different indirect FIB deposition conditions. Elemental analyses by means of energy dispersive x-ray spectroscopy and electron energy filtered TEM imaging verify the deposition of SiO(2) and Pt layers. Relatively uniform Pt and SiO(2) coatings on individual GaP nanowires can be achieved with overall thickness deviation of about 10% for deposition up to 25-30?nm thick Pt or SiO(2) shells. It should be possible to extend this approach to any nanowire/nanotube system, and to a wide range of coatings in any desired layer sequences.     

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.