Gas-phase separations of protein and peptide ion fragments generated by collision-induced dissociation in an ion trap

Ion mobility/time-of-flight mass spectrometry techniques have been used to examine distributions of fragment ions generated by collision-induced dissociation (CID) in a quadrupole ion trap. The mobility-based separation step prior to mass-to-charge (m/z) analysis reduces spectral congestion and provides information that complements m/z-based assignments of peaks. The approach is demonstrated by examining fragmentation patterns of insulin chain B (a 30-residue peptide), and ubiquitin (a protein containing 76 amino acids). Some fragments of ubiquitin show evidence for multiple stable conformations.     

A tandem ion trap/ion mobility spectrometer

A tandem quadrupole ion trap/ion mobility spectrometer (QIT/IMS) has been constructed for structural analysis based on the gas-phase mobilities of mass-selected ions. The instrument combines the ion accumulation, manipulation, and mass-selection capabilities of a modified ion trap mass spectrometer with gas-phase electrophoretic separation in a custom-built ion mobility drift cell. The quadrupole ion trap may be operated as a conventional mass spectrometer, with ion detection using an off-axis dynode/multiplier arrangement, or as an ion source for the IMS drift cell. In the latter case, pulses of ions are ejected from the trap and transferred to the drift cell where mobility in the presence of helium buffer gas is determined by the collision cross section of the ion. Ions traversing the drift cell are detected by an in-line electron multiplier and the data processed with a multichannel scaler. Preliminary data are presented on instrumental performance characteristics and the application of QIT/ IMS to structural and conformational studies of aromatic ions and protonated amine/crown ether noncovalent complexes generated via ion/molecule reactions in the ion trap.     

Thermally assisted collision-induced dissociation in a quadrupole ion trap mass spectrometer

Thermally assisted collision-induced dissociation (TA-CID) provides increased dissociation in comparison with CID performed at ambient temperature in a quadrupole ion trap mass spectrometer. Heating the bath/collision gas during CID increases the initial internal energy of the ions and reduces the collisional cooling rate. Thus, using the same CID parameters, the parent ion can be activated to higher levels of internal energy, increasing the efficiency of dissociation and the number of dissociation pathways. The increase in the number of dissociation pathways can provide additional structural information. A consequence of the increase in initial internal energy is the ability to use less power to effect collisional activation. This allows lower q(z) values to be used and, thus, a greater mass range of product ions to be observed. TA-CID alleviates the problems associated with traditional CID and results in more available information than traditional CID.     

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.     

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.     

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.     

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.     

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.     

Interfacing the orbitrap mass analyzer to an electrospray ion source

The orbitrap mass analyzer employs the trapping of pulsed ion beams in an electrostatic quadro-logarithmic field. This field is created between an axial central electrode and a coaxial outer electrode. Stable ion trajectories combine rotation around the central electrode with harmonic oscillations along it. The frequencies of axial oscillations and hence mass-to-charge ratios of ions are obtained using fast Fourier transform of the image current detected on the two split halves of the outer electrode. This work proves that such a trap could be coupled to a continuous, electrospray, ion source. Such a coupling necessitated the development of an rf-only quadrupole for external accumulation of ions and their injection in very short (< 1 micros) ion bunches. Along with good sensitivity, this mass spectrometer provides mass resolving power up to 150,000 fwhm, mass accuracies within a few parts per million, and relative mass range up to 8-fold. The maximum number of ions available for analysis is limited by the space-charge capacity of the accumulation quadrupole.     

MALDI ion trap mass spectrometer with charge detector for large biomolecule detection

Up to now, all commercial matrix-assisted laser desorption/ionization (MALDI) mass spectrometers still can not efficiently analyze very large biomolecules. In this work, we report the development of a novel MALDI ion trap mass spectrometer which can enrich biomolecular ions to enhance the detection sensitivity. A charge detector was installed to measure the large ions directly. With this design, we report the first measurement of IgM with the mass-to-charge ratio (m/z) at 980?000. In addition, quantitative measurements of the number of ions can be obtained. A step function frequency scan was first developed to get a clear signal in the m/z range from 200,000 to 1,000,000.     

Combined Ion Mobility/Time-of-Flight Mass Spectrometry Study of Electrospray-Generated Ions

A commercially available ion mobility spectrometer was interfaced to a custom-built linear time-of-flight (TOF) mass spectrometer for the purpose of examining electrospray-generated plumes. Ionic species that were separated in the ion mobility spectrometer could be selectively determined with the TOF mass spectrometer. Tetraalkylammonium salts, a peptide, and proteins were examined. Their ion mobility spectra typically comprised a few peaks; some of these mobility-resolved species produced characteristic electrospray ions, while others of lower relative mobility did not. The TOF mass spectra of cytochrome c, injected from the ion mobility spectrometer at an indicated temperature of 90 °C or lower, showed signs that were characteristic of protein-solvent clustering.     

Atmospheric pressure photoionization fourier transform ion cyclotron resonance mass spectrometry for complex mixture analysis

We have coupled atmospheric pressure photoionization (APPI) to a home-built 9.4-T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Analysis of naphtho[2,3-a]pyrene and crude oil mass spectra reveals that protonated molecules, deprotonated molecules, and radical molecular ions are formed simultaneously in the ion source, thereby complicating the spectra (>12 000 peaks per mass spectrum and up to 63 peaks of the same nominal mass), and eliminating the "nitrogen rule" for nominal mass determination of number of nitrogens. Nevertheless, the ultrahigh mass resolving power and mass accuracy of FT-ICR MS enable definitive elemental composition assignments, even for doublets as closely spaced as 1.1 mDa (SH3(13)C vs (12)C4). APPI efficiently ionizes nonpolar compounds that are unobservable by electrospray and allows nonpolar sulfur speciation of petrochemical mixtures.     

Mobility labeling for parallel CID of ion mixtures

An ion mobility/mass spectrometry technique has been developed to record collision-induced dissociation patterns for multiple ions in a parallel fashion. In this approach, a mixture of ions is separated in a drift tube on the basis of differences in mobilities through a buffer gas. As the ions exit the drift tube, they are accelerated into a collision cell and the ensuing fragment ions are dispersed by differences in mass-to-charge (m/z) ratios in a time-of-flight mass spectrometer. Fragment ions that are formed in the collision cell have drift times that are coincident with their antecedent parent ions, allowing the origin of all fragments formed from the mixture of ions to be determined. The approach is demonstrated by examining fragmentation patterns of the [M + H]+ parent and a series of a-, b-, and y-type fragments of [D-Ala2,3]methionine enkephalin.     

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.     

Coupling high-pressure MALDI with ion mobility/orthogonal time-of-flight mass spectrometry

A new ion mobility/time-of-flight mass spectrometer employing a high-pressure MALDI source has been designed and tested. The prototype instrument operates at a source/drift cell pressure of 1-10 Torr helium, resulting in a mobility resolution of approximately 25. A small time-of-flight mass spectrometer (20 cm) with a mass resolution of up to 200 has been attached to the drift cell to identify (in terms of mass-to-charge ratio) the separated ions. A simple tripeptide mixture has been separated in the drift tube and mass identified as singly protonated species. The ability to separate peptide mixtures, e.g., tryptic digest of a protein, is illustrated and compared to results obtained on a high-vacuum time-of-flight instrument.     

Rapidly alternating transmission mode electron-transfer dissociation and collisional activation for the characterization of polypeptide ions

Cation transmission/electron-transfer reagent anion storage mode electron-transfer ion/ion reactions and beam-type collisional activation of the polypeptide ions are performed in rapid succession in the high-pressure collision cell (Q2) of a quadrupole/time-of-flight tandem mass spectrometer (QqTOF), where the electron-transfer reagent anions are accumulated. Duty cycles for both electron-transfer dissociation (ETD) and collision-induced dissociation (CID) experiments are improved relative to ion trapping approaches since there are no discrete ion storage and reaction steps for ETD experiments and no discrete ion storage step and frequency tuning for CID experiments. For this technique, moderately high resolution and mass accuracy are also obtained due to mass analysis via the TOF analyzer. This relatively simple approach has been demonstrated with a triply charged tryptic peptide, a triply charged tryptic phosphopeptide, and a triply charged tryptic N-linked glycopeptide. For the tryptic peptide, the sequence is identified with more certainty than would be available from a single method alone due to the complementary information provided by these two dissociation methods. Because of the complementary information derived from both ETD and CID dissociation methods, peptide sequence and post-translational modification (PTM) sites for the phosphopeptide are identified. This combined ETD and CID approach is particularly useful for characterizing glycopeptides because ETD generates information about both peptide sequence and locations of the glycosylation sites, whereas CID provides information about the glycan structure.     

Multiplexed ion mobility spectrometry-orthogonal time-of-flight mass spectrometry

Ion mobility spectrometry (IMS) coupled to orthogonal time-of-flight mass spectrometry (TOF) has shown significant promise for the characterization of complex biological mixtures. The enormous complexity of biological samples (e.g., from proteomics) and the need for both biological and technical analysis replicates imposes major challenges for multidimensional separation platforms with regard to both sensitivity and sample throughput. A major potential attraction of the IMS-TOF MS platform is separation speeds exceeding that of conventional condensed-phase separations by orders of magnitude. Known limitations of the IMS-TOF MS platforms that presently mitigate this attraction include the need for extensive signal averaging due to factors that include significant ion losses in the IMS-TOF interface and an ion utilization efficiency of less than approximately 1% with continuous ion sources (e.g., ESI). We have developed a new multiplexed ESI-IMS-TOF mass spectrometer that enables lossless ion transmission through the IMS-TOF as well as a utilization efficiency of >50% for ions from the ESI source. Initial results with a mixture of peptides show a approximately 10-fold increase in signal-to-noise ratio with the multiplexed approach compared to a signal averaging approach, with no reduction in either IMS or TOF MS resolution.     

Enhanced analyte detection using in-source fragmentation of field asymmetric waveform ion mobility spectrometry-selected ions in combination with time-of-flight mass spectrometry

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (FAIMS) is used for the selective transmission of differential mobility-selected ions prior to in-source collision-induced dissociation (CID) and time-of-flight mass spectrometry (TOFMS) analysis. The FAIMS-in-source collision induced dissociation-TOFMS (FISCID-MS) method requires only minor modification of the ion source region of the mass spectrometer and is shown to significantly enhance analyte detection in complex mixtures. Improved mass measurement accuracy and simplified product ion mass spectra were observed following FAIMS preselection and subsequent in-source CID of ions derived from pharmaceutical excipients, sufficiently close in m/z (17.7 ppm mass difference) that they could not be resolved by TOFMS alone. The FISCID-MS approach is also demonstrated for the qualitative and quantitative analysis of mixtures of peptides with FAIMS used to filter out unrelated precursor ions thereby simplifying the resulting product ion mass spectra. Liquid chromatography combined with FISCID-MS was applied to the analysis of coeluting model peptides and tryptic peptides derived from human plasma proteins, allowing precursor ion selection and CID to yield product ion data suitable for peptide identification via database searching. The potential of FISCID-MS for the quantitative determination of a model peptide spiked into human plasma in the range of 0.45-9.0 μg/mL is demonstrated, showing good reproducibility (%RSD < 14.6%) and linearity (R(2) > 0.99).     

Surface-induced dissociation of ion mobility-separated noncovalent complexes in a quadrupole/time-of-flight mass spectrometer

A custom in-line surface-induced dissociation (SID) device has been incorporated into a commercial ion mobility quadrupole/time-of-flight mass spectrometer in order to provide an alternative and potentially more informative activation method than the commonly used collision-induced dissociation (CID). Complicated sample mixtures can be fractionated by ion mobility (IM) and then dissociated by CID or SID for further structural analysis. Interpretation of SID spectra for cesium iodide clusters was greatly simplified with IM prior to dissociation because products originating from different precursors and overlapping in m/z but separated in drift time can be examined individually. Multiple conformations of two protein complexes, source-activated transthyretin tetramer and nativelike serum amyloid P decamer, were separated in ion mobility and subjected to CID and SID. CID spectra of the mobility separated conformations are similar. However, drastic differences can be observed for SID spectra of different conformations, implying different structures in the gas phase. This work highlights the potential of utilizing IM-SID to study quaternary structures of protein complexes and provides information that is complementary to our recently reported SID-IM approach.     

Matrix-assisted laser desorption/ionization coupled with quadrupole/orthogonal acceleration time-of-flight mass spectrometry for protein discovery, identification, and structural analysis

The design and operation of a novel UV-MALDI ionization source on a commercial QqoaTOF mass spectrometer (Applied Biosystem/MDS Sciex QSTAR Pulsar) is described. Samples are loaded on a 96-well target plate, the movement of which is under software control and can be readily automated. Unlike conventional high-energy MALDI-TOF, the ions are produced with low energies (5-10 eV) in a region of relatively low vacuum (8 mTorr). Thus, they are cooled by extensive low-energy collisions before selection in the quadrupole mass analyzer (Q1), potentially giving a quasi-continuous ion beam ideally suited to the oaTOF used for mass analysis of the fragment ions, although ion yields from individual laser shots may vary widely. Ion dissociation is induced by collisions with argon in an rf-only quadrupole cell, giving typical low-energy CID spectra for protonated peptide ions. Ions separated in the oaTOF are registered by a four-anode detector and time-to-digital converter and accumulated in "bins" that are 625 ps wide. Peak shapes depend upon the number of ion counts in adjacent bins. As expected, the accuracy of mass measurement is shown to be dependent upon the number of ions recorded for a particular peak. With internal calibration, mass accuracy better than 10 ppm is attainable for peaks that contain sufficient ions to give well-defined Gaussian profiles. By virtue of its high resolution, capability for accurate mass measurements, and sensitivity in the low-femotomole range, this instrument is ideally suited to protein identification for proteomic applications by generation of peptide tags, manual sequence interpretation, identification of modifications such as phosphorylation, and protein structural elucidation. Unlike the multiply charged ions typical of electrospray ionization, the singly charged MALDI-generated peptide ions show a linear dependence of optimal collision energy upon molecular mass, which is advantageous for automated operation. It is shown that the novel pulsing technique of this instrument that increases the sensitivity for precursor ions scans is applicable to the identification of peptides labeled with isotope-coded affinity tags.