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.     

C60 secondary ion mass spectrometry with a hybrid-quadrupole orthogonal time-of-flight mass spectrometer

A hybrid quadrupole orthogonal time-of-flight mass spectrometer optimized for matrix-assisted laser desorption ionization (MALDI) and electrospray ionization has been equipped with a C 60 cluster ion source. This configuration is shown to exhibit a number of characteristics that improve the performance of traditional time-of-flight secondary ion mass spectrometry (TOF-SIMS) experiments for the analysis of complex organic materials and, potentially, for chemical imaging. Specifically, the primary ion beam is operated as a continuous rather than a pulsed beam, resulting in up to 4 orders of magnitude greater ion fluence on the target. The secondary ions are extracted at very low voltage into 8 mTorr of N 2 gas introduced for collisional focusing and cooling purposes. This extraction configuration is shown to yield secondary ions that rapidly lose memory of the mechanism of their birth, yielding tandem mass spectra that are identical for SIMS and MALDI. With implementation of ion trapping, the extraction efficiency is shown to be equivalent to that found in traditional TOF-SIMS machines. Examples are given, for a variety of substrates that illustrate mass resolution of 12,000-15,600 with a mass range for inorganic compounds to m/ z 40,000. Preliminary chemical mapping experiments show that with added sensitivity, imaging in the MS/MS mode of operation is straightforward. In general, the combination of MALDI and SIMS is shown to add capabilities to each technique, providing a robust platform for TOF-SIMS experiments that already exists in a large number of laboratories.     

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.     

Ion isolation in a continuous zero angle reflecting time-of-flight mass spectrometer

A continuous zero angle reflecting time-of-flight mass spectrometer capable of tandem mass spectrometry measurements with high resolution and high sensitivity has been developed. The instrument design features two pulsed-ion mirrors in a coaxial geometry. Ions can be reflected back and forth with the mirrors, which increases the net flight length and permits kinetic energy focusing for enhanced resolution. The instrument also contains an electrostatic particle guide which increases ion transmission efficiency and can be used in a bipolar pulsed mode to isolate ions of interest for structural study.     

A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies

We report the design and first applications of a tandem mass spectrometer (a quadrupole time-of-flight mass spectrometer) optimized for the transmission and analysis of large macromolecular assemblies. Careful control of the pressure gradient in the different pumping stages of the instrument has been found to be essential for the detection of macromolecular particles. Such assemblies are, however, difficult to analyze by tandem-MS approaches, because they give rise to signals above m/z 3,000-4,000, the limit for commercial quadrupoles. By reducing the frequency of the quadrupole to 300 kHz and using it as a narrow-band mass filter, we show that it is possible to isolate ions from a single peak at m/z 22,000 in a window as narrow as 22 m/z units. Using cesium iodide cluster signals, we show that the mass range in the time-of-flight (TOF) analyzer extends beyond m/z 90,000, in theory to more than m/z 150,000. We also demonstrate that the resolution of the instrument is greater than 3,000 at m/z 44,500. Tandem-MS capabilities are illustrated by separating components from heterooligomeric assemblies formed between tetrameric transthyretin, thyroxine, retinol-binding protein, and retinol. Isolation of a single charge state at m/z 5,340 in the quadrupole and subsequent collision-induced dissociation (CID) in the gas-filled collision cell leads to the formation of ions from individual subunits and subcomplexes, identified by their mass and charge in the TOF analyzer.     

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.     

Multipole-storage-assisted dissociation for the characterization of large proteins and simple protein mixtures by ESI-FTICR-MS

In Fourier transform ion cyclotron resonance mass spectrometry, collisionally activated dissociation (CAD) typically is accomplished within the analyzer ion cell. An alternative approach of multipole-storage-assisted dissociation (MSAD) has previously been demonstrated by inducing collisional fragmentation in the external multipole that is usually employed for ion accumulation. To explore the utility of MSAD for interrogating intact proteins and simple protein mixtures in a multiplexed manner, we have investigated the means of controlling the collisional energy and the fragmentation pattern for this experimental approach. With protein samples in the low micromolar concentration range, the two major experimental parameters affecting MSAD in the hexapole region were found to be the dc offset voltage and accumulation time. While low-energy MSAD of intact proteins yields fragment ions similar to sustained off resonance irradiation collision-activated dissociation (SORI-CAD), high-energy MSAD induces sequential fragmentation for intact proteins to yield a rich variety of singly charged ions in the m/z 600-1200 Da region. Each of the seven proteins (Mr range of 8.5-116 kDa) examined in this study exhibited their own characteristic MSAD fragmentation pattern, which could be used as a signature of the presence of a given protein, even in a mixture. In addition, any MSAD fragment can be isolated and dissociated further by SORI-CAD in an MS3-type experiment inside the FTICR analyzer cell. This presents a novel way to interrogate the identities of these fragment ions as well as obtain amino acid sequence tag information that can be used to identify proteins from mixtures.     

Ways to improve the analytical characteristics of magnetic-sector time-of-flight mass analyzers

Ways to improve the characteristics of analyzers and the parameters of ion beams in magnetic-sector time-of-flight mass spectrometers with axisymmetric electrostatic fields are considered. It is shown that the analyzer transmission can be improved by eliminating the edge effects at the analyzer entrance and exit. The limiting ion current through the analyzer is determined, above which the mass resolution decreases by an order of magnitude. A new method is proposed for compensating for the intrinsic space charge of ions. Necessary requirements for the precision of manufacturing mass analyzers are refined.     

Tandem ion trap design with enhanced mass analysis capabilities for large populations of ions

A new arrangement consisting of two separate radio frequency (rf) quadrupole ion traps is used to analyze large populations of ions over a wide mass-to-charge (m/z) range. The setup consists of an "accumulation" trap that is maintained at a higher pressure than the second high-performance "analyzer" trap. The two traps are scanned simultaneously, with a mass difference between that determines the residence time and mass range of ions in the analytical trap. Initially, all ions are trapped in the accumulation trap and then mass-selectively ejected into the analyzer trap. As ions arrive in the analyzer trap, they cool through collisions with the buffer gas and then are mass selectively ejected toward the detector. This concurrent linked mass scanning reduces the total number of ions present in the analyzer trap during mass analysis, thereby reducing space charge effects and leading to improved resolution and mass accuracy of analytical spectra.     

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.     

Collisional cooling of large ions in electrospray mass spectrometry

Collisional cooling of ions in the rf-only multipole guides has become a method of choice for coupling electrospray sources to various mass analyzers. Normally parameters of such ion guides (length, pressure) provide enough thermalization and focusing for ions in a wide mass range. Noncovalent complexes, however, have more compact conformations than denatured biomolecules of similar mass and, therefore may not be transmitted efficiently through standard ion guides, as demonstrated by theoretical analysis, simulations, and experiments. Several methods of improving collisional cooling for large compact ions have been developed on a quadrupole time-of-flight instrument, which include operating the ion guides at higher pressure and trapping ions to increase the cooling time. Improved transmission of heavy ions obtained with those methods is studied in experiments with proteasome 20S, an oligomeric protein noncovalent complex with molecular weight around 692,000, and a few other compounds.     

Achievement of energy focus for distance-of-flight mass spectrometry with constant momentum acceleration and an ion mirror

Distance-of-flight mass spectrometry (DOF-MS) has not yet been implemented, though it has many potential advantages in a variety of applications. Impeding the implementation of DOF-MS is the development of the required array detectors and working out the equivalents to the focusing methods now used in time-of-flight (TOF) mass analyzers. Ideally, a batch of ions composed of a variety of m/z values, despite initial distributions of space and energy, would be spatially focused at their respective flight distances at the same time. First-order energy focusing, including ion turnaround, is shown to be accomplished by the use of an ion mirror in conjunction with constant momentum acceleration of the initial ion packet. The initial spatial dispersion is maintained throughout the flight path. With zero initial spatial ion spread, energy focusing to achieve resolutions in the tens of thousands is shown to be feasible with ions from the elemental and isotope ratio mass regions through the extremely high m/z range. With moderate spatial spread taken into account, the DOF-MS approach is shown to achieve resolutions competitive with quadrupole and ion trap mass analyzers. Advantages of DOF-MS include all the advantages of TOF-MS plus simpler detector electronics and the improved signal-to-noise ratio and dynamic range afforded by array detection.     

Electrostatic axially harmonic orbital trapping: a high-performance technique of mass analysis

This work describes a new type of mass analyzer which employs trapping in an electrostatic field. The potential distribution of the field can be represented as a combination of quadrupole and logarithmic potentials. In the absence of any magnetic or rf fields, ion stability is achieved only due to ions orbiting around an axial electrode. Orbiting ions also perform harmonic oscillations along the electrode with frequency proportional to (m/z)-1/2. These oscillations are detected using image current detection and are transformed into mass spectra using fast FT, similarly to FTICR. Practical aspects of the trap design are presented. High-mass resolution up to 150,000 for ions produced by laser ablation has been demonstrated, along with high-energy acceptance and wide mass range.     

Expanding the library of secondary ions that distinguish lignin and polysaccharides in time-of-flight secondary ion mass spectrometry analysis of wood

Extracted pine (Pinus spp.) wood and the holocellulose and cellulose fractions of pine were analyzed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). The main sources of variation among wood constituents were elucidated by principal component analysis (PCA). Peaks characteristic of lignin or polysaccharides were identified through the combination of high mass resolution analyses of pine fractions and high lateral resolution image analyses distinguishing the lignin-rich middle lamella from the secondary cell wall layers in solid wood cross-sections. A collection of peaks was compiled which (1) extends the library of characteristic lignin and polysaccharide secondary ions in wood, (2) can be applied to both high and nominal mass resolution spectra, and (3) is free from peaks that contraindicate between wood components. The removal of additional peaks to avoid mass interferences with common contaminants was also successful. Many of the characteristic peaks were high-intensity fingerprint ions below m/z 100, which provided for rapid analysis of the lignin and polysaccharide biopolymers in woody samples. The analysis also identified important mass interferences with previously reported wood ions.     

Laser microprobe with fourier transform ion cyclotron resonance mass spectrometer for surface analysis

Fourier transform ion cyclotron resonance laser microprobe mass spectrometry (FTICR LMMS) uses focused laser irradiation of solids with a spot of 5 microm and a FTICR mass analyzer for local analysis with high mass resolution. A new ion source design has been developed to improve the extraction and transfer of ions generated in an external laser microprobe source. Calculations predicted trapping of ions initially emitted with angles up to 40 degrees and 60 degrees from the surface and from a distance of 1 mm above the sample, respectively. The analytical performances of the method have been verified on two sets of test samples. First, detection of chemisorbed benzotriazole on copper, average of two monolayers, has been shown with less sample consumption than typically required in static secondary ion mass spectrometry with a time-of-flight analyzer. Second, experiments on a thermal plate for offset printing have shown the feasibility of analysis and quantification of dyes embedded in a polymer matrix.     

Superimposition of a magnetic field around an ion guide for electron ionization time-of-flight mass spectrometry

A new electron ionization source was developed for orthogonal acceleration time-of-flight mass spectrometry (TOFMS) based on the superimposition of a magnetic field around a radio frequency-only (rf-only) ion guide. The cylindrically symmetric magnetic field compresses the electron beam from the electron source into a long narrow volume along the ion guide axis. The magnetic field also helps to maintain a narrow energy distribution of electrons that penetrate the full length of the ion guide despite the influence of the radial rf field. Ionization occurs inside the ion guide with improved efficiency resulting from efficient use of electrons, prolonged interaction time, and nontraditionally large ionization volume. At the same time, the rf field effectively focuses ions radially and confines them to the axis of the ion guide by collisional focusing, leading to high ion transmission efficiency. Furthermore, the source can also be operated in a trap-and-pulse mode to improve the ion sampling duty cycle of orthogonal acceleration TOFMS. To validate the design concept of this new ion source, a simple prototype using a single set of cylindrical rods was constructed and retrofitted to an orthogonal acceleration TOFMS. A significant increase in ion signal intensity was observed by operating the source in a pulsed ion extraction mode. Low detection limits (for example, 12 fg for toluene) were determined at 12.5 spectra s(-1) in the full spectrum mode.     

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 mass spectrometry with a multiple pass quadrupole mass analyzer

The peak shape narrows and the resolution improves if the ions are simply reflected back and forth through a conventional quadrupole mass analyzer. CO(+) and N(2)(+) at m/z = 28 are separated to 50% valley with half of the original signal remaining. These two ions can be resolved to baseline (m/Δm) = 5000 with 1% of the original signal remaining.     

Ion dynamics in plasma sheath under the effect of E×B and collisional forces

Using the fluid model, we investigated the velocity, kinetic energy and the density distribution of the ions in collisional and collisionless magnetized plasma sheath. Considering an external magnetic field, the ion movement under the effect of magnetic, electric and collisional forces has been analyzed numerically. The nonexistence of fluctuations in ions kinetic energy in collisionless strong magnetized plasma sheath and increasing the ions velocity in depth direction due to the collisions in some positions in the sheath are shown. The fluctuations of ion velocity in weak magnetized plasma sheath are shown too when ions enter the sheath with oblique incident angle.     

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.