High-Resolution Differential Ion Mobility Separations/Orbitrap Mass Spectrometry without Buffer Gas Limitations.

Strong orthogonality between differential ion mobility spectrometry (FAIMS) and mass spectrometry (MS) makes their hybrid a powerful approach to separate isomers and isobars. Harnessing that power depends on high resolution in both dimensions. The ultimate mass resolution and accuracy are delivered by Fourier Transform MS increasingly realized in Orbitrap MS, whereas FAIMS resolution is generally maximized by buffers rich in He or H2 that elevate ion mobility and lead to prominent non-Blanc effects. However, turbomolecular pumps have lower efficiency for light gas molecules and their flow from the FAIMS stage complicates maintaining the ultrahigh vacuum (UHV) needed for Orbitrap operation. Here we address this challenge via two hardware modifications: (i) a differential pumping step between FAIMS and MS stages and (ii) reconfiguration of vacuum lines to isolate pumping of the high vacuum (HV) region. Either greatly ameliorates the pressure increases upon He or H2 aspiration. This development enables free optimization of FAIMS carrier gas without concerns about MS performance, maximizing the utility and flexibility of FAIMS/MS platforms.

Development and characterization of a field-deployable ion-trap mass spectrometer with an atmospheric pressure interface.

A field-deployable quadrupole ion-trap mass spectrometer with an atmospheric pressure interface is designed, built, and characterized. The instrument enclosure (48 cm × 43 cm × 42 cm) includes a roughing pump and a helium lecture bottle; the total weight of the instrument is 68 kg. Peak power consumption during the instrument operation is ∼500 W. The instrument has a mass range of m/z 30-2500, across which it provides better than unit mass resolution. The typical peak width at half height is 0.3 Th for a scan speed of 4000 Th/s. Operation of the instrument with electrospray and atmospheric-pressure matrix-assisted laser desorption ionization (AP-MALDI) ion sources is demonstrated. AP-MALDI analysis of low femtomole amounts of peptides reveals that the sensitivity of the instrument is on par with modern commercially available quadrupole ion-trap mass spectrometers. Tandem mass spectrometry capabilities of the instrument include simultaneous isolation and fragmentation of several different compounds. Two ways to reduce the size, weight, and power consumption of the portable instrument were explored, and results of these initial studies are presented. One of the ways includes utilization of hydrogen as a buffer gas for operation of the ion-trap mass analyzer in combination with a metal hydride method for storage of hydrogen in a compact rechargeable cartridge. Furthermore, careful selection of the inlet capillary dimensions allowed to eliminate the first "1 Torr" stage of the differential pumping without any significant loss of the instrument sensitivity. The elimination of this first pumping stage removed two turbo drag pumps, which substantially decreased the instrument's maximum power consumption (to ∼300 W in peak use, and ∼150 W during standby), as well as its size (to 30 cm × 43 cm × 50 cm) and weight (to 35 kg).

Peptide fragmentation induced by radicals at atmospheric pressure.

A novel ion dissociation technique, which is capable of providing an efficient fragmentation of peptides at essential atmospheric pressure conditions, is developed. The fragmentation patterns observed often contain c-type fragments that are specific to electron capture dissociation/electron transfer dissociation (ECD/ETD), along with the y-/b-type fragments that are specific to collision-activated dissociation (CAD). In the presented experimental setup, ion fragmentation takes place within a flow reactor located in the atmospheric pressure region between the ion source and the mass spectrometer. According to a proposed mechanism, the fragmentation results from the interaction of ESI-generated analyte ions with the gas-phase radical species produced by a corona discharge source.

On the mechanism of ion formation from the aqueous solutions irradiated with 3 microm IR laser pulses under atmospheric pressure.

The mechanism of atmospheric pressure (AP) laser ionization of water and water/glycerol liquid samples at a 3-microm wavelength is studied experimentally. For the ion desorption, an in-house built Yb : YAG-pumped optical parametric oscillator (OPO) infrared (IR) laser has been coupled with AP MALDI ion source interfaced to an ion trap mass spectrometer (MS). It has been shown that water is primarily responsible for ion generation in water/glycerol samples, while glycerol increases the solution viscosity and decreases the water evaporation rate and sample losses. In contrast to AP UV-MALDI, the electric field in the case of AP IR-MALDI does not assist in ion production. It was found that the absence of the electrical field provides the optimum ionization condition both for water and water/glycerol liquid samples at the 3-microm laser irradiation. A two-stage ion formation mechanism, which includes the initial emission of microdroplets and release of molecular ions at the second stage, can explain the experimentally observed ion signal dependencies upon the voltage applied between MS inlet and the MALDI sample plate. Postionization using additional corona discharge APCI increases the observed signal by approximately 50%, which indicates that some portion of the analyte is desorbed in the form of neutral molecules.

Orthogonal extraction ion mobility spectrometry.

Ion Mobility Spectrometry is a powerful tool for the study of molecular conformations, separation of mass isomers, and analysis of complex mixtures and suppression of chemical background. The factors that limit the capabilities of the technique include its relatively low resolving power and duty cycle. New principle of gas-phase ion separation, based on ion focusing under the influence of electrostatic field and stationary in time gas flow, is proposed. Both analytical calculations and a numerical simulation show that a diffusion-limited resolution of several hundred can be achieved. The new type of ion mobility analyzer is called orthogonal extraction IMS. The proposed ortho-IMS can be interfaced with commercial mass spectrometers and offers the theoretical resolution of several hundred and ion transmission close to 100%.

Interfacing liquid chromatography with atmospheric pressure MALDI-MS.

Two different strategies for coupling liquid chromatography with atmospheric pressure matrix assisted laser desorption/ionization (AP MALDI) are presented. The first method is flow-injection liquid AP UV-MALDI. Compared with previous similar research, the detection limit was improved 10 times to 8.3 fmol using a solution of 50 nM peptide with 25 mM alpha-cyano-4-hydroxycinnamic acid. The applicability of this method to measure oligosaccharides, actinomycin antibiotics, antibiotics, phosphopeptides, and proteins is demonstrated. The upper mass limit achieved with the current instrumentation is 6,500 Da (doubly charged cytochrome c). The feasibility of a second strategy based on single-droplet IR AP MALDI is demonstrated here. Aqueous peptide solutions were successfully measured by this method.

Mass spectrometry of N-linked oligosaccharides using atmospheric pressure infrared laser ionization from solution.

An atmospheric pressure (AP) infrared (IR) laser ionization technique, implemented on a quadrupole ion trap mass spectrometer, was used to analyze underivatized, N-linked oligosaccharides in solution. Experiments were conducted on an atmospheric pressure infrared ionization from solution (AP-IRIS) ion source which differed from previous AP IR matrix-assisted laser desorption/ionization (MALDI) interfaces in that the ion source operated in the absence of an extraction electric field with a higher power 2.94 microm IR laser. The general term 'IRIS' is used as the mechanism of ionization differs from that of MALDI, and is yet to be fully elucidated. The AP-IRIS ion source demonstrated femtomole-level sensitivity for branched oligosaccharides. AP-IRIS showed approximately 16 times improved sensitivity for oligomannose-6 and the core-fucosylated glycan M3N2F over optimal results obtainable on a AP UV-MALDI with a 2,4,6-trihydroxyacetophenone matrix. Comparison between IR and UV cases also showed less fragmentation in the IR spectrum for a glycan with a conserved trimannosyl core, core-substituted with fucose. A mixture of complex, high-mannose and sialylated glycans resulted in positive ion mass spectra with molecular ion peaks for each sugar. Tandem mass spectrometry of the sodiated molecular ions in a mixture of glycans revealed primarily glycosidic (B, Y) cleavages. The reported results show the practical utility of AP-IRIS while the ionization mechanism is still under investigation.

Atmospheric pressure MALDI-fourier transform mass spectrometry.

The coupling of atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) with Fourier transform mass spectrometry (FTMS) is described, and its significance for the high-resolution analysis of complex peptide mixtures is demonstrated. High kinetic energy and extensive metastable decay characteristic of ions generated by vacuum MALDI have been known to constitute a possible obstacle to high-resolution analysis by FTMS. Since the initial coupling of laser desorption techniques with FTMS was realized two decades ago, several different solutions have been proposed to control the energy of the ions and fulfill the promise of high sensitivity and high resolution offered by this analytical method. Initial results obtained on quadrupole time-of-flight and ion trap analyzers have shown that ions generated by MALDI at atmospheric pressure are intrinsically less energetic than those provided by vacuum MALDI. Our report indicates that this characteristic is particularly beneficial for FTMS applications in which a sharp reduction of metastable decay can make larger ion currents available for detection and possible tandem experiments. In our hands, AP MALDI-FTMS has enabled the analysis of complex peptide mixtures with resolution and accuracy comparable to those obtained by analogous electrospray ionization-FTMS experiments, with no evidence of either metastable decomposition or significant formation of matrix adducts. Analysis of a trypsin digest of bovine serum albumin provided signal-to-noise ratios and limits of detection similar to those obtained by ion trap analyzers, but with unmatched resolution and accuracy. AP MALDI has been shown to provide stable precursor ions in amounts that allowed for informative tandem experiments. Finally, the potential of AP MALDI-FTMS for the high-resolution screening of complex mixtures was demonstrated by the analysis of isobaric peptides differing in mass by less than 0.04 Da.

Atmospheric pressure MALDI with pulsed dynamic focusing for high-efficiency transmission of ions into a mass spectrometer.

The atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) technique described to date has proven to be a convenient and rapid method for soft ionization of biomolecules. However, this technique, like other AP ionization methods, has so far suffered from a low efficiency in transmitting ions from atmospheric pressure into the vacuum of the mass spectrometer (MS). In this work, a novel technique we termed pulsed dynamic focusing, or PDF, which improves the ion transmission efficiency and sensitivity of AP-MALDI by over an order of magnitude, is described. Pulsed dynamic focusing operates on the basis of pulsing a high-voltage extraction field to zero, when ions are just outside of the MS entrance, to allow the intake gas flow of the MS to effectively entrain the ions into the MS. Results from application of the PDF technique to an AP-MALDI ion trap MS demonstrated that in comparison to static AP-MALDI operation (1). up to 2.1 times more ions from a given laser shot could be transferred into the MS, (2). applying higher voltages in combination with the switching scheme yielded up to 1.6-times-higher ion intensities, and (3). a 3-times-larger laser spot area could be utilized. The combination of these factors produced an enhancement in throughput and sensitivity, as measured by the ions detected per unit time, of over 12 times for a digest sample of bovine serum albumin. In addition, the PDF technique proved to make AP-MALDI less sensitive to laser positioning, creating a more robust ion source in comparison to static AP-MALDI.

Detection of cyclic lipopeptide biomarkers from Bacillus species using atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry.

A novel approach to microbial detection using atmospheric pressure matrix-assisted laser desorption/ionization with an ion trap mass spectrometer to analyze whole cell bacteria is introduced. This new approach was tested with lyophilized spores and cultures of Bacillus globigii (BG) grown on agar media for 4 days or longer. At each stage of growth, it was found that biomarkers, identified as cyclic lipopeptides known as fengycin and surfactin, could be detected by pulsed ultraviolet laser irradiation of intact BG cells (approximately 5 mg) cocrystallized with alpha-cyano-4-hydroxycinnamic acid. Furthermore, definitive amino acid sequence information was obtained by performing tandem mass spectrometry on the precursor ions of the cyclic lipopeptides. The investigation was broadened to include the examination of aerosolized BG spores collected from the atmosphere and directly deposited onto double-sided tape. Subsequent analysis of the recovered spores resulted in the production of mass peaks consistent with fengycin. Other Bacillus species were analyzed for comparison and showed mass spectral peaks also identified as originating from various cyclic lipopeptides. Further studies were conducted using a pulsed infrared laser as the excitation source to analyze BG cells (approximately 5 mg) suspended in a matrix of 0.03 M ammonium citrate and glycerol resulting in the production of ions characteristic of fengycin and surfactin.

Atmospheric pressure laser desorption/ionization on porous silicon.

A recently developed commercial atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) source (MassTech, Inc.) was modified to adopt commercially available DIOS plates (Mass Consortium Corp.) for the studies of laser desorption from the surface of porous silicon under atmospheric pressure conditions. The feasibility of atmospheric pressure laser desorption/ionization from the surface of porous silicon (AP-DIOS) was demonstrated. The advantages of this new AP-DIOS technique include reasonably good sensitivity (subpicomole range for standard peptide mixtures), simplicity of sample preparation, uniformity of target spots and the absence of matrix peaks in the spectra. The AP-DIOS source was interfaced with a commercial ion trap (LCQ Classic, Thermo Finnigan) which additionally provides a unique MS(n) capability. The AP-DIOS spectrum of 250 fmol of unseparated tryptic digest of bovine serum albumin (BSA) was compared with that of AP-MALDI for the same compound. AP-DIOS offers significantly better coverage for the digest components in the mass range 200-1000 Da. The combined data of both techniques enabled us to nearly double the number of matched peaks in BSA digest analysis compared with AP-DIOS or AP-MALDI analysis separately.

Desorption/ionization of biomolecules from aqueous solutions at atmospheric pressure using an infrared laser at 3 microm.

A new atmospheric pressure (AP) infrared (IR) matrix-assisted laser desorption/ionization (MALDI) ion source was developed and interfaced with a Thermo Finnigan LCQ ion trap mass spectrometer. The source utilized a miniature all-solid-state optical parametric oscillator (OPO)-based IR laser system tunable in the lambda = 1.5-4 microm spectral range and a nitrogen ultraviolet (UV) laser (lambda = 337 nm) for use in comparative studies. The system demonstrated comparable performance at 3 microm and 337 nm wavelengths if UV matrices were used. However, AP IR-MALDI using a 3 microm wavelength showed good performance with a much broader choice of matrices including glycerol and liquid water. AP IR-MALDI mass spectra of peptides in the mass range up to 2000 Da were obtained directly from aqueous solutions at atmospheric conditions for the first time. A potential use of the new AP IR-MALDI ion source includes direct MS analysis of biological cells and tissues in a normal atmospheric environment as well as on-line coupling of mass spectrometers with liquid separation techniques.