Ion mobility spectrometry (IMS) has become an important method for the detection of many compounds because of its high sensitivity and amenability to miniaturization for field-portable monitoring; applications include detection of narcotics, explosives, and chemical warfare agents. High-field asymmetric waveform ion mobility spectrometry (FAIMS) differs from IMS in that the electric fields are applied using a high-frequency periodic asymmetric waveform, rather than a dc voltage. Furthermore, in FAIMS the compounds are separated by the difference in the mobility of ions at high electric field relative to low field, rather than by compound to compound differences in mobility at low electric field (IMS). We report here the first cylindrical-geometry-FAIMS interface with mass spectrometry (FAIMS-MS) and the MS identification of the peaks observed in a FAIMS compensation voltage (CV) spectrum. Using both an electrometer-based-FAIMS (FAIMS-E) and FAIMS-MS, several variables that affect the sensitivity of ion detection were examined for two (polarity reversed) asymmetric waveforms (modes 1 and 2) each of which yields a unique spectrum. An increase in the dispersion voltage (DV) was found to improve the sensitivity and separation observed in the FAIMS CV spectrum. This increase in sensitivity and the unexpected dissimilarity in modes 1 and 2 suggest that atmospheric pressure ion focusing is occurring in the FAIMS analyzer. The sensitivity and peak locations in the CV spectra were affected by temperature, gas flow rates, operating pressure, and analyte concentration.

The focusing of ions at atmospheric pressure and room temperature in a high-field asymmetric waveform ion mobility spectrometer (FAIMS) has been investigated. FAIMS operates with the application of a high-voltage, high-frequency asymmetric waveform across parallel plates. This establishes conditions wherein an ion migrates towards one of the plates because of a difference in the ion mobility at the low and high electric field conditions during application of the waveform. The migration can be stopped by applying a dc compensation voltage (CV) which serves to create a “balanced” condition wherein the ion experiences no net transverse motion. This method has also been called “transverse field compensation ion mobility spectrometry” and “field ion spectrometry®.” If this experiment is conducted using a device with cylindrical geometry, rather than with flat plates, an ion focusing region can exist in the annular space between the two concentric cylinders. Ion trajectory modeling showed that the behavior of the ions in the cylindrical geometry FAIMS analyzer was unlike any previously described atmospheric pressure ion optics system. The ions appeared to be trapped, or focused by being caught between two opposing forces. Requirements for establishing this focus for a given ion were identified: the applied waveform must be asymmetric, the electric field must be sufficiently high that the mobility of the ion deviates from its low-field value during the high-voltage portion of the asymmetric waveform, and finally, the electric field must be nonuniform in space (e.g., cylindrical or spherical geometry). Experimental observations with a prototype FAIMS device, which was designed to measure the radial distribution of ions in the FAIMS analyzer region, have confirmed the results of ion trajectory modeling.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is a new technique that separates gas-phase ions at atmospheric pressure (760 Torr) and room temperature. A FAIMS instrument acts as an ion filter and can be set to continuously transmit one type of ion. Despite the stringent requirement for a flow of clean, dry gas in the FAIMS analyzer region, a method of coupling electrospray to FAIMS has been developed. The identity of the electrospray ions separated by FAIMS was determined using mass spectrometry (FAIMS-MS). The theory of FAIMS is discussed, and electrospray FAIMS-MS spectra of several compounds in modes P1, P2, N1, and N2 are presented. Ions appearing in P1 and N1 modes tend to have mobilities that increase as a function of increasing electric field strength, whereas ions appearing in P2 and N2 modes tend to have mobilities that decrease. In general, low-mass ions are focused in P1 and N1 modes, whereas larger ions (e.g., proteins) are focused in P2 and N2 modes. Short-chain peptides, (Gly)(n) where n = 1-6, are shown to cross over from P1 mode into P2 mode as the chain length increases. The removal of the low-mass solvent cluster ions, combined with a reduction of the background noise in electrospray FAIMS-MS, results in an improved signal-to-noise ratio for mass spectra of larger ions (e.g., cyctochrome c) when compared with conventional electrospray-MS. Preliminary results also suggest that various charge states of cytochrome c can be distinguished by FAIMS, implying that the ion mobility of these species at high electric field strength is sensitive to the structure of the protein ion. The linearity of response of electrospray FAIMS-MS was investigated using leucine enkephalin and shows the calibration curve to be linear for ∼3 orders of magnitude.

Approaches to separation and characterization of ions based on their mobilities in gases date back to the 1960s. Conventional ion mobility spectrometry (IMS) measures the absolute mobility, and field asymmetric waveform IMS (FAIMS) exploits the difference between mobilities at high and low electric fields. However, in all previous IMS and FAIMS experiments ions experienced an essentially free rotation; thus the separation was based on the orientationally averaged cross-sections Omega(avg) between ions and buffer gas molecules. Virtually all large ions are permanent electric dipoles that will be oriented by a sufficiently strong electric field. Under typical FAIMS conditions this will occur for dipole moments >400 D, found for many macroions including most proteins above approximately 30 kDa. Mobilities of aligned dipoles depend on directional cross-sections Omega(dir) (rather than Omega(avg)), which should have a major effect on FAIMS separation parameters. Here we report the FAIMS behavior of electrospray-ionization-generated ions for 10 proteins up to approximately 70 kDa. Those above 29 kDa exhibit a strong increase of mobility at high field, which is consistent with predicted ion dipole alignment. This effect expands the useful FAIMS separation power by an order of magnitude, allowing separation of up to approximately 10(2) distinct protein conformers and potentially revealing information about Omega(dir) and ion dipole moment that is of utility for structural characterization. Possible approaches to extending dipole alignment to smaller ions are discussed.

The continuous gas-phase ion separation and atmospheric pressure focusing properties of high-field asymmetric waveform ion mobility spectrometry (FAIMS) offer significant advantages for the mass spectrometric analysis of tryptic digests of proteins. In this study, tryptic peptides of pig hemoglobin were examined by ESI-FAIMS-MS using a newly designed FAIMS device. The new, hemispherical geometry of the inner electrode served to deliver the ions, via the gas flows, to the center axis of the FAIMS analyzer, improving the sensitivity relative to previous prototypes. Mass spectra collected using this new FAIMS showed significantly less chemical background noise than conventional ESI-MS, while maintaining approximately the same absolute sensitivity as that observed with ESI-MS. As a consequence of the ion separation in FAIMS, the identification of the tryptic fragments was simplified and some peptides, such as the triply protonated WAGVANALAHK3+, that were obscured by the intense background of ESI-MS, were readily detected using ESI-FAIMS-MS. In addition, the FAIMS device was shown to separate isobaric ions at m/z 532.4. Correlations between CV and mass-to-charge ratio, as well as CV and ionic collision cross section, were evaluated for 38 peptide ions identified in the tryptic digest. The correlation between the CV of the peptide and the mass-to-charge ratio is very poor, indicating good orthogonality between the separation by FAIMS and the separation by mass spectrometry.

Thiol-stabilized Au(25)L(18) monolayer protected clusters (MPCs) were found to be active for the reduction of 4-nitrophenol. Results suggest that these MPCs are stable catalysts and do not lose their structural integrity during the catalytic process. High stability under the reaction conditions enables the recyclability of these MPCs.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) separates gas-phase analyte ions from chemical background, offering substantial improvements in the detection of peptides from complex protein digests. For a digest of enolase 1 (baker's yeast), the focusing and separation offered by FAIMS produced an average intensity gain of 3.5 for the tryptic ions and reductions in background intensity of 5- to 10-fold when compared with ESI-MS. The increased signal-to-background in the ESI-FAIMS-MS experiment resulted in a greater number of identifiable peptides and therefore greater sequence coverage. Compensation voltage (CV) maps for a total of 282 tryptic peptides from thirteen proteins, generated according to charge-state, mass-to-charge ratios, and chain length, show that a majority of tryptic peptides can be detected by operating FAIMS at a few discrete values of CV rather than scanning CV across a wide range. The ability to reduce scanning requirements has potential benefits for coupling FAIMS with LC-MS. In select cases, FAIMS can be used to eliminate isobaric MS overlap between tryptic peptides; however, the primary advantage of FAIMS in an LC-FAIMS-MS analysis is foreseen to be the attenuation of chemical background noise rather than the separation of individual peptides. Using FAIMS to reduce mass spectral noise will offer improved detection of peptides from low abundance proteins in complex biological samples.

SUMMARYThe psychoactive and analgesic cannabinoids (e.g. D 9 -tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short-chain fatty acyl-coenzyme A (CoA) precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g )1 fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short-and medium-chained fatty acids. This activity and the trichome-specific expression of CsAAE1 suggest that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids.