Barbara Ells

Separation of leucine and isoleucine by electrospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry

An electrospray ionization (ESI) source was used to generate gas-phase molecular anions of the amino acids leucine and isoleucine ((M-H)−; m/z −130), which were separated by high-field asymmetric waveform ion mobility spectrometry (FAIMS) and detected by quadrupole mass spectrometry (MS). This combination of ESI-FAIMS-MS enabled selective determination of either amino acid in mixtures that contained at least a 625-fold excess of the other. Comparisons with conventional ESI-MS showed a 50-fold improvement in the signal to background ratio for a 1 µM solution of leucine.

Application of ESI-FAIMS-MS to the analysis of tryptic peptides

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.

Elongated conformers of charge states +11 to +15 of bovine ubiquitin studied using ESI-FAIMS-MS

Recent advancements in high-field asymmetric waveform ion mobility spectrometry (FAIMS) have led to significant improvements in the application of this technology to the study of protein conformers. Compared with previous work, the maximum value of the separation voltage (i.e., the dispersion voltage) has increased, thereby enabling multiple, elongated conformers of individual charge states of bovine ubiquitin to be separated in the gas phase (e.g., four conformers of each of the +11 and +12 charge states were separated). The use of a carrier gas mixture of 40% nitrogen and 60% helium changed the separation selectivity compared with pure nitrogen and enhanced the signal intensity, especially for the +14 and +15 charge states (the latter was not detected in a nitrogen carrier gas). Conformer cross sections were determined using the FAIMS/energy-loss method and found to be similar within a given charge state. The cross sections for conformers of charge states +13, +14, and +15 plateau at about 2000 Å2 suggesting that the structure of bovine ubiquitin is essentially unfolded after the addition of the 13th proton.

Evaluation of carrier gases for use in high-field asymmetric waveform ion mobility spectrometry.

Effects of carrier gas type (N2, O2, CO2, N2O, and SF6) on changes in the ratio of high-to low-field ion mobility, Kh/K, of cesium, gramicidin S, tetrahexylammonium, heptadecanoic acid, and aspartic acid in fields of up to 67 Td are presented. The theory of the mobility of ions at high E/N in different gases is discussed. Plots of Kh/K as a function of the ionic energy parameter, E/N, for the five ions in each of the gases were derived from experimental data collected using a high-field asymmetric waveform ion mobility spectrometer. The change in the ratio of high-to low-field ion mobility of cesium in carrier gases of O2 and N2 showed excellent agreement with literature values. The behavior of cesium in O2 and N2 is used to illustrate that the ratio Kh/K as a function of effective temperature is invariant with gas type as long as the well depth of the interaction potential significantly exceeds thermal energy. From these results, it appears that the well depth of the interaction potential of the heavier ions studied here, including gramicidin S, tetrahexylammonium, and heptadecanoic acid, with bath gases such as N2 and O2, is shallow relative to thermal energy.

Investigation of bovine ubiquitin conformers separated by high-field asymmetric waveform ion mobility spectrometry: Cross section measurements using energy-loss experiments with a triple quadrupole mass spectrometer

High-field asymmetric waveform ion mobility spectrometry (FAIMS) was used to separate gas-phase conformers of bovine ubiquitin produced by electrospray ionization. These conformers were sampled by a triple quadrupole mass spectrometer where energy-loss experiments, following the work of Douglas and co-workers, were used to determine their cross sections. The measured cross sections for some conformers were readily altered by the voltages applied to the interface ion optics, therefore very gentle mass spectrometer interface conditions were required to preserve gas-phase conformers separated by FAIMS. Cross sections for 19 conformers (charge states +5 through +13) were measured. Two conformers for the +12 charge state, which were readily separated in FAIMS, were found to have similar cross sections. Based on a method to calibrate the collision gas thickness, the cross sections measured using the FAIMS/energy-loss method were compared with literature values determined using drift tube ion mobility spectrometry. The comparison illustrated that the conformers of bovine ubiquitin that were identified using drift tube ion mobility spectrometry were also observed using the FAIMS device.

Separation of o-, m- and p-phthalic acids by high-field asymmetric waveform ion mobility spectrometry (FAIMS) using mixed carrier gases

Baseline separation of the three isomers of phthalic acid was achieved in a mixed gas system containing a 95 : 5 mixture of N(2) and CO(2), even though the acids could not be distinguished by high-field asymmetric waveform ion mobility spectrometry (FAIMS) when either pure N(2) or pure CO(2) was used as the carrier gas. Pseudomolecular anions of o-, m- and p-phthalic acids were generated by electrospray ionization and detected, following separation by FAIMS, using a quadrupole mass spectrometer. Addition of small amounts of CO(2) to an N(2) carrier gas also caused the compensation voltages to increase by as much as 12 V, accompanied by 2-7-fold improvements in the measured ion current and dramatic reductions in both adduct ion formation and parent ion fragmentation. Copyright 2000 John Wiley & Sons, Ltd.

Atmospheric Pressure Ion Trapping in a Tandem FAIMS-FAIMS Coupled to a TOFMS: Studies with Electrospray Generated Gramicidin S Ions

A tandem FAIMS-FAIMS system for ion trapping at room temperature and atmospheric pressure is described. The first FAIMS device consisted of a side-to-side configuration (sFAIMS) suitable for ion separation, whereas the second FAIMS device was appropriate for ion trapping (tFAIMS). Ions pre-selected by the sFAIMS entered the tFAIMS and were captured by virtual trapping fields at the hemispherical tip of the inner electrode. The use of the sFAIMS, with wider electrode diameters, and consequently better ion separation efficiency than the tFAIMS, lowered the number of background ions captured in the trapping region of tFAIMS, and thus reduced the space charge effects in the trap. This tandem device was coupled to a laboratory built time-of-flight mass spectrometer and was evaluated using the electrospray generated [M + 2H]2+ ion of gramicidin S. The half-time (t1/2) of the exponential decay of the ion cloud in tFAIMS, determined by monitoring the residual intensity of ions extracted from the ion trapping region of tFAIMS after various delay times, was about 2 s.