High-resolution differential ion mobility separations using helium-rich gases

Abstract

Analyses of complex mixtures and characterization of ions increasingly involve gas-phase separations by ion mobility spectrometry (IMS) and particularly differential or field asymmetric waveform IMS (FAIMS) based on the difference of ion mobility in strong and weak electric fields. The key advantage of FAIMS is substantial orthogonality to mass spectrometry (MS), which makes FAIMS/MS hybrid a powerful analytical platform of broad utility. However, the potential of FAIMS has been constrained by limited resolution. Here, we report that the use of gas mixtures comprising up to 75% He dramatically increases the FAIMS separation capability, with the resolving power for peptides and peak capacity for protein digests reaching and exceeding 100. The resolution gains extend to small molecules, where previously unresolved isomers can now be separated. These performance levels open major new applications of FAIMS in proteomic and other biomolecular analyses.

Figure 1

Mass-selected FAIMS spectra for the 1:1 Leu/IIe mixture (solid lines) in He/N₂ with ≤50% He (left) and ≥50% He (right). The values of resolution r are given for separated isomers. Pure Leu or IIe yield single peaks matching “L” and “I”, respectively (dashed lines in the right panel for IIe at 50–70% He).

Figure 2

Mass-selected FAIMS spectra for bradykinin 2+ (left column) and syntide 2 with z = 2–4 (right column) at 50–75% He. Samples were 20 μM solutions of pure peptides. For well-shaped peaks, we show the widths (V/cm) with the resolving power values underneath.

Figure 3

Successive replicates of the peak d for Syntide 3+ in Figure 2 at 75% He, measured in a window of 3.2 V/cm width at the scan speed of 1.06 V/(cm × min). The E_C values and peak widths are given for each case.

Figure 4

Total FAIMS spectra for the BSA tryptic digest (1.5 μM by the initial protein content) in He/N₂ with 50–75% He. The separation widths are given in V/cm.

Figure 5

FAIMS spectra for selected ions extracted from the data in Figure 4 at 50% He (a) and 75% He (b). We color-code the features by the charge state as labeled, giving the m/z values and (where possible) peak widths (V/cm) underneath. These species were chosen to illustrate the separation peak capacity and are not necessarily the most intense ones. The peak heights are arbitrary and adjusted for the picture clarity. We sought to mirror in (a) the most features found in (b), but some did not fit in because of lower peak capacity in (a).

Figure 6

Correlation of FAIMS separation properties at 50% and 75% He for the peptides in Figure 5b (including those absent from Figure 5a), with the same color coding. The line is the first-order regression for the whole data set, and linear correlation coefficients are listed for the whole set and each z separately.