Peak deconvolution in high-field asymmetric waveform ion mobility spectrometry (FAIMS) to characterize macromolecular conformations

Abstract

Protonated poly(ethylene glycol), produced by electrospray ionization (ESI), with molecular weights ranging from 0.3 to 5 kDa and charge states from 1+ to 7+ were characterized using high-field asymmetric waveform ion mobility spectrometry (FAIMS). Results for all but some of the 3+ and 4+ charge states are consistent with a single gas-phase conformer or family of unresolved conformers for each of these charge states. The FAIMS compensation voltage scans resulted in peaks that could be accurately fit with a single Gaussian for each peak. The peak widths increase linearly with compensation voltage for maximum ion transmission but do not depend on m/z or molecular weight. Fitting parameters obtained from the poly(ethylene glycol) data were used to analyze conformations of oxidized and reduced lysozyme formed from different solutions. For oxidized lysozyme formed from a buffered aqueous solution, a single conformer (or group of unresolved conformers) was observed for the 7+ and 8+ charge states. Two conformers were observed for the 9+ and 10+ charge states formed from more denaturing solutions. Data for the fully reduced form indicate the existence of up to three different conformers for each charge state produced directly by ESI and a general progression from a more extended to a more folded structure with decreasing charge state. These results are consistent with those obtained previously by proton-transfer reactivity and drift tube ion mobility experiments, although more conformers were identified for the fully reduced form of lysozyme using FAIMS.

Fig. 1

FAIMS CV scans of protonated PEG 1+ formed by ESI from a 1.0 × 10⁻⁴ M PEG 600 in 49.5/49.5/1%, by volume, water/methanol/acetic acid solution and protonated PEG 3+ through 6+ from a 1.0 × 10⁻⁴ M PEG 3400 solution in 49.5/49.5/1%, by volume, water/methanol/acetic acid. The solid lines are the best fit Gaussian distributions to the experimental data. Data for the 2+ and 7+ significantly overlap those for the 5+ and 6+, respectively, and were omitted for clarity.

Fig. 2

The peak widths of Gaussian distributions as a function of CV for PEG 1+ (grey filled triangles), 2+ (solid diamonds), 3+ (open circles), 4+ (black filled triangles), 5+ (squares), 6+ (open diamonds), and 7+ (filled circles). The trend line is calculated using linear regression of all plotted PEG peak width data.

Fig. 3

The peak widths of Gaussian distributions as a function of m/z for PEG 1+ (grey filled triangles), 2+ (solid diamonds), 3+ (open circles), 4+ (black filled triangles), 5+ (squares), 6+ (open diamonds), and 7+ (filled circles).

Fig. 4

The m/z of PEG ions as a function of CV of ion transmission for PEG 1+ (grey filled triangles), 2+ (solid diamonds), 3+ (open circles), 4+ (black filled triangles), 5+ (squares), 6+ (open diamonds), and 7+ (filled circles).

Fig. 5

ESI mass spectra of 3.3 × 10⁻⁵ M lysozyme from (a) a 200 mM ammonium bicarbonate aqueous solution, (b–d) 33.2/66.5/0.3%, by volume, water/methanol/acetic acid solution with: (b) DTT at 20 °C, (c) 30 min at 100 °C, and (d) both DTT and 30 min at 100 °C. The mass of the ions formed from solutions with DTT and 30 min at 100 °C (d) indicates that all four disulfide bonds are reduced. Less than 10% reduction of a single disulfide bond was detected in ESI mass spectra from the other solutions (a–c). Adducts in (a) corresponding to attachment of sodium and ammonia are observed for both the 7+ and 8+ change states formed from buffered solutions. Asterisk (*) indicates instrumental noise peaks.

Fig. 6

CV scans for fully oxidized lysozyme 7+ to 10+ charge states formed by ESI from either a 200 mM ammonium bicarbonate aqueous solutions (7+ and 8+) or a 33.2/66.5/0.3%, by volume, water/methanol/acetic acid solution with 2.0 mM DTT reducing agent added (9+ and 10+) (no reduced lysozyme present). For the 7+ and 8+ a single Gaussian distribution (solid line) fit the CV data. The sum of two Gaussian distributions (solid line) fit the CV data for 9+ and 10+. The individual Gaussian distributions (thin solid lines) that make up the total fit for the 9+ and 10+ are plotted lower for clarity.

Fig. 7

CV scans for fully reduced lysozyme 10+ through 16+ formed from 33.2/66.5/0.3%, by volume, water/methanol/acetic acid solution with DTT and heated to 100 °C for 30 min. The sum of two Gaussian distributions fit the CV data for 10+. For the 11+ to 16+, three Gaussian distributions fit the CV data and a single Gaussian distribution fit to the CV data for 17+ (solid lines). Individual Gaussian distributions (thin solid lines) used in the sum for 10+ through 16+ are plotted lower for clarity.

Fig. 8

CV scans for: (a) fully oxidized and (b) fully reduced lysozyme 10+ from 33.2/66.5/0.3%, by volume, water/methanol/acetic acid solution. The summed Gaussian distribution (solid line) for both scans is comprised of two individual Gaussian distributions (thin solid lines) which are individually plotted lower for clarity. Partial mass spectra (right) show isotope distributions (calculated distributions indicated by “×”) for the 10+ change state indicating an 8 Da mass change upon reduction of the four disulfide bonds.