High-resolution separation of bioisomers using ion cloud profiling

Abstract

Elucidation of complex structures of biomolecules plays a key role in the field of chemistry and life sciences. In the past decade, ion mobility, by coupling with mass spectrometry, has become a unique tool for distinguishing isomers and isoforms of biomolecules. In this study, we develop a concept for performing ion mobility analysis using an ion trap, which enables isomer separation under ultra-high fields to achieve super high resolutions over 10,000. The potential of this technology has been demonstrated for analysis of isomers for biomolecules including disaccharides, phospholipids, and peptides with post-translational modifications.

Fig. 1. Instrumental setup, principal, and performance characterization of the ion cloud profiling technology.

Schematics of a the miniature MS system used in this work and b its key components for isomer structural analysis. c Simulated ion trajectories for two isomeric ions characterized by reduced damping coefficients: $b^{\prime }$= 0.0010 (blue) and 0.0012 (purple). Here, $b^{\prime }=2b/\Omega m$, where $\Omega$ is the angular frequence of the RF field, m is ion mass, b is the damping coefficient of the ions. Insets, zoom-in plots of the ion trajectories at the beginning (1) and ejection (2) stages of the AC excitation. Isomeric ions are ejected sequentially according to their DCSs when their oscillation amplitudes exceed the trap geometry, r₀, as indicated by the blue dashed lines. d Ion cloud profiling spectra of three biomolecules, lactose (m/z 365, CCS 177.6 Ų), phosphatidylcholine (PC) 18:1/16:0 (m/z 761, CCS 296.2 Ų), and an acetylated peptide (m/z 542, CCS 357.9 Ų), superimposed in one spectrum. The CCS values of lactose and peptide are measured by timsTOF (Bruker Daltonics, Bremen, Germany). The CCS value of phosphatidylcholine is taken from Groessl’s work. The resolution here is defined as, $R=V_{AC}/△V_{AC}$, where $V_{AC}$ and $△V_{AC}$ are the AC ejection amplitude of analyte ions and the full width at half maximum (FWHM) of the peak, respectively.

Fig. 2. Structural analysis of glycans.

a Structures of four isomeric disaccharides, which have isomerization of composition, connectivity, and configuration in pairs. b Ion cloud profiling spectra of the four disaccharides: trehalose (red), maltose (orange), cellose (green), lactose (blue), and the mixture of these four (black). c Ion cloud profiling spectrum of lactose and cellose mixture. d Tandem MS spectra of lactose and cellose mixture (top), pure lactose (middle), and pure trehalose (bottom). Lactose (blue) and cellose (green) have identical mass to charge ratio, m/z 365, and fragment, m/z 305, in tandem MS spectra. e Calibration curves for pure lactose and cellose. For quantitative analysis of pure sample, concentrations varied from 5 μM to 50 μM. Each value represents the mean ± s.d. (N = 10). f Calibration curves for the mixture of lactose and cellose. For quantitative analysis of mixture, concentration of cellose was 5 μM, and concentration ratios of lactose to cellose varied from 0.5 to 10. Each value represents the mean ± s.d. (N = 15). Source data are provided as a Source Data file.

Fig. 3. Structural analysis of lipids and peptides.

a Structure of phospholipids with isomerization of sn position, carbon-carbon double bond location, and cis/trans structure due to the double bond. Ion cloud profiling spectra of b PC 18:1(9Z)/16:0 (blue), PC 16:0/18:1(9Z) (green), and their mixture (red); c PC 18:1 (11Z)/18:1(11Z) (blue), PC 18:1 (9Z)/18:1(9Z) (green), and their mixture (red); d PC 18:1 (9E)/18:1(9E) (blue), PC 18:1 (9Z)/18:1(9Z) (green), and their mixture (red). e Structure of peptide, SGKLRASHKG, with different types of PTMs. Ion cloud profiling spectra of the peptide with f methylation in K3 (blue), K9 (green), and their mixture (red); g acetylation in K3 (blue), K9 (green), and their mixture (red); h phosphorylation in S1 (blue), S7 (green), and their mixture (red).