Search

Rationale: Atmospheric pressure drift tube ion mobility is a powerful addition to the Orbitrap mass spectrometer enabling direct separation of isomers. Apart from offering high resolving power in a compact design, it also facilitates optimization of the separation gas, as shown here for a series of biologically relevant isomer pairs., Methods: An Excellims MA3100 High-Resolution Atmospheric Pressure Ion Mobility Spectrometer (HR-IMS) was coupled to a Thermo Scientific™ Q Exactive™ Focus hybrid quadrupole-Orbitrap™ mass spectrometer, using an Excellims Directspray™ Electrospray Ionization source and a gas mixture setup to provide various drift gases (air, CO 2 and mixtures). This instrument combination was used to separate isomers of eight pairs of metabolites and gangliosides, optimizing drift gas conditions for best separation of each set., Results: All but one of the isomers pairs provided could be partially or fully separated by the HR-IMS-MS combination using ion mobility drift times. About half of the separated compounds showed significantly better analytical separation when analyzed in a mixture of CO 2 and air rather than air or CO 2 alone. Resolving power of up to 102 was achieved using the 10 cm atmospheric drift tube ion mobility add-on for the Orbitrap mass spectrometer., Conclusions: The present analysis demonstrates the usefulness of using atmospheric drift tube IMS on an Orbitrap mass spectrometer to characterize the isomeric composition of samples. It also highlights the potential benefits of being able to quickly optimize the drift gas composition to selectively maximize the mobility difference for isomer separation., (© 2019 John Wiley & Sons, Ltd.)

Nearly all compounds comprise numerous isotopologues ensuing from stable natural isotopes for constituent elements. The consequent isotopic envelopes in mass spectra can reveal the ion stoichiometry but not geometry. We found those envelopes to split in differential ion mobility (FAIMS) spectra in a manner dependent on the ion geometry and buffer gas composition. The resulting multidimensional matrix of isotopic shifts is specific to isomers, providing a fundamentally new approach to the characterization of chemical structure. The physical origins of the effect remain to be clarified but likely ensue from the transposition of center of mass of the ion within its geometry frame affecting the partition of energy in above-thermal collisions between the translational and rotational degrees of freedom. The additivity of shifts, holding with no exception so far, may be the key to unraveling the foundations of observed behavior.

Histone proteins are subject to dynamic post-translational modifications (PTMs) that cooperatively modulate the chromatin structure and function. Nearly all functional PTMs are found on the N-terminal histone domains (tails) of ∼50 residues protruding from the nucleosome core. Using high-definition differential ion mobility spectrometry (FAIMS) with electron transfer dissociation, we demonstrate rapid baseline gas-phase separation and identification of tails involving monomethylation, trimethylation, acetylation, or phosphorylation in biologically relevant positions. These are by far the largest variant peptides resolved by any method, some with PTM contributing just 0.25% to the mass. This opens the door to similar separations for intact proteins and in top-down proteomics.

The rising profile of ion mobility spectrometry (IMS) in proteomics has driven the efforts to predict peptide cross-sections. In the simplest approach, these are derived by adding the contributions of all amino acid residues and post-translational modifications (PTMs) defined by their intrinsic size parameters (ISPs). We show that the ISPs for PTMs can be calculated from properties of constituent atoms, and introduce the "impact scores" that govern the shift of cross-sections from the central mass-dependent trend for unmodified peptides. The ISPs and scores tabulated for 100 more common PTMs enable predicting the domains for modified peptides in the IMS/MS space that would guide subproteome investigations. Graphical Abstract ᅟ.

Differential or field asymmetric waveform ion mobility spectrometry (FAIMS) operating at high electric fields fully resolves isotopic isomers for a peptide with labeled residues. The naturally present isotopes, alone and together with targeted labels, also cause spectral shifts that approximately add for multiple heavy atoms. Separation qualitatively depends on the gas composition. These findings may enable novel strategies in proteomic and metabolomic analyses using stable isotope labeling.