Despite often minute concentrations in vivo, D-amino acid containing peptides (DAACPs) are crucial to many life processes. Standard proteomics protocols fail to detect them as D/L substitutions do not affect the peptide parent and fragment masses. The differences in fragment yields are often limited, obstructing the investigations of important but low abundance epimers in isomeric mixtures. Separation of D/L-peptides using ion mobility spectrometry (IMS) was impeded by small collision cross section differences (commonly ~1%). Here, broad baseline separation of DAACPs with up to ~30 residues employing trapped IMS with resolving power up to ~340, followed by time-of-flight mass spectrometry is demonstrated. The D/L-pairs co-eluting in one charge state were resolved in another, and epimers merged as protonated species were resolved upon metalation, effectively turning the charge state and cationization mode into extra separation dimensions. Linear quantification down to 0.25% proved the utility of high resolution IMS-MS for real samples with large inter-isomeric dynamic range. Very close relative mobilities found for DAACP pairs using traveling-wave IMS (TWIMS) with different ion sources and faster IMS separations showed the transferability of results across IMS platforms. Fragmentation of epimers can enhance their identification and further improve detection and quantification limits, and we demonstrate the advantages of online mobility separated collision-induced dissociation (CID) followed by high resolution mass spectrometry (TIMS-CID-MS) for epimer analysis.

Comprehensive characterization of proteomes comprising the same proteins with distinct post-translational modifications (PTMs) is a staggering challenge. Many such proteoforms are isomers (localization variants) that require separation followed by top-down or middle-down mass spectrometric analyses, but condensed-phase separations are ineffective in those size ranges. The variants for "middle-down" peptides were resolved by differential ion mobility spectrometry (FAIMS), relying on the mobility increment at high electric fields, but not previously by linear IMS on the basis of absolute mobility. We now use complete histone tails with diverse PTMs on alternative sites to demonstrate that high-resolution linear IMS, here trapped IMS (TIMS), broadly resolves the variants of ∼50 residues in full or into binary mixtures quantifiable by tandem MS, largely thanks to orthogonal separations across charge states. Separations using traveling-wave (TWIMS) and/or involving various time scales and electrospray ionization source conditions are similar (with lower resolution for TWIMS), showing the transferability of results across linear IMS instruments. The linear IMS and FAIMS dimensions are substantially orthogonal, suggesting FAIMS/IMS/MS as a powerful platform for proteoform analyses.

The structural elucidation of native macromolecular assemblies has been a subject of considerable interest in native mass spectrometry (MS), and more recently in tandem with ion mobility spectrometry (IMS-MS), for a better understanding of their biochemical and biophysical functions. In the present work, we describe a new generation trapped ion mobility spectrometer (TIMS), with extended mobility range (K 0 = 0.185−1.84 cm 2 •V −1 •s −1 ), capable of trapping high-molecular-weight (MW) macromolecular assemblies. This compact 4 cm long TIMS analyzer utilizes a convex electrode, quadrupolar geometry with increased pseudopotential penetration in the radial dimension, extending the mobility trapping to high-MW species under native state (i.e., lower charge states). The TIMS capabilities to perform variable scan rate (Sr) mobility measurements over short time (100−500 ms), high-mobility resolution, and ion-neutral collision cross-section (CCS N 2 ) measurements are presented. The trapping capabilities of the convex electrode TIMS geometry and ease of operation over a wide gas flow, rf range, and electric field trapping range are illustrated for the first time using a comprehensive list of standards varying from CsI clusters (n = 6−73), Tuning Mix oligomers (n = 1−5), common proteins (e.g., ubiquitin, cytochrome C, lysozyme, concanavalin (n = 1−4), carbonic anhydrase, β clamp (n = 1−4), topoisomerase IB, bovine serum albumin (n = 1−3), topoisomerase IA, alcohol dehydrogenase), IgG antibody (e.g., avastin), protein−DNA complexes, and macromolecular assemblies (e.g., GroEL and RNA polymerase (n = 1−2)) covering a wide mass (up to m/z 19 000) and CCS range (up to 22 000 Å 2 with <0.6% relative standard deviation (RSD)).

In the present work, the conformational dynamics and folding pathways of i-motif DNA were studied in solution and in the gas-phase as a function of the solution pH conditions using circular dichroism (CD), photoacoustic calorimetry analysis (PAC), trapped ion mobility spectrometry - mass spectrometry (TIMS-MS), and molecular dynamics (MD). Solution studies showed at thermodynamic equilibrium the existence of a two-state folding mechanism, whereas during the pH = 7.0 → 4.5 transition a fast and slow phase (ΔHfast+ ΔHslow = 43 ± 7 kcal mol−1) with a volume change associated with the formation of hemiprotonated cytosine base pairs and concomitant collapse of the i-motif oligonucleotide into a compact conformation were observed. TIMS-MS experiments showed that gas-phase, kinetically trapped i-motif DNA intermediates produced by nanoESI are preserved, with relative abundances depending on the solution pH conditions. In particular, a folded i-motif DNA structure was observed in nanoESI-TIMS-MS for low charge states in both positive and negative ion mode (e.g., z = +/− 3 to +/−5) at low pH conditions. As solution pH increases, the cytosine deprotonation leads to the loss of cytosine-cytosine+ (C•CH+) base pairing in the CCC strands and in those conditions we observe partially unfolded i-motif DNA conformations in nanoESI-TIMS-MS for higher charge states (e.g., z = − 6 to −9). Collisional induced activation prior TIMS-MS showed the existence of multiple local free energy minima, associated with the i-motif DNA unfolding at z = −6 charge state. For the first time, candidate gas-phase structures are proposed based on mobility measurements of the i-motif DNA unfolding pathway. Moreover, the inspection of partially unfolded i-motif DNA structures (z = −7 and z = −8 charge states) showed that the presence of inner cations may or may not induce conformational changes in the gas-phase. For example, incorporation of ammonium adducts does not lead to major conformational changes while sodium adducts may lead to the formation of sodium mediated bonds between two negatively charged sides inducing the stabilization towards more compact structures in new local, free energy minima in the gas-phase.