Ion mobility spectrometry (IMS) can delineate gas-phase ions and probe their geometries. Coupling with electrospray ionization and MS has brought IMS to structural biology, revealing the macromolecular folding and subunit connectivity. However, the orientational averaging of ion-molecule collision cross sections (Ω) in the linear and field asymmetric waveform IMS (FAIMS) diminishes the resolution and structural specificity. In the novel low-field differential (LOD) IMS, a field too weak for ion heating (and thus FAIMS) aligns strong macrodipoles, capturing their magnitudes and directional Ω across the dipole (Ω⊥). However, the bisinusoidal waveforms (from FAIMS) have compromised the resolution, measurement accuracy, and correlation to the ion properties. Large ions amenable to LODIMS have low mobility and diffuse slowly, allowing the waveform frequencies down to ∼10 kHz. The low field and frequency permit generating the ideal rectangular waveforms with a flexible frequency and duty cycle by direct switching (impractical for FAIMS) in a miniature low-power format. This new IMS stage is evaluated for the exemplary large protein albumin (66 kDa) previously studied using the bisinusoidal waveform. The flat voltages and greater form factor initiate the differential IMS effect at lower fields, expand the separation space, and enable the quantification of Ω⊥ values by varying the duty cycle.

Along with mass spectrometry (MS), ion mobility separations (IMS) are advancing to ever larger biomolecules. The emergence of electrospray ionization (ESI) and native MS enabled the IMS/MS analyses of proteins up to ∼100 kDa in the 1990s and whole protein complexes and viruses up to ∼10 MDa since the 2000s. Differential IMS (FAIMS) is substantially orthogonal to linear IMS based on absolute mobility K and offers exceptional resolution, unique selectivity, and steady filtering readily compatible with slower analytical methods such as electron capture or transfer dissociation (ECD/ETD). However, the associated MS stages had limited FAIMS to ions with m/z < 8000 and masses under ∼300 kDa. Here, we integrate high-definition FAIMS with the Q-Exactive Orbitrap UHMR mass spectrometer that can handle m/z up to 80,000 and MDa-size ions in the native ESI regime. In the initial evaluation, the oligomers of monoclonal antibody adalimumab (148 kDa) are size-selected up to at least the nonamers (1.34 MDa) with m/z values up to ∼17,000. This demonstrates the survival and efficient separation of noncovalent MDa assemblies in the FAIMS process, opening the door to novel analyses of the heaviest macromolecules.

Specific lipid isomers are functionally critical, but their structural rigidity and usually minute geometry differences make separating them harder than other biomolecules. Such separations by ion mobility spectrometry (IMS) were recently enabled by new high-definition methods using dynamic electric fields, but major resolution gains are needed. Another problem of identifying many isomers with no unique fragments in ergodic collision-induced dissociation (CID) was partly addressed by the direct ozone-induced dissociation (OzID) that localizes the double bonds, but a low reaction efficiency has limited the sensitivity, dynamic range, throughput, and compatibility with other tools. Typically lipids are analyzed by MS as singly charged protonated, deprotonated, or ammoniated ions. Here, we explore the differential IMS (FAIMS) separations with OzID for exemplary lipids cationized by polyvalent metals. These multiply charged adducts have much greater FAIMS compensation voltages (UC) than the 1+ ions, with up to 10-fold resolution gain enabling baseline isomer separations even at a moderate resolving power of the SelexION stage. Concomitantly OzID speeds up by many orders of magnitude, producing a high yield of diagnostic fragments already in 1 ms. These capabilities can be ported to the superior high-definition FAIMS and high-pressure OzID systems to take lipidomic analyses to the next level.

Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) has become a versatile tool to fractionate complex mixtures, distinguish structural isomers, and elucidate molecular geometries. Along with the whole MS field, IMS/MS advances to ever larger species. A topical proteomic problem is the discovery and characterization of d-amino acid-containing peptides (DAACPs) that are critical to neurotransmission and toxicology. Both linear IMS and FAIMS previously disentangled d/l epimers with up to similar to 30 residues. In the first study using all three most powerful IMS methodologies & horbar;trapped IMS, cyclic IMS, and FAIMS & horbar;we demonstrate baseline resolution of the largest known d/l peptides (CHH from Homarus americanus with 72 residues) with a dynamic range up to 100. This expands FAIMS analyses of isomeric modified peptides, especially using hydrogen-rich buffers, to the similar to 50-100 residue range of small proteins. The spectra for d and l are unprecedentedly strikingly similar except for a uniform shift of the separation parameter, indicating the conserved epimer-specific structural elements across multiple charge states and conformers. As the interepimer resolution tracks the average for smaller DAACPs, the IMS approaches could help search for yet larger DAACPs. The a priori method to calibrate cyclic (including multipass) IMS developed here may be broadly useful.

Differential ion mobility spectrometry (FAIMS) had emerged in the 2000s as a novel tool for postionization separations in conjunction with mass spectrometry (MS). High-definition FAIMS introduced a decade ago has enabled resolution of peptide, lipid, and other molecular isomers with minute structural variations and recently the isotopic shift analyses where the spectral pattern for stable isotopes fingerprints the ion geometry. Those studies, including all isotopic shift analyses, were in the positive mode. Here, we achieve the same high resolution for anions exemplified by phthalic acid isomers. The resolving power and magnitude of isotopic shifts are in line with the metrics for analogous haloaniline cations, establishing high-definition negative-mode FAIMS with structurally specific isotopic shifts. Different shifts (including the new 18O) remain additive and mutually orthogonal, demonstrating the generality of those properties across the elements and charge states. Expanding to common (not halogenated) organic compounds is a key step toward the broad use of FAIMS isotopic shift methodology.

We had reported the isotopic envelopes in differential IMS (FAIMS) separations depending on the ion structure. However, this new approach to distinguish isomers was constrained by the unit-mass resolution commingling all nominally isobaric isotopologues. Here, we directly couple high-definition FAIMS to ultrahigh-resolution (Orbitrap) MS and employ the resulting platform to explore the FAIMS spectra for isotopic fine structure. The peak shifts therein for isotopologues of halogenated anilines with 15N and 13C (split by 6 mDa) in N2/CO2 buffers dramatically differ, more than for the 13C, 37Cl, or 81Br species apart by 1 or 2 Da. The shifts in FAIMS space upon different elemental isotopic substitutions are orthogonal mutually and to the underlying separations, forming fingerprint multidimensional matrices and 3-D trajectories across gas compositions that redundantly delineate all isomers considered. The interlocking instrumental and methodological upgrades in this work take the structural isotopic shift approach to the next level.

Ion mobility spectrometry (IMS) has become a mainstream approach to fractionate complex mixtures, separate isomers, and assign the molecular geometries. All modalities were grouped into linear IMS (based on the absolute ion mobility, K) and field asymmetric waveform IMS (FAIMS) relying on the evolution of K at a high normalized electric field (E/N) that induces strong ion heating. In the recently demonstrated low-field differential (LOD) IMS, the field is too weak for significant heating but locks the macromolecular dipoles to produce novel separations controlled by the relevant directional collision cross sections (CCSs). Here, we show LODIMS for mass-selected species, exploring the dipole alignment across charge states for the monomers and dimers of an exemplary protein, the alcohol dehydrogenase. Distinct conformational families for aligned species are revealed with directional CCS estimated from the field-dependent trend lines. We set up a model to extract the fractions of pendular conformers as a function of field intensity and translate them into dipole moment distributions. These developments make a critical step toward establishing LODIMS as a new tool for top-down proteomics and integrative structural biology.

Continuing advances in proteomics highlight the ubiquity and biological importance of proteoforms─proteins with varied sequence, splicing, or distribution of post-translational modifications (PTMs). The preeminent example is histones, where the PTM pattern encodes the combinatorial language controlling the DNA transcription central to life. While the proteoforms with distinct PTM compositions are distinguishable by mass, the isomers with permuted PTMs commonly coexisting in cells generally require separation before mass-spectrometric (MS) analyses. That was accomplished on the bottom-up and middle-down levels using chromatography or ion mobility spectrometry (IMS), but proteolytic digestion obliterates the crucial PTM connectivity information. Here, we demonstrate baseline IMS resolution of intact isomeric proteoforms, specifically the acetylated H4 histones (11.3 kDa). The proteoforms with a single acetyl moiety on five alternative lysine residues (K5, K8, K12, K16, K20) known for distinct functionalities in vivo were constructed by two-step native chemical ligation and separated using trapped IMS at the resolving power up to 350 on the Bruker TIMS/ToF platform. Full resolution for several pairs was confirmed using binary mixtures and by unique fragments in tandem MS employing collision-induced dissociation. This novel capability for top-down proteoform characterization is poised to open major new avenues in proteomics and epigenetics.

Since its inception in 1980s, differential or field asymmetric waveform ion mobility spectrometry (FAIMS) has been implemented at or near ambient gas pressure. We recently developed FAIMS at 15-30 Torr with mass spectrometry and utilized it to analyze amino acids, isomeric peptides, and protein conformers. The separations broadly mirrored those at atmospheric pressure, save for larger proteins that (as predicted) exhibited dipole alignment at ambient but not low pressure. Here we reduce the pressure down to 4.7 Torr, allowing normalized electric fields up to 543 Td-double the maximum in prior FAIMS or IMS studies of polyatomic ions. Despite the collisional heating to ∼1000 °C at the waveform peaks, the proteins of size from ubiquitin to albumin survived intact. The dissociation of macromolecules in FAIMS appears governed by the average ion temperature over the waveform cycle, unlike the isomerization controlled by the peak temperature. The global separation trends in this "superhot" regime extend those at moderately low pressures, with distinct conformers and no alignment as theorized. Although the scaling of the compensation voltage with the field fell below cubic at lower fields, the resolving power increased and the resolution of different proteins or charge states substantially improved.

Mass spectrometry (MS) and isotopes were intertwined for a century, with stable isotopes central to many MS identification and quantification protocols. In contrast, the analytical separations including ion mobility spectrometry (IMS) largely ignored isotopes, partly because of insufficient resolution. We recently delineated various halogenated aniline isomers by structurally specific splitting in FAIMS spectra. While this capability hinges on the 13C shifts, all preceding studies leveraged 37Cl or 81Br to enhance the differentiation. However, such abundant heavy isotopes are absent from typical organic compounds. With single I isotope, iodinated organics generate similar isotopic envelopes dominated by the 13C atoms. Here, we distinguish the three monoiodoaniline isomers based on the shifts solely for one or two 13C atoms. The differentiation may be somewhat improved using multipoint peak position descriptions for more reproducible shifts. The interisomer order of shifts differs from those for chlorinated or brominated analogues, showcasing the specificity of approach. We also investigated the mass scaling of isotopic shifts, encountering divergent trends for different structural families.

The preponderance and functional importance of isomeric biomolecules have become topical in biochemistry. Therefore, one must distinguish and identify all such forms across compound classes, over a wide dynamic range as minor species often have critical activities. With all the power of modern mass spectrometry for compositional assignments by accurate mass, the identical precursor and often fragment ion masses render this task a steep challenge. This is recognized in proteomics and epigenetics, where proteoforms are disentangled and characterized employing novel separations and non-ergodic dissociation mechanisms. This issue is equally pertinent to lipidomics, where the lack of isomeric depth has thwarted the deciphering of functional networks. Here we introduce a new platform, where the isomeric lipids separated by high-resolution differential ion mobility spectrometry (FAIMS) are identified using ozone-induced dissociation (OzID). Cationization by metals (here K+, Ag+, and especially Cu+) broadly improves the FAIMS resolution of isomers with alternative C═C double bond (DB) positions or stereochemistry, presumably via metal attaching to the DB and reshaping the ion around it. However, the OzID yield diminishes for Ag+ and vanishes for Cu+ adducts. Argentination still strikes the best compromise between efficient separation and diagnostic fragmentation for optimal FAIMS/OzID performance.

Life was originally assumed to utilize the l-amino acids only. Since 1980s, the d-amino acid-containing peptides (DAACPs) were detected in animals, often at extremely low levels with tremendous functional specificity. As the unguided proteomic algorithms based on peptide masses are oblivious to DAACPs, many more are believed to be hidden in organisms and novel methods to tackle DAACPs are sought. Linear ion mobility spectrometry (IMS) can distinguish and characterize the d/l-epimers but is restricted by poor orthogonality to MS as in other contexts. We now bring to this area the newer technique of differential IMS (FAIMS). The orthogonality of MS to high-resolution FAIMS exceeded that to linear IMS by 6×, the greatest factor found for biomolecules so far. Hence, FAIMS has achieved the 2.5× resolution of trapped IMS on average despite a lower resolving power, fully separating all 18 pairs of representative epimer species with masses of ∼400-5,000 Da and charge states of 1-6. A constant isomer resolution over these ranges allows projecting success for yet larger DAACPs.

The isotopic molecular envelopes due to stable isotopes for most elements were a staple of mass spectrometry since its origins, often leveraged to identify and quantify compounds. However, all isomers share one MS envelope. As the molecular motion in media also depends on the isotopic composition, separations such as liquid chromatography (LC) and ion mobility spectrometry (IMS) must also feature isotopic envelopes. These were largely not observed because of limited resolution, except for the (structurally uninformative) shifts in LC upon H/D exchange. We recently found the isotopic shifts in FAIMS for small haloanilines (∼130-170 Da) to hinge on the halogen position, opening a novel route to isomer characterization. Here, we extend the capability to heavier species: dibromoanilines (DBAs, ∼250 Da) and tribromoanilines (TBAs, ∼330 Da). The 13C shifts for DBAs and TBAs vary across isomers, some changing sign. While 81Br shifts are less specific, the 2-D 13C/81Br shifts unequivocally differentiate all isomers. The trends for DBAs track those for dichloroanilines, with the 13C shift order preserved for most isomers. The peak broadening due to merged isotopomers is also isomer-specific. The absolute shifts for TBAs are smaller than those for lighter haloanilines, but differentiate isomers as well because of compressed uncertainties. These results showcase the feasibility of broadly distinguishing isomers in the more topical ∼200-300 Da range using the isotopic shifts in IMS spectra.

Glycosylation is a ubiquitous post-translational modification (PTM) that strongly affects the protein folding and function. Glycosylation patterns are impacted by many diseases, making promising biomarkers. Glycans are also the most complex PTMs, exhibiting isomers (linkage, anomers, and those with isomeric moieties). Permuted with localization variants that occur for all PTMs, these produce numerous isomeric glycoforms. Characterizing them by mass spectrometry and ion mobility spectrometry (IMS) has been a challenge. High-definition differential IMS (FAIMS) had robustly disentangled isomeric peptides involving other PTMs but was not evaluated for glycopeptides that featured multilevel isomerism. Here, we apply it to representative mucin glycopeptides with O-linked glycans: three GalNAc localization variants, a pair with α/β GalNAc anomers, and another with GalNAc/GlcNAc isomers. The first two classes were separated baseline with the resolution exceeding previous benchmarks by 10-fold, and the last pair was partly resolved. The recently demonstrated straightforward coupling to ultrahigh-resolution MS and electron-transfer dissociation makes high-definition FAIMS an attractive tool for glycoproteomics.

Biological functions of many proteins are governed by post-translational modifications (PTMs). In particular, the rich PTM complement in histones controls the gene expression and chromatin structure with major health implications via a combinatoric language. Deciphering that "histone code" is the great challenge for proteomics given an astounding number of possible proteoforms, including isomers with different PTM positions. These must be disentangled on the top- or middle-down level to preserve the key PTM connectivity, which condensed-phase separations failed to achieve. We reported the capability of ion mobility spectrometry (IMS) methods to resolve such isomers for model histone tails. Here, we advance to biological samples, showing middle-down analyses of histones from mouse embryonic stem cells via online chromatography to fractionate proteoforms with distinct PTM sets, differential or field asymmetric waveform IMS (FAIMS) to resolve the isomers, and Orbitrap mass spectrometry with electron transfer dissociation to identify the resolved species.

Ion mobility spectrometry (IMS) with mass spectrometry has grown into a powerful approach to simplify complex mixtures, disentangle isomers, and elucidate their geometries. Two established branches are linear IMS based on the absolute mobility K at moderate normalized electric field E/N and field asymmetric waveform IMS (FAIMS) relying on the evolution of K at high E/N causing strong ion heating. Here, we introduce low-field differential IMS (LODIMS), where the field is too weak for significant heating but suffices to lock the permanent macromolecular ion dipoles, producing novel separations based solely on their alignment. The method is demonstrated for a prototypical large protein-albumin. Its oligomers start separating at fields of just 1 kV/cm (4 Td), or ∼5% of those typical for FAIMS. Negligible ion heating at such fields allows preserving fragile species, in particular the noncovalent complexes up to pentamers (332 kDa) destroyed in FAIMS and not detected without it. The separation parameter (compensation field, EC) in this regime scales with the field linearly versus cubically in FAIMS. The dipole moments obtained from threshold fields for alignment and directional cross sections estimated from the slope of said linear EC dependence appear reasonable.

Strong orthogonality between differential ion mobility spectrometry (FAIMS) and mass spectrometry (MS) makes their hybrid a powerful approach to separate isomers and isobars. Harnessing that power depends on high resolution in both dimensions. The ultimate mass resolution and accuracy are delivered by Fourier Transform MS increasingly realized in Orbitrap MS, whereas FAIMS resolution is generally maximized by buffers rich in He or H2 that elevate ion mobility and lead to prominent non-Blanc effects. However, turbomolecular pumps have lower efficiency for light gas molecules and their flow from the FAIMS stage complicates maintaining the ultrahigh vacuum (UHV) needed for Orbitrap operation. Here we address this challenge via two hardware modifications: (i) a differential pumping step between FAIMS and MS stages and (ii) reconfiguration of vacuum lines to isolate pumping of the high vacuum (HV) region. Either greatly ameliorates the pressure increases upon He or H2 aspiration. This development enables free optimization of FAIMS carrier gas without concerns about MS performance, maximizing the utility and flexibility of FAIMS/MS platforms.

Since inception in the 1980s, differential or field asymmetric waveform ion mobility spectrometry (FAIMS) was implemented at or near the ambient gas pressure (AP). Recently, we developed FAIMS at 15-30 Torr within a mass spectrometer and demonstrated it for small and medium sized ions, including peptides. The overall separation properties mirrored those at AP, reflecting the shared underlying physics. Here we extend these analyses to macromolecules, namely, multiply charged proteins generated by electrospray ionization. The spectra for smaller proteins (ubiquitin, cytochrome c, myoglobin) again resemble those at AP, producing features for one or a few adjacent well-defined conformers with type C behavior. Large proteins (single aldolase domain and albumin) now follow, with no broad bands for type A or B species that dominated at 1 atm. Those unique behaviors were ascribed to pendular ions with electric dipoles reversibly locked by the strong field in FAIMS. Disappearance of those bands shows loss of alignment predicted by first-principles theory, further supporting dipole locking at AP. The capability to modulate dipole alignment by varying gas pressure at constant normalized field provides the basis for determining the ion dipole moment and direction within the molecular frame from the pressure of onset and characteristics of spectral drift. This new approach to alter FAIMS separations of proteins could make a powerful tool for structural biology and be useful for proteomics and imaging.