Structure-Guided Design of Conformationally Constrained Cyclohexane Inhibitors of Severe Acute Respiratory Syndrome Coronavirus-2 3CL Protease.

A series of nondeuterated and deuterated dipeptidyl aldehyde and masked aldehyde inhibitors that incorporate in their structure a conformationally constrained cyclohexane moiety was synthesized and found to potently inhibit severe acute respiratory syndrome coronavirus-2 3CL protease in biochemical and cell-based assays. Several of the inhibitors were also found to be nanomolar inhibitors of Middle East respiratory syndrome coronavirus 3CL protease. The corresponding latent aldehyde bisulfite adducts were found to be equipotent to the precursor aldehydes. High-resolution cocrystal structures confirmed the mechanism of action and illuminated the structural determinants involved in binding. The spatial disposition of the compounds disclosed herein provides an effective means of accessing new chemical space and optimizing pharmacological activity. The cellular permeability of the identified inhibitors and lack of cytotoxicity warrant their advancement as potential therapeutics for COVID-19.

Differential Ion Mobility Separations of d/l Peptide Epimers.

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.

Delineation of Isomers by the 13C Shifts in Ion Mobility Spectra.

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.

Structurally Informative Isotopic Shifts in Ion Mobility Spectra for Heavier Species.

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.

High-Resolution Ion Mobility Separations of Isomeric Glycoforms with Variations on the Peptide and Glycan Levels.

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/ß 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.

Middle-Down Proteomic Analyses with Ion Mobility Separations of Endogenous Isomeric Proteoforms.

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.

High-Resolution Differential Ion Mobility Separations/Orbitrap Mass Spectrometry without Buffer Gas Limitations.

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.

Elemental Dependence of Structurally Specific Isotopic Shifts in High-Field Ion Mobility Spectra.

Nearly all molecules incorporate at least one element with stable isotopes, yielding ubiquitous isotopologic envelopes in mass spectra. Those envelopes split in differential or field asymmetric waveform ion mobility (FAIMS) spectra depending on the ion geometry, enabling a new general approach to isomer delineation as we demonstrated for chloroanilines. Here, we report that analogous bromoanilines exhibit qualitatively distinct isotopic shifts under identical conditions, some changing signs depending on the gas. This dramatic elemental specificity conveys the breadth and diversity of structural isotopic effect in FAIMS, suggesting unique information-rich patterns for compounds involving various elements and feasibility of enhancing the structural elucidation by atom substitution. We also introduce the capability to make or ensure structural assignments employing major isomer-specific peak broadening due to unresolved isotopomer mixtures.

Differential Ion Mobility Separations/Mass Spectrometry with High Resolution in Both Dimensions.

Strong orthogonality to mass spectrometry makes differential ion mobility spectrometry (FAIMS) a powerful tool for isomer separations. However, high FAIMS resolution has been achieved overall only with buffers rich in He or H2. That obstructed coupling to Fourier transform mass spectrometers operating under ultrahigh vacuum, but exceptional m/ z resolution and accuracy of FTMS are indispensable for frontline biological and environmental applications. By raising the waveform amplitude to 6 kV, we enabled high FAIMS resolution using solely N2 and thus straightforward integration with any MS platform: here Orbitrap XL with the electron transfer dissociation (ETD) option. The initial evaluation for complete histone tails (50 residues) with diverse post-translational modifications on alternative sites demonstrates a broad capability to separate and confidently identify the PTM localization variants in the middle-down range.

Identification of Isomers by Multidimensional Isotopic Shifts in High-Field Ion Mobility Spectra.

Nearly all molecules incorporate elements with stable isotopes. The resulting isotopologue envelopes in mass spectra tell the exact stoichiometry but nothing about the geometry. Chromatography and electrophoresis at high resolution also can distinguish isotopologues, again without revealing structural information. In high-definition differential ion mobility (FAIMS) spectra, these envelopes universally split in a structure-specific manner, providing a new general approach to isomer delineation. Here, we show that the peak shifts from instances of the same isotope are equal and can be averaged into characteristic elemental shifts, namely 13C and 37Cl for dichloroanilines (DCA). Matrices of these shifts, including the gas composition dimension, are unique to the structure. Hence, all six DCA isomers (with four making two unresolved pairs) are readily delineated in the 13C/37Cl maps with He/CO2 buffer gases. Mixtures of coeluting isomers are also distinguished from pure components.

Linear and Differential Ion Mobility Separations of Middle-Down Proteoforms.

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.

Molecular Structure Characterization by Isotopic Splitting in Nonlinear Ion Mobility Spectra.

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.

Characterization of Complete Histone Tail Proteoforms Using Differential Ion Mobility Spectrometry.

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.

Localization of Post-Translational Modifications in Peptide Mixtures via High-Resolution Differential Ion Mobility Separations Followed by Electron Transfer Dissociation.

Precise localization of post-translational modifications (PTMs) on proteins and peptides is an outstanding challenge in proteomics. While electron transfer dissociation (ETD) has dramatically advanced PTM analyses, mixtures of localization variants that commonly coexist in cells often require prior separation. Although differential or field asymmetric waveform ion mobility spectrometry (FAIMS) achieves broad variant resolution, the need for standards to identify the features has limited the utility of approach. Here we demonstrate full a priori characterization of variant mixtures by high-resolution FAIMS coupled to ETD and the procedures to systematically extract the FAIMS spectra for all variants from such data. Graphical Abstract ᅟ.