Differential Ion Mobility Mass Spectrometry Research Articles

Differential ion mobility mass spectrometry in immunopeptidomics identifies neoantigens carrying colorectal cancer driver mutations

Understanding the properties of human leukocyte antigen (HLA) peptides (immunopeptides) is essential for precision cancer medicine, while the direct identification of immunopeptides from small biopsies of clinical tissues by mass spectrometry (MS) is still confronted with technical challenges. Here, to overcome these hindrances, high-field asymmetric waveform ion mobility spectrometry (FAIMS) is introduced to conduct differential ion mobility (DIM)-MS by seamless gas-phase fractionation optimal for scarce samples. By established DIM-MS for immunopeptidomics analysis, on average, 42.9 mg of normal and tumor colorectal tissues from identical patients (n = 17) were analyzed, and on average 4921 immunopeptides were identified. Among these 44,815 unique immunopeptides, two neoantigens, KRAS-G12V and CPPED1-R228Q, were identified. These neoantigens were confirmed by synthetic peptides through targeted MS in parallel reaction monitoring (PRM) mode. Comparison of the tissue-based personal immunopeptidome revealed tumor-specific processing of immunopeptides. Since the direct identification of neoantigens from tumor tissues suggested that more potential neoantigens have yet to be identified, we screened cell lines with known oncogenic KRAS mutations and identified 2 more neoantigens that carry KRAS-G12V. These results indicated that the established FAIMS-assisted DIM-MS is effective in the identification of immunopeptides and potential recurrent neoantigens directly from scarce samples such as clinical tissues.

Separation of disaccharide epimers, anomers and connectivity isomers by high resolution differential ion mobility mass spectrometry

Glycans are ubiquitous, structurally diverse molecules that have specific and general roles involving metabolism, structure, and cell-to-cell signaling. Functional specificity depends strongly on the complexity of structures that polysaccharides can adopt based on their subunit composition, length, extent of branching, glycosidic bond connectivity and anomeric configuration. However, a rapid and comprehensive characterization of glycan isomers can be challenging owing to limitations associated with their separation. Here, ten composition, anomeric and connectivity disaccharide isomers were separated and detected using high-resolution differential ion mobility-mass spectrometry (DMS-MS, also known as FAIMS). Focus was primarily directed to compositional isomers corresponding to epimers that differ by the axial or equatorial position of a single hydroxyl group. DMS resolving power was enhanced 14-fold primarily by increasing the fraction of helium in the ion carrier gas and lowering the flow rate. At relatively high disaccharide concentrations, DMS-MS of each disaccharide resulted in complex and unique multi-peak spectra with up to ten fully and partially resolved peaks for β-1,4-mannobiose (Man-1,4β-Man), which can be attributed to the DMS separation and subsequent dissociation of ionic non-covalently bound oligomers into monomer ions. Each DMS spectrum has at least one differentiating peak that is not in the other spectra, indicating that DMS can be used to fully or partially resolve composition, configuration and connectivity isomers. At relatively low disaccharide concentrations, mixtures of disaccharide epimers can also be readily separated by DMS. The integration of high-resolution, ambient pressure DMS with complementary reduced-pressure ion mobility and MS-based glycomics and glycoproteomics workflows may be useful for improving the characterization of glycans and glycosylated biomolecules.

An atmospheric pressure ion funnel with a slit entrance for enhancing signal and resolution in high resolution differential ion mobility mass spectrometry.

Differential ion mobility (DMS) is a versatile ion separation method that is often integrated with mass spectrometry (MS). In DMS, extremely high electric fields are used such that ion mobility depends non-linearly on electric field and thus, ion separations can be more orthogonal to MS than lower field ion mobility-based methods. DMS can have sufficiently high resolution to be used for enantiomer analysis of small molecules and to separate protein ions with peak widths comparable to those obtained for peptides. However, the performance of high resolution DMS-MS can be limited owing to the substantial loss of ions (>10-fold) that can occur upon their transfer from atmospheric pressure (where DMS separation typically occurs) to vacuum through a narrow conductance limited inlet (e.g. capillary) to the MS. Here, results from simulated ion trajectory simulations suggest that in high resolution DMS most ions can be lost by 'crashing' onto the narrow capillary inlet after exiting the DMS separation channel. To enhance DMS sensitivity and resolving power, an integrated DMS-MS interface concept is reported that consists of a slit electrode and a 12-electrode atmospheric pressure ion funnel (APIF). By using an APIF with slit entrance, the simulated ion transmission efficiencies increase by up to 257% for singly charged ions ([DMMP + H]+, [tryptophan + H]+, and [(2-dodecanone)2 + H]+) and by 209% for [ubiquitin + 12H]12+, without compromising resolving power. The use of APIF improves the ion focussing from the DMS exit to the MS capillary to improve sensitivity, and the slit ensures that ion dispersion in the analytically relevant direction perpendicular to the DMS electrodes is restricted to enhance resolution. By narrowing the slit of the DMS-Slit-APIF interface, the DMS resolving power can be increased further by at least 20%. Overall, these results indicate that the integrated DMS-Slit-APIF interface is promising for improving the sensitivity and resolution for many different types of DMS-MS experiments.

Alkali Metal Cationization of Tumor-associated Antigen Peptides for Improved Dissociation and Measurement by Differential Ion Mobility-Mass Spectrometry.

Tandem mass spectrometry (MS/MS) is a highly sensitive and selective method for the detection of tumor-associated peptide antigens. These short, nontryptic sequences may lack basic residues, resulting in the formation of predominantly [peptide + H]+ ions in electrospray. These singly charged ions tend to undergo inefficient dissociation, leading to issues in sequence determination. Addition of alkali metal salts to the electrospray solvent can drive the formation of [peptide + H + metal]2+ ions that have enhanced dissociation characteristics relative to [peptide + H]+ ions. Both previously identified tumor-associated antigens and predicted neoantigen sequences were investigated. The previously reported rearrangement mechanism in MS/MS of sodium-cationized peptides is applied here to demonstrate complete C-terminal sequencing of tumor-associated peptide antigens. Differential ion mobility spectrometry (DIMS) is shown to selectively enrich [peptide + H + metal]2+ species by filtering out singly charged interferences at relatively low field strengths, offsetting the decrease in signal intensity associated with the use of alkali metal cations.

Separation of Isobaric Mono- and Dimethylated RGG-Repeat Peptides by Differential Ion Mobility-Mass Spectrometry.

Methylation of arginine residues in proteins, an enzyme-mediated post-translational modification (PTM), is important for mRNA processing and transport and for the regulation of many protein-protein interactions. However, proteolytic peptides resulting from alternative sites of post-translational methylation have identical masses and cannot be readily separated by standard liquid chromatography-mass spectrometry. Unlike acetylation or phosphorylation, methylation of arginine does not strongly affect the charge states of peptide ions, multiple instances of methylation can occur on a single amino acid residue, and the relative mass of the modification is <1% that of the typical proteolytic peptide. High field asymmetric waveform ion mobility spectrometry (FAIMS) is an orthogonal separation method to liquid chromatography that can rapidly separate gaseous ions prior to detection by mass spectrometry. Here, we report that FAIMS can be used to separate arginine-methylated peptides that differ by the position of a single methyl group for both mono- and dimethylated variants. Although the resolution of separation for these arginine-methylated peptides improved with increasing amounts of helium in the FAIMS carrier gas as expected, we found that the site of methylation can strongly affect the dependence of the electric field used for ion transmission on the extent of helium in the carrier gas. Thus, certain isobaric peptides can be cotransmitted at high helium concentrations whereas lower concentrations can be used for successful separations of such peptide mixtures. The capability to rapidly resolve isobaric arginine-methylated peptides should be useful in the future for the detailed analysis of protein arginine methylation in biological samples.

Rapid separation of isomeric perfluoroalkyl substances by high-resolution differential ion mobility mass spectrometry

The analysis of persistent organic pollutants, such as perfluoroalkyl substances (PFAS) including perfluorooctanoic acid (C7F15COOH, PFOA) and perfluorooctane sulfonate (C8F17SO3−, PFOS) by hyphenated chromatography-mass spectrometry methods is crucial in ensuring water quality. One of the challenges in PFOA and PFOS analysis is the separation of linear and branched isomers that have the same mass-to-charge ratio but can have different toxicological properties. Current methods that are used for separating isomeric PFAS require relatively long analysis times (min to h) that can limit throughput. An emerging technique for the direct analysis of isomeric compounds is differential mobility spectrometry (DMS), which can rapidly separate gas phase ions prior to detection by mass spectrometry (MS). However, an ion mobility-based method for the analysis of PFAS has not been reported in the literature. Herein, high-resolution DMS-MS is used to separate and detect isomeric PFAS compounds for the first time in a separation process that occurs in milliseconds. The resolution of isomeric peaks increased by over 200% and 500% for PFOA and PFOS isomers, respectively, using a DMS carrier gas composed of 50:50% He:N2 by volume compared to 100% N2, which was crucial in the separation of PFAS isomers under these conditions. Linear, secondary-branched, and tertiary-branched isomers of PFOA and PFOS including those that differ by the position of a single perfluoromethyl group can be resolved by DMS-MS. The DMS compensation field required to transmit different isomers increases as the extent of branching increases. For isomeric PFASs with a single branching point, the compensation field for optimal transmission also increases as the perfluoromethyl group is positioned closer to the carboxylate and sulfonate groups under these conditions. These results indicate that high-resolution DMS-MS should be a useful approach for the rapid analysis of isomeric PFAS.

Metal-ion free chiral analysis of amino acids as small as proline using high-definition differential ion mobility mass spectrometry

The chiral analysis of enantiomers is important because bioactivity can depend strongly on stereochemistry as ligand-protein binding motifs are typically chiral. Ion mobility mass spectrometry-based methods are emerging for the rapid and sensitive chiral analysis of molecules. However, such methods are typically limited by the use of metal-bound trimers, which can be challenging to form owing to ion suppression and the need for extensive pre-screening experiments to identify suitable metal ions. Moreover, the chiral separation of very small molecules, such as cysteine and proline, using ion mobility has remained challenging. Here, using electrospray ionisation high-resolution differential ion mobility mass spectrometry (ESI-DMS-MS), we demonstrate that the enantiomers of benchmark amino acids as small as proline can be rapidly distinguished without the use of metal ions for the first time. ESI-DMS-MS of proton-bound diastereomeric dimer complexes, containing enantiomers of amino acids and a ‘chiral selector’ (N-tert-butoxycarbonyl-O-benzyl-l-serine; BBS) corresponding to [L/D-X(BBS)+H]+ (X = cysteine and proline) resulted in the separation of L and D-enantiomers. By use of DMS-MS and standard solutions of chiral mixtures, these data indicate that the enantiomeric excess of proline can be accurately quantified by differential ion mobility mass spectrometry. Overall, these results provide further evidence that DMS-MS can be used for the rapid and accurate ‘metal-ion free’ chiral analysis of many other biologically important molecules.

Chiral recognition of amino acid enantiomers using high-definition differential ion mobility mass spectrometry

Enantiomeric analysis of small molecules is important in many research fields, including in drug development. Here, chiral recognition of amino acid enantiomers using differential ion mobility spectrometry (DMS) mass spectrometry (MS) is demonstrated. Diastereomeric proton bound complexes were formed between enantiomers of amino acids (tryptophan and phenylalanine) and N-tert-butoxycarbonyl-O-benzyl-l-serine (BBS) by electrospray ionization for analysis by DMS-MS and collision-induced dissociation (CID). If the DMS resolution is sufficiently high, ionic diastereomers (as opposed to enantiomers) can in principle be separated without the use of a chiral gas. Peaks corresponding to the L- and D-enantiomers for both tryptophan and phenylalanine in the DMS-MS spectra were resolved by increasing the ratio of He in the carrier gas from 100% N2 to 50:50 He:N2%. In contrast, CID spectra of the corresponding diastereomeric dimer complex ions were nearly identical, indicating that chiral recognition by CID was not possible under these conditions. For mixtures of L- and D-tryptophan, a linear calibration curve can be obtained by plotting the enantiomeric excess measured by DMS-MS vs. the known values in solution (slope of 1.000, intercept of −0.010 and R2 of 0.997). That is, enantiopurity can be quantified using a separation process that occurs in milliseconds. Thus, DMS-MS analysis of proton bound diastereomeric dimers is a powerful approach for the rapid enantiomeric analysis of relatively small molecules.

Comprehensive mapping of O-glycosylation in flagellin from Campylobacter jejuni 11168: A multienzyme differential ion mobility mass spectrometry approach.

Glycosylation of flagellin is essential for the virulence of Campylobacter jejuni, a leading cause of bacterial gastroenteritis. Here, we demonstrate comprehensive mapping of the O‐glycosylation of flagellin from Campylobacter jejuni 11168 by use of a bottom‐up proteomics approach that incorporates differential ion mobility spectrometry (also known as high field asymmetric waveform ion mobility spectrometry or FAIMS) together with proteolysis with proteinase K. Proteinase K provides complementary sequence coverage to that achieved following trypsin proteolysis. The use of FAIMS increased the number of glycopeptides identified. Novel glycans for this strain were identified (pseudaminic acid and either acetamidino pseudaminic acid or legionaminic acid), as were novel glycosylation sites: Thr208, Ser343, Ser348, Ser349, Ser395, Ser398, Ser423, Ser433, Ser436, Ser445, Ser448, Ser451, Ser452, Ser454, Ser457 and Thr465. Multiply glycosylated peptides were observed, as well as variation at individual residues in the nature of the glycan and its presence or absence. Such extreme heterogeneity in the pattern of glycosylation has not been reported previously, and suggests a novel dimension in molecular variation within a bacterial population that may be significant in persistence of the organism in its natural environment. These results demonstrate the usefulness of differential ion mobility in proteomics investigations of PTMs.

Confirmation of diosmetin 3-O-glucuronide as major metabolite of diosmin in humans, using micro-liquid-chromatography-mass spectrometry and ion mobility mass spectrometry.

Diosmin is a flavonoid often administered in the treatment of chronic venous insufficiency, hemorrhoids, and related affections. Diosmin is rapidly hydrolized in the intestine to its aglicone, diosmetin, which is further metabolized to conjugates. In this study, the development and validations of three new methods for the determination of diosmetin, free and after enzymatic deconjugation, and of its potential glucuronide metabolites, diosmetin-3-O-glucuronide, diosmetin-7-O-glucuronide, and diosmetin-3,7-O-glucuronide from human plasma and urine are presented. First, the quantification of diosmetin, free and after deconjugation, was carried out by high-performance liquid chromatography coupled with tandem mass spectrometry, on an Ascentis RP-Amide column (150 × 2.1 mm, 5 μm), in reversed-phase conditions, after enzymatic digestion. Then glucuronide metabolites from plasma were separated by micro-liquid chromatography coupled with tandem mass spectrometry on a HALO C18 (50 × 0.3 mm, 2.7 μm, 90 Å) column, after solid-phase extraction. Finally, glucuronides from urine were measured using a Discovery HSF5 (100 × 2.1 mm, 5 μm) column, after simple dilution with mobile phase. The methods were validated by assessing linearity, accuracy, precision, low limit of quantification, selectivity, extraction recovery, stability, and matrix effects; results in agreement with regulatory (Food and Drug Administration and European Medicines Agency) guidelines acceptance criteria were obtained in all cases. The methods were applied to a pharmacokinetic study with diosmin (450 mg orally administered tablets). The mean C max of diosmetin in plasma was 6,049.3 ± 5,548.6 pg/mL. A very good correlation between measured diosmetin and glucuronide metabolites concentrations was obtained. Diosmetin-3-O-glucuronide was identified as a major circulating metabolite of diosmetin in plasma and in urine, and this finding was confirmed by supplementary experiments with differential ion-mobility mass spectrometry.

Differential Mobility Spectrometry of Isomeric Protonated Dipeptides: Modifier and Field Effects on Ion Mobility and Stability

The ability to resolve isomeric protonated dipeptides was investigated with the new technique of differential ion mobility mass spectrometry that uses "modifier" molecules to enhance differential mobility. Two pairs of protonated peptides [glycine-alanine (GlyAla) and alanine-glycine (AlaGly), glycine-serine (GlySer) and serine-glycine (SerGly)] and eight different modifiers (water, 2-propanol, 1,5-hexadiene, 2-chloropropane, chlorobenzene, dichloromethane, acetonitrile, and cyclohexane) were used in the initial study. Separation of the protonated peptides was found to be dependent on the mass and proton affinity of the modifier and combinations of functionalities present in the modifier and the analyte ion. Six of the eight modifiers (water, 2-propanol, chlorobenzene, cyclohexane, dichloromethane, and acetonitrile) were able to separate the protonated isomeric peptide pairs, and generally, modifiers with electron-rich groups performed the best. In the presence of some modifiers, a reduction of ion current was observed under the highest field conditions (>115 Td). Dopant-catalyzed isomerization, likely by proton-transport catalysis, and field-induced fragmentation may have contributed to these losses. Two high vapor pressure modifiers, 1,5-hexadiene and 2-chloropropane, significantly influenced ion formation leading to the formation of stable cluster populations that could be observed in the mass spectrometer. Although not a major concern, both fragmentation and influence of modifier evaporation warrant further studies in order to fully understand and possibly eliminate them.