Trapped Ion Mobility Spectrometry-mass Spectrometry Research Articles

Investigating direct current potentials that affect native protein conformation during trapped ion mobility spectrometry-mass spectrometry.

Trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOFMS) has emerged as a tool to study protein conformational states. In TIMS, gas-phase ions are guided across the IM stages by applying direct current (DC) potentials (D1-6), which, however, might induce changes in protein structures through collisional activation. To define conditions for native protein analysis, we evaluated the influence of these DC potentials using the metalloenzyme bovine carbonic anhydrase (BCA) as primary test compound. The variation of DC potentials did not change BCA-ion charge and heme content but affected (relative) charge-state intensities and adduct retention. Constructed extracted-ion mobilograms and corresponding collisional cross-section (CCS) profiles gave useful insights in (alterations of) protein conformational state. For BCA, the D3 and D6 potential (which are applied between the deflection transfer and funnel 1 [F1] and the accumulation exit and the start of the ramp, respectively) had most profound effects, showing multimodal CCS distributions at higher potentials indicating gradual unfolding. The other DC potentials only marginally altered the CCS profiles of BCA. To allow for more general conclusions, five additional proteins of diverse molecular weight and conformational stability were analyzed, and for the main protein charge states, CCS profiles were constructed. Principal component analysis (PCA) of the obtained data showed that D1 and D3 exhibit the highest degree of correlation with the ratio of folded and unfolded protein (F/U) as extracted from the mobilograms obtained per set D potential. The correlation of D6 with F/U and protein charge were similar, and D2, D4, and D5 showed an inverse correlation with F/U but were correlated with protein charge. Although DC boundary values for induced conformational changes appeared protein dependent, a set of DC values could be determined, which assured native analysis of most proteins.

Impact of Source Conditions on Collision Cross Section Determination by Trapped Ion Mobility Spectrometry.

Collision cross section (CCS) values determined in ion mobility-mass spectrometry (IM-MS) are increasingly employed as additional descriptors in metabolomics studies. CCS values must therefore be reproducible and the causes of deviations must be carefully known and controlled. Here, we analyzed lipid standards by trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) to evaluate the effects of solvent and flow rate in flow injection analysis (FIA), as well as electrospray source parameters including nebulizer gas pressure, drying gas flow rate, and temperature, on the ion mobility and CCS values. The stability of ion mobility experiments was studied over 10 h, which established the need for a delay-time of 20 min to stabilize source parameters (mostly pressure and temperature). Modifications of electrospray source parameters induced shifts of ion mobility peaks and even the occurrence of an additional peak in the ion mobility spectra. This behavior could be essentially explained by ion-solvent cluster formation. Changes in source parameters were also found to impact CCS value measurements, resulting in deviations up to 0.8%. However, internal calibration with the Tune Mix calibrant reduced the CCS deviations to 0.1%. Thus, optimization of source parameters is essential to achieve a good desolvation of lipid ions and avoid misinterpretation of peaks in ion mobility spectra due to solvent effects. This work highlights the importance of internal calibration to ensure interoperable CCS values, usable in metabolomics annotation.

Isomer-specific quantification of human milk oligosaccharides based on Girard reagent P-d0/d5 derivatization and trapped ion mobility spectrometry-mass spectrometry

Human milk oligosaccharides (HMOs), the third most abundant components in breastmilk, show high structural diversity with many isomers. Here, we proposed a trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) strategy for the differentiating and isomer-specific quantification of HMOs combined with Girard reagent P-d0/d5 (GP-d0/d5) derivatization. Three functional HMO isomer pairs including LNT/LNnT, 3'-SL/6'-SL, and 2'-FL/3'-FL were investigated by the proposed method. The GP-derivatized HMO isomers showed enhanced ionization efficiency (8-fold) and achieved mobility separation with ΔCCS % ≥ 3.7% in TIMS-MS. With the GP-d5 derivatized HMOs as an internal standard, HMOs in real samples could be quantified by derivatization with GP-d0, and each isomeric species could be further determined based on their corresponding mobility peaks. The recoveries of spiked HMOs are obtained at 72.4 − 109%. Thus, an isomer-specific quantification strategy for HMO isomers was developed based on the GP-d0/d5 derivatization and TIMS-MS.

Differentiation of steroid isomers by steroid analogues adducted trapped ion mobility spectrometry-mass spectrometry.

Steroids are one of the important indicators of health and disease. However, due to the high similarity of steroid structures, there are several potential obstacles in the differentiation of steroids, especially for their isomers. Herein, we described a trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) approach based on the steroid analogue adduction for isomer-specific identification of steroids. The application of dexamethasone (DEX) to form heterodimers with steroids enhanced the separation of their isomers in TIMS. Two isomer pairs including 17-hydroxyprogesterone/11-deoxycorticosterone and androsterone/epiandrosterone were successfully separated as the heterodimers with DEX by TIMS. The stability of DEX-adducted heterodimers is comparable with steroid dimers. Owing to the high separation efficiency and stability, the relative quantification of steroid isomers was demonstrated with the proposed method.

Exploiting ion-mobility mass spectrometry for unraveling proteome complexity.

Ion mobility spectrometry-mass spectrometry (IMS-MS) is experiencing rapid growth in proteomic studies, driven by its enhancements in dynamic range and throughput, increasing the quantitation precision, and the depth of proteome coverage. The core principle of ion mobility spectrometry is to separate ions in an inert gas under the influence of an electric field based on differences in drift time. This minireview provides an introduction to IMS operation modes and a description of advantages and limitations is presented. Moreover, the principles of trapped IMS-MS (TIMS-MS), including parallel accumulation-serial fragmentation are discussed. Finally, emerging applications linked to TIMS focusing on sample throughput (in clinical proteomics) and sensitivity (single-cell proteomics) are reviewed, and the possibilities of intact protein analysis are discussed.

Drug adulteration analysis based on complexation with cyclodextrin and metal ions using ion mobility spectrometry

Drug adulteration and contamination are serious threats to human health therefore, their accurate monitoring is very important. Allopurinol (Alp) and theophylline (Thp) are commonly used drugs for the treatment of gout and bronchitis, while their isomers hypoxanthine (Hyt) and theobromine (Thm) have no effect and affect the efficacy of the drug. In this work, the drug isomers of Alp/Hyt and Thp/Thm are simply mixed with α-, β-, γ-cyclodextrin (CD) and metal ions and separated using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). TIMS-MS results showed that Alp/Hyt and Thp/Thm isomers could interact with CD and metal ions and form corresponding binary or ternary complexes to achieve their TIMS separation. Different metal ions and CDs showed different separation effect for the isomers, among which Alp and Hyt could be successfully distinguished from the complexes of [Alp/Hyt+γ-CD + Cu–H]+ with separation resolution (RP–P) of 1.51; whereas Thp and Thm could be baseline separated by [Thp/Thm+γ-CD + Ca–H]+ with RP–P of 1.96. Besides, chemical calculations revealed that the complexes were in the inclusion forms, and microscopic interactions were somewhat different, making their mobility separation. Moreover, relative and absolute quantification was investigated with an internal standard to determine the precise isomers content, and good linearity (R2 > 0.99) was obtained. Finally, the method was applied for the adulteration detection where different drugs and urine were analyzed. In addition, due to the advantages of fast speed, simple operation, high sensitivity, and no chromatographic separation required, the proposed method provides an effective strategy for the drug adulteration detection of isomers.

Simultaneous chirality separation of amino acids and their derivative by natamycin based on mobility measurements

The separation of chiral amino acids (AAs) and their derivatives has always been a research difficulty in the field of biochemistry due to the high similarity of enantiomeric structures. In this work, a simple and quick method using natamycin (Nat) as chiral selector has been developed to simultaneously separate chiral AAs and their derivatives of carbobenzoxy/benzyl-AAs (Cbz/Bzl-AAs) by trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). Specifically, 12 groups of the Cbz-AAs and Bzl-AAs can get baseline mobility separation by simple mixing with Nat to form binary diastereomeric complex ions [Nat+(Cbz-D/L-AA)+H]+ and [Nat+(Bzl-D/L-AA)+H]+. While for the remained 5 groups of Bzl-D/L-AAs and 16 groups of D/L-AAs with unsatisfying separation, by further adding P-toluenesulfonic acid (PTS), the formed ternary complexes can allow their baseline chiral separation. Specifically, Bzl-D-AAs and Bzl-L-AAs get much improved separation effect by the formed diastereomeric complexes of [Nat+(Bzl-AA)2+PTS2+H]+, which the Rp-p was improved from 0 to 2.40; while the D/L-AAs can get baseline separation by the formed diastereomeric complexes of [Nat + AA + PTS + H]+, [Nat + AA+(PTS)2+H]+, and [Nat+(AA)2+(PTS)2+H]+, with the Rp-p ranged from 0.44 to 3.53. Definitely, PTS is the first time reported as the ligand to improve the separation effect for the enantiomers, and with the higher assembly of chiral analyte, Nat, and PTS might enable better chiral separation for the chiral amino acid and their derivatives. Moreover, method validation of relative quantification and accuracy for the D/L-AA and their derivatives were measured in a mixture, yielding R2 greater than 0.99 and RSD% ≤ 2.68%. Overall, Nat and PTS as chiral selector and ligand can be widely used for chiral AAs and their derivatives mobility separation, and potentially for the separation of other AA-related chiral molecules.

Exploring the Conformational and Binding Dynamics of HMGA2·DNA Complexes Using Trapped Ion Mobility Spectrometry-Mass Spectrometry.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is an intrinsically disordered DNA-binding protein expressed during embryogenesis. In the present work, the conformational and binding dynamics of HMGA2 and HMGA2 in complex with a 22-nt (DNA22) and a 50-nt (DNA50) AT-rich DNA hairpin were investigated using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) under native starting solvent conditions (e.g., 100 mM aqueous NH4Ac) and collision-induced unfolding/dissociation (CIU/CID) as well as solution fluorescence anisotropy to assess the role of the DNA ligand when binding to the HMGA2 protein. CIU-TIMS-CID-MS/MS experiments showed a significant reduction of the conformational space and charge-state distribution accompanied by an energy stability increase of the native HMGA2 upon DNA binding. Fluorescence anisotropy experiments and CIU-TIMS-CID-MS/MS demonstrated for the first time that HMGA2 binds with high affinity to the minor groove of AT-rich DNA oligomers and with lower affinity to the major groove of AT-rich DNA oligomers (minor groove occupied by a minor groove binder Hoechst 33258). The HMGA2·DNA22 complex (18.2 kDa) 1:1 and 1:2 stoichiometry suggests that two of the AT-hook sites are accessible for DNA binding, while the other AT-hook site is probably coordinated by the C-terminal motif peptide (CTMP). The HMGA2 transition from disordered to ordered upon DNA binding is driven by the interaction of the three basic AT-hook residues with the minor and/or major grooves of AT-rich DNA oligomers.

Trapped ion mobility spectrometry-mass spectrometry improves the coverage and accuracy of four-dimensional untargeted lipidomics

Lipids play vital roles in many physiological and pathological processes in living organisms. Due to the high structural diversity and the numerous isomers and isobars of lipids, high-coverage and high-accuracy lipidomic analysis of complex biological samples remain the bottleneck to investigate lipid metabolism. Here, we developed the trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) based four-dimensional untargeted lipidomics to support accurate lipid identification and quantification in biological samples. We first demonstrated that the TIMS based multi-dimensional separation improved the differentiations of isomeric and isobaric lipids, and increased the purity of precursor ion isolation and the quality of MS/MS spectra. Hyphenation of TIMS and PASEF technologies significantly improved the coverages of MS/MS spectra. These technological advantages jointly improved the coverage and accuracy of lipid identification in untargeted lipidomics. We further demonstrated that the CCS values of lipids acquired using TIMS were highly consistent with those from drift tube ion mobility spectrometry (DTIMS). Lipid identification and quantification results of NIST human plasma samples were also verified with inter-laboratory reports. Finally, we applied the TIMS-MS based untargeted lipidomics to characterize the spatial distributions of 1393 distinctive lipids in the mouse brain, and demonstrated that diverse lipid distributions and compositions among brain regions contributed to different functions of brain regions. Altogether, TIMS-MS based four-dimensional untargeted lipidomics significantly improved the coverage and accuracy of untargeted metabolomics, thereby facilitating a system-level understanding of lipid metabolism in biological organisms.

Chiral distinction of phenyl-substituted ethanediol enantiomers by measuring the ion mobility of their ternary complexes

The distinction of enantiomers is increasingly critical in scientific research and chemical industries, because of their differences in the molecular structure and properties. R/S-Phenyl-1,2-ethanediol (PED), R,R/S,S-Hydrobenzoin (HB), and R/S-1,1,2-Triphenyl-1,2-ethanediol (TPED) are representative aromatic alcohol catalyst-class enantiomers, which directly affect the type and yield of reactants, that their chiral distinction and separation are important. In this work, the three groups of the enantiomers are recognized by measuring the ion mobility spectrometry (IMS) difference of their ternary complexes, which formed by simple non-covalent interaction with chiral selectors and metal ions. Their ion mobility was detected using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). Different chiral selectors include α-, β-, γ-Cyclodextrins (CDs), rifamycin (Rif), and natamycin (Nat) etc were investigated, and different metal ions were studied as ligands. Interestingly, the target enantiomers could not interact with the chiral selectors directly, but with the addition of metal ions, the ternary complexes of enantiomers-chiral selectors-metal ions can be formed. The mobility results reveal that the peak to peak resolution (Rp-p) was increased with the number of their benzene rings goes, which can up to 2.08. Simply, R/S-PED enantiomers can be successfully distinguished by measuring the IMS of the formed ternary complexes of [β-CD + PED + Cr-2H]+; R,R/S,S-HB enantiomers can be distinguished by measuring the IMS of [Rif + HB + Ba-H]+ and [Rif + HB + Ca-H]+; And R/S-TPED enantiomers can be distinguished by measuring the IMS of [Nat + TPED + Mn-H]+, [Nat + TPED + Co-H]+, [Nat + TPED + Mg-H]+, [Nat + TPED + Ca-H]+, [Nat + TPED + Ba-H]+ and [α-CD + TPED + Al-2H]+, respectively. Moreover, the tandem mass spectrometry experimental results showed that the binding stability of the R- and S-enantiomer ternary complexes has obvious difference and is related to different metal ion, indicating the ternary enantiomer complexes have different rigid structures. In addition, the relative quantification of R/S-enantiomer complexes were performed, good linear coefficient (R2) greater than 0.99 was obtained. The method based on IMS analysis can represent an effective approach for the distinction of phenyl-Substituted ethanediol enantiomers, and the CDs, Rif, Nat can be promising chiral selectors for enantiomers distinction.

Recognition of Cis-Trans and Chiral Proline and Its Derivatives by Ion Mobility Measurement of Their Complexes with Natamycin and Metal Ion.

Discrimination of isomers is an important and valuable feature in many analytical applications, and the identification of chiral isomers and cis-trans isomers is the current research focus. In this work, a simple method for direct, simultaneous recognition of d-/l-proline (P), d-/l-/cis-/trans-4-hydroxyproline (4-HP), and d-/l-/cis-/trans-N-tert-butoxycarbony (N-Boc-4-HP) was investigated by means of trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The isomers with cis-/trans-/d-/l-configuration can be directly recognized based on their mobility upon reaction with natamycin (Nat) and metal ions through noncovalent interactions. The results indicate that the recognition of the enantiomers has certain specificity, and the structural difference of the enantiomers was increased in a complex with Nat and metal ions. Herein, d-/l-P can be recognized through the ternary complexes [P + Nat + Mg - H]+, [P + 2Nat + Ca - H]+, [P + 2Nat + Mn - H]+, and [P + Nat + Cu - H]+. Similarly, c-4-HPL, c-4-HPD, t-4-HPL, and t-4-HPD can be recognized by [4-HP + Nat + Ca - H]+, [4-HP + 2Nat + Ca - H]+, and [4-HP + Nat + Cu - H]+, while N-Boc-c-4-HPL, N-Boc-c-4-HPD, N-Boc-t-4-HPL, and N-Boc-t-4-HPD were recognized through the enantiomer complexes [N-Boc-4-HP + Nat + Li]+, [N-Boc-4-HP + Nat + 2Na - H]+, [N-Boc-4-HP + Nat + K]+, [N-Boc-4-HP + Nat + Mn - H]+, and [N-Boc-4-HP + Nat + Ba - H]+. Moreover, tandem mass spectrometry (MS/MS) results indicated that different collision energies were obtained for the same fragment ions, which implied that the enantiomer complexes that contributed to their mobility separation shared identical interaction mode but had different gas-phase rigid geometries. Furthermore, the relative quantification for the enantiomers was performed, and the results were supported by a satisfactory coefficient (R2 > 0.99). The developed method can provide a promising and powerful strategy for the separation of chiral proline and its d-/l-/cis-/trans derivatives, bearing the advantages of higher speed, better accuracy, high selectivity, and no need for chemical derivatization and chromatographic separation.

Structure-elucidation of human CCL5 by integrating trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) with Structure Relaxation Approximation (SRA) analysis

Ion mobility spectrometry-mass spectrometry offers the potential to characterize structures of transient protein assemblies and protein isoforms by means of their orientationally-averaged momentum transfer cross-sections. A commonly observed phenomenon is the compaction of a protein in the ion mobility measurement, that is, the cross section measured for the protein by ion mobility spectrometry is smaller than the cross section expected for its native structure. Consequently, this compaction means that at least some structural changes of the protein must have occurred during the ion mobility measurement. A major challenge is then to identify which aspects of the solution structure are retained in the ion mobility measurement and which ones are not. Here, we apply our recently developed Structure Relaxation Approximation (SRA) method in conjunction with trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) to probe compaction of the human protein chemokine (C-C motif) ligand 5 (also CCL5). Ion mobility spectra are recorded for various charge states and solution conditions of CCL5 under both “soft” and collisionally-activated conditions. Our data show that the SRA reproduces the overall trends in the experimental spectra: (1) the compaction of the CCL5 structure as seen in the experiments; (2) the general increase in the cross section for the various charge states; and (3) the increase in cross section after collisional-activation. The SRA attributes the compaction of the CCL5 structure mainly to the folding of the unstructured N-terminus onto the central Greek key motif of CCL5. By contrast, the SRA indicates that native residue-residue contacts present in the NMR structure are largely retained. Additionally, our analysis indicates that accurate treatment of proton transfer processes during the structural relaxation process would significantly improve the structural interpretation of ion mobility data by the SRA.

Exploring structural signatures of the lanthipeptide prochlorosin 2.8 using tandem mass spectrometry and trapped ion mobility-mass spectrometry.

Lanthipeptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by intramolecular thioether cross-links formed between a dehydrated serine/threonine (dSer/dThr) and a cysteine residue. Prochlorosin 2.8 (Pcn2.8) is a class II lanthipeptide that exhibits a non-overlapping thioether ring pattern, for which no biological activity has been reported yet. The variant Pcn2.8[16RGD] has been shown to bind tightly to the αvβ3 integrin receptor. In the present work, tandem mass spectrometry, using collision-induced dissociation (CID) and electron capture dissociation (ECD), and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) were used to investigate structural signatures for the non-overlapping thioether ring pattern of Pcn2.8. CID experiments on Pcn2.8 yielded bi and yj fragments between the thioether cross-links, evidencing the presence of a non-overlapping thioether ring pattern. ECD experiments of Pcn2.8 showed a significant increase of hydrogen migration events near the residues involved in the thioether rings with a more pronounced effect at the dehydrated residues as compared to the cysteine residues. The high-resolution mobility analysis, aided by site-directed mutagenesis ([P8A], [P11A], [P12A], [P8A/P11A], [P8A/P12A], [P11A/P12A], and [P8A/P11A/P12A] variants), demonstrated that Pcn2.8 adopts cis/trans-conformations at Pro8, Pro11, and Pro12 residues. These observations were complementary to recent NMR findings, for which only the Pro8 residue was evidenced to adopt cis/trans-orientations. This study highlights the analytical power of the TIMS-MS/MS workflow for the structural characterization of lanthipeptides and could be a useful tool in our understanding of the biologically important structural elements that drive the thioether cyclization process.

Comprehensive Screening of Polycyclic Aromatic Hydrocarbons and Similar Compounds Using GC-APLI-TIMS-TOFMS/GC-EI-MS.

In the present work, a novel workflow based on complementary gas-phase separations for the identification of isomeric PAHs from complex mixtures is described. This is the first report on the coupling of gas chromatography (GC), atmospheric pressure laser ionization (APLI), and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) for the characterization of polycyclic aromatic hydrocarbons. Over a hundred known unknowns are uniquely identified based on the molecular ion retention indices I (5%), mobility (RSD < 0.6% and R = 50-90 with Sr = 0.18 V/ms), mobility-based theoretical candidate assignment (<3%), accurate mass chemical formula assignment (<2 ppm), and electron impact fragmentation pattern and database search. The advantages of theoretical modeling of PAHs and similar compounds were evaluated using candidate structures ranked by retention indices and fragmentation pattern from GC-EI-MS data sets. Over 20 PAH isomeric and deuterated standards were utilized for the GC-APLI-TIMS-TOF MS workflow validation. Noteworthy is the analytical capability for untargeted screening of isomeric and isobaric compounds with additional characterization metrics not available in traditional GC-EI-MSn workflows.

Structural resolution of disaccharides through halogen anion complexation using negative trapped ion mobility spectrometry

Carbohydrates are an indispensable part of early life evolution. The determination of their structures is a key step to analyze their critical roles in biological systems. A variation of composition, glycosidic linkage, and (or) configuration between carbohydrate isomers induces structure diversity and brings challenges for their structural determination. Ion mobility spectrometry (IMS), an emerging gas-phase ion separation technology, has been considered as a promising tool for performing carbohydrate structure elucidation. In this work, eight disaccharides were analyzed by trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) in the negative ion mode as the complexed form of [M + X]-, where M = disaccharide, and X = Cl, Br, and I. As compared to the positive ion analysis of the selected disaccharide in a sodiated form, a reversal charge state provided the ability to eliminate or even reverse the collision cross section (CCS) difference between disaccharide isomers. By the combination of TIMS analysis and the calculation of density functional theory, the only observed two conformers of ions [lactulose + I]- may result from different adduction sites for an iodide anion. Based on the comparison of different halogen adducts, the [M + I]- ion form exhibited more powerful ability for isomeric disaccharide differentiation with an average resolution (RP-P) of 1.17, which results in a 34.5% improvement as compared to the corresponding chloride adducts. This result indicates that the use of negative charge states, especially the complexation of an iodide anion, could be a supplemental strategy to commonly used positive ion analysis for carbohydrate separation.

Effective separation of carbohydrate isomers using metal cation and halogen anion complexes in trapped ion mobility spectrometry

Comprehensive analysis of carbohydrates, one of the critical steps towards the fundamental understanding of biological systems, has been hindered by the identifications of their complex isomeric structures differing in monosaccharide constituents, linkage positions, and configurations. While ion mobility spectrometry-mass spectrometry (IMS-MS) based methods have shown their utilities in performing glycomics measurements, there is still a lack of analytical methods allowing effective isomeric carbohydrate separation. Here, we systematically investigated the effect of alkaline earth cations (Ca2+ and Ba2+) and halogen anions (Cl−, Br− and I−) addition on the separation of 21 pairs of isomeric oligosaccharides using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). [M + Ca + I]+ complex (M is an oligosaccharide) has shown experimentally to be the optimum complex for the separation of all 21 pairs of isomeric oligosaccharides used in this study with a calculated average resolution (r) ~ 1.82 and resolving power (R) ~ 100–150. The separation capability of this complex was further demonstrated by using mixtures of 15 oligosaccharides. The optimum [M + Ca + I]+ complex can be potentially used as an effective complex for enhancing the IMS separation resolution of isomeric carbohydrates.

Effective Chiral Discrimination of Amino Acids through Oligosaccharide Incorporation by Trapped Ion Mobility Spectrometry.

Chiral analysis is critical to many research fields due to different biological functions of enantiomers in living systems. Although the use of ion mobility spectrometry (IMS) has become an alternative technology in the area of chiral measurements, there is still a lack of a general chiral selector for IMS-based chiral recognition, especially for small chiral molecules. Here, a new method using oligosaccharides as the chiral selector has been developed to discriminate chiral amino acids (AAs) by trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). We analyzed 21 chiral amino acids, including small molecules (e.g., alanine and cysteine). Our data showed that the use of nonreducing tetrasaccharides was effective for the separation of chiral AAs, which differentiated 21 chiral AAs without using metal ions. By further incorporating a copper ion, the separation resolution could be improved to 1.64 on average, which accounts for an additional 52% improvement on top of the already achieved separation in metal-free analysis. These results indicate that the use of tetrasaccharides is an effective strategy for the separation of AA enantiomers by TIMS. The method developed in this study may open up a new strategy for effective IMS-based chiral analysis.

Following Structural Changes by Thermal Denaturation Using Trapped Ion Mobility Spectrometry-Mass Spectrometry.

The behavior of biomolecules as a function of the solution temperature is often crucial to assessing their biological activity and function. While heat-induced changes of biomolecules are traditionally monitored using optical spectroscopy methods, their conformational changes and unfolding transitions remain challenging to interpret. In the present work, the structural transitions of bovine serum albumin (BSA) in native conditions (100 mM aqueous ammonium acetate) were investigated as a function of the starting solution temperature (T ∼ 23-70 °C) using a temperature-controlled nanoelectrospray ionization source (nESI) coupled to a trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) instrument. The charge state distribution of the monomeric BSA changed from a native-like, narrow charge state ([M + 12H]12+ to [M + 16H]16+ at ∼23 °C) and narrow mobility distribution toward an unfolded-like, broad charge state (up to [M + 46H]46+ at ∼70 °C) and broad mobility distribution. Inspection of the average charge state and collision cross section (CCS) distribution suggested a two-state unfolding transition with a melting temperature Tm ∼ 56 ± 1 °C; however, the inspection of the CCS profiles at the charge state level as a function of the solution temperature showcases at least six structural transitions (T1-T7). If the starting solution concentration is slightly increased (from 2 to 25 μM), this method can detect nonspecific BSA dimers and trimers which dissociate early (Td ∼ 34 ± 1 °C) and may disturb the melting curve of the BSA monomer. In a single experiment, this technology provides a detailed view of the solution, protein structural landscape (mobility vs solution temperature vs relative intensity for each charge state).

Dynamics of the E. coli β-Clamp Dimer Interface and Its Influence on DNA Loading

The ring-shaped sliding clamp proteins have crucial roles in the regulation of DNA replication, recombination, and repair in all organisms. We previously showed that the Escherichia coli β-clamp is dynamic in solution, transiently visiting conformational states in which Domain 1 at the dimer interface is more flexible and prone to unfolding. This work aims to understand how the stability of the dimer interface influences clamp-opening dynamics and clamp loading by designing and characterizing stabilizing and destabilizing mutations in the clamp. The variants with stabilizing mutations conferred similar or increased thermostability and had similar quaternary structure as compared to the wild type. These variants stimulated the ATPase function of the clamp loader, complemented cell growth of a temperature-sensitive strain, and were successfully loaded onto a DNA substrate. The L82D and L82E I272A variants with purported destabilizing mutations had decreased thermostability, did not complement the growth of a temperature-sensitive strain, and had weakened dimerization as determined by native trapped ion mobility spectrometry-mass spectrometry. The β L82E variant had a reduced melting temperature but dimerized and complemented growth of a temperature-sensitive strain. All three clamps with destabilizing mutations had perturbed loading on DNA. Molecular dynamics simulations indicate altered hydrogen-bonding patterns at the dimer interface, and cross-correlation analysis showed the largest perturbations in the destabilized variants, consistent with the observed change in the conformations and functions of these clamps.

Fractionation and molecular characterization of natural organic matter (NOM) by solid-phase extraction followed by FT-ICR MS and ion mobility MS

Electrospray ionization (ESI) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has been widely used for the characterization of dissolved organic matter (DOM) extracted by solid-phase extraction (SPE) from various environmental waters. It is known that common SPE generally has a relatively low recovery and the non-polar and weakly polar components are less likely to be ionized due to ionization discrimination. However, the molecular selectivity during SPE is not clear so far. In this study, the Suwannee River natural organic matter (SRNOM) was fractionated by multi-step SPE with different solvents and the fractions were characterized by negative ESI FT-ICR MS and trapped ion mobility spectrometry-mass spectrometry (IMS-MS). The sequential solvent elution increased the extraction recovery of DOM in water by SPE and enabled the characterization of a weakly polar component, which cannot be detected in common SPE separation. The weakly polar component accounts for 5.7% in TOC for the SRNOM, which has complex but different molecular composition with methanol- and/or water-eluted fractions. Lipid-like compounds were enriched in this fraction. Compared with the polar molecules directly eluted by one-step SPE from the SRNOM, the weakly polar fractions exhibit larger molecular size. The results are helpful for understanding of the molecular composition of SRNOM, as well as other environmental waters. In addition, the study demonstrates that the combination of FT-ICR MS and IMS-MS has potential to characterize the chemical composition of a complex mixture, like DOM, to a new depth. Graphical abstract.