Ion Mobility Mass Spectrometry Research Articles

Sn-Position-Resolved Quantification of Aminophospholipids by Isotopic N,N-Dimethyl Leucine Labeling and High-Resolution Ion Mobility Mass Spectrometry.

Aminophospholipids (APLs), composed of phosphatidylethanolamines (PEs) and phosphatidylserines (PSs), are vital components of mammalian cell membranes and lipoproteins, participating in both homeostasis and cellular signaling. Their structural changes, including the permutation of fatty acid connectivity (sn-positions), due to dysfunctional metabolic processes have been linked to many diseases. However, the accurate quantification of APLs with unambiguous fatty acyl assignment through routine label-free LC-MS/MS lipidomic analysis remains a major challenge. In this study, we explore the functionalization of the free primary amine groups of APLs using amine-reactive isotopic N,N-dimethyl leucine (iDiLeu) and employ high-resolution ion mobility MS (IM-MS) to develop a novel method for sensitive discernment and accurate quantification of APL sn-isomers. With high-resolution demultiplexing (HRdm) providing IM resolving power >200, labeled sn-isomeric pairs of APLs (ΔCCS ≈ 1%) demonstrate excellent, near baseline separation. In addition to greatly enhanced sensitivity, 5-plex iDiLeu labeling enables the construction of an internal 4-point calibration curve and therefore absolute quantification of APL sn-isomers in a single run. This strategy enabled precise annotation and quantification of 239 APLs including 60 pairs of sn-isomers in the mouse cortex. Additionally, we were able to find ratio changes in multiple APL sn-isomer pairs between wild type and APP/PS1 Alzheimer's disease (AD) model mice at different ages, indicating their strong correlation to AD progression. This strategy could provide universal utility in unraveling the alteration of APL sn-isomers, which have long been considered as the "dark matter" of traditional lipidomic analyses, leading to more precise elucidation of molecular mechanisms of various diseases.

Probing the Intramolecular Folding of Nucleic Acids with Native Ion Mobility Mass Spectrometry: Strategies and Caveats.

The goal of native mass spectrometry is to obtain information on noncovalent interactions in solution through mass spectrometry measurements in the gas phase. Characterizing intramolecular folding requires using structural probing techniques such as ion mobility spectrometry. However, inferring solution structures of nucleic acids is difficult because the low-charge state ions produced from aqueous solutions at physiological ionic strength get compacted during electrospray. Here we explored whether native supercharging could produce higher charge states that would better reflect solution folding, and whether the voltage required for collision-induced unfolding (CIU) could reflect preserved intramolecular hydrogen bonds. We studied pH-responsive i-motif structures with different loops, and unstructured controls. We also implemented a multivariate curve resolution procedure to extract physically meaningful pure components from the CIU data and reconstruct unfolding curves. We found that the relative unfolding voltages reflect to some extent, but not always unambiguously, the number of intramolecular hydrogen bonds that were present in solution. Reaching phosphate charging densities over 0.25 makes it easier to discriminate between structures, and the use of native supercharging agents is thus essential. We also uncovered several caveats in data interpretation: (1) when different structures (for example the i-motif with and without hairpin) unfold via different pathways, the unfolding voltages do not necessarily reflect the number of hydrogen bonds, (2) unstructured controls also undergo unfolding, and the base composition influences the unfolding voltage, (3) changing the solution pH also unexpectedly changed the unfolding voltage, and (4) the ion mobility patterns become more complicated when two structures are present simultaneously, such as an i-motif and a harpin, because of opposite effects on the collision cross section upon activation.

Photochemical and Collision-Induced Cross-Linking of Asp, Glu, Asn, and Gln Residues in Peptide-Nitrile Imine Conjugate Ions in the Gas Phase.

Peptide conjugates furnished with a 2,5-diaryltetrazolecarbonyl tag at the C-terminal lysine, which we call peptide-tet-K, were found to undergo efficient cross-linking of Asp, Glu, Asn, and Gln residues to transient nitrile-imine intermediates produced by photodissociation and collision-induced dissociation (CID) of the tetrazole ring in gas-phase ions. UV photodissociation (UVPD) at 213 nm achieved cross-linking conversion yields of 37 and 61% for DAAAK-tet-K and EAAAK-tet-K, respectively. The yields for NAAAK-tet-K and QAAAK-tet-K were 29 and 57%, respectively. Even higher cross-link yields were found for CID-MS3 of stable denitrogenated ions, (peptide-tet-K-N2 + H)+, that were in the 69-83% range. Different types of cross-links were distinguished by CID-MSn that showed a distinct series of backbone fragment ions, loss of N-terminal groups, and loss of phenylhydrazine from the modified nitrile imines. The Asp and Glu side-chain carboxyl groups were major participants in cross-linking that resulted in proton and oxygen transfer to the nitrile imine group. Other types of cross-linking involved Asn and Gln CONH2 groups and backbone amides. Cyclic ion mobility-mass spectrometry was used to separate NAAAK-tet-K and QAAAK-tet-K conformers and products of their collision-induced denitrogenation. Linear nitrile-imine and cross-linked ion structures were identified by comparing the experimental collision cross sections (CCSexp) to those for structures obtained by combined Born-Oppenheimer molecular dynamics and density functional theory (DFT) calculations. The formation of cross-links was found to be energetically favorable and involved proton-facilitated nucleophilic attack at the nitrile-imine carbon atom.

Structure and Stabilities of Solution and Gas Phase Protein Complexes.

Collision-induced unfolding (CIU) has provided new levels of understanding of the stabilities and structure(s) for gas phase protein and protein complex ions formed by electrospray ionization (ESI). Variable-temperature (vT-ESI) data provide complementary information about temperature-induced folding/unfolding (TIU) reactions of solution phase ions. Results obtained by using CIU and TIU provide complementary information about stabilities of gas phase versus solution phase ions. Such comparisons may provide the most direct experimental approach to answer a long-standing question from Fred McLafferty: "For how long, under what conditions, and to what extent, can solution structure be retained without solvent?" Answers to this question require greater understanding of the (i) structure(s), stabilities, and dynamics of proteins/protein complexes in solution prior to ESI; (ii) effects of water removal by droplet fission and "freeze-drying" by evaporation of water from the nanodroplet; and (iii) potential reactions and structural changes that may occur as the ions traverse the heated capillary, the final stage in the transition to solvent-free gas phase ions. Here, we employ vT-ESI coupled with ion mobility-mass spectrometry (IM-MS) as a means to provide more detailed answers to the above-mentioned questions. Apo- and metalated-metallothionein-2A (MT), a cysteine-rich metal binding protein, and various proteoforms of transthyretin (TTR), a homotetrameric (56 kDa) retinol and thyroxine transporter protein complex were studied to examine distinct features of CIU and TIU across two different types of protein complexes. The results in this work shed light on the capabilities of CIU, TIU, and average charge state (Zavg) for probing the rugged energy landscape of native proteins and highlights the effects of water and cofactors (metal ions) on the structure and stabilities of proteins and protein complexes.

Hybrid Mass Spectrometry Applied across the Production of Antibody Biotherapeutics.

Post expression from the host cells, biotherapeutics undergo downstream processing steps before final formulation. Mass spectrometry and biophysical characterization methods are valuable for examining conformational and stoichiometric changes at these stages, although typically not used in biomanufacturing, where stability is assessed via bulk property studies. Here we apply hybrid MS methods to understand how solution condition changes impact the structural integrity of a biopharmaceutical across the processing pipeline. As an exemplar product, we use the model IgG1 antibody, mAb4. Flexibility, stability, aggregation propensity, and bulk properties are evaluated in relation to perfusion media, purification stages, and formulation solutions. Comparisons with Herceptin, an extensively studied IgG1 antibody, were conducted in a mass spectrometry-compatible solution. Despite presenting similar charge state distributions (CSD) in native MS, mAb4, and Herceptin show distinct unfolding patterns in activated ion mobility mass spectrometry (aIM-MS) and differential scanning fluorimetry (DSF). Herceptin's greater structural stability and aggregation onset temperature (Tagg) are attributed to heavier glycosylation and kappa-class light chains, unlike the lambda-class light chains in mAb4. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed that mAb4 undergoes substantial structural changes during purification, marked by high flexibility, low melting temperature (Tm), and prevalent repulsive protein-protein interactions but transitions to a compact and stable structure in high-salt and formulated environments. Notably, in formulation, the third constant domain (CH3) of the heavy chain retains flexibility and is a region of interest for aggregation. Future work could translate features of interest from comprehensive studies like this to targeted approaches that could be utilized early in the development stage to aid in decision-making regarding targeted mutations or to guide the design space of bioprocesses and formulation choices.

Accelerated Amyloid Aggregation Dynamics of Intrinsically Disordered Proteins in Heavy Water.

We explored the influence of D2O on the fibrillation kinetics and structural dynamics of amyloid intrinsically disordered proteins (IDPs), including α-synuclein, amyloid-β 1-42, and K18. Our findings revealed that fibrillation of IDPs was accelerated in D2O compared to that in H2O, exhibiting faster kinetics in contrast to the structured protein, insulin. Structural investigations using electrospray ionization ion mobility mass spectrometry and small-angle X-ray scattering combined with molecular dynamics simulations demonstrated that IDPs did not show significant structural changes that could influence accelerated fibrillation in D2O. Umbrella sampling of protein protofibrils verified that an increased level of hydrogen bonding of D2O and enhanced hydrophobic interactions stabilized β-sheet structured fibrils in D2O. These findings indicate that stabilizing β-sheet fibrils and a more hydrophobic microenvironment in D2O result in enhanced and faster fibrillation of IDPs. The study highlights the importance of considering D2O's differential impact on protein interactions when conducting structural and kinetic analyses, particularly for native peptides and proteins.

Synthesis of β-cyclodextrin magnetic-graphene-oxide pyrenyl (β-mGOP) gels for high-throughput enrichment and detection of various trace mycotoxins in food using UHPLC-cIMS

In this research, β-cyclodextrin magnetic-graphene-oxide pyrenyl gel composite (β-mGOP) was employed to remove trace mycotoxins from food products. The β-mGOP gels exhibited a three-dimensional macroporous structure that facilitated the rapid diffusion of target molecules into the nanoporous structure of the covalent organic frameworks embedded within the gel. The adsorption process of mycotoxins onto the β-mGOP gels conformed to the pseudo-second-order kinetics and the Freundlich adsorption model. These findings demonstrated exceptional rapid adsorption kinetics without pore collapse, making the material suitable as fillers for solid-phase extraction adsorbents. Additionally, a novel screening technique utilizing β-mGOP gels in conjunction with cyclic ion mobility mass spectrometry was developed to illustrate the efficacy of β-mGOP gels in capturing various trace substances in food and enhancing detection resolution. The versatile, uncomplicated, and scalable characteristics of β-mGOP gels indicate their promising potential for future applications in high-throughput analysis of trace contaminants in food.

Abstract 4131674: ADP-Ribosylation In a Mouse Model of Atherosclerosis: a Potential Novel Link Between Dyslipidemia and Inflammation in Cardiovascular Disease

Background: Inflammation and lipid accumulation are major features of atherosclerosis, a leading cause of death and morbidity worldwide. Our previous study recognized ADP-ribosylation, a post-translational modification, as a novel regulator of macrophage activation. We also have established mass spectrometry-based ADP-ribosylation proteomics. Using this technology, we evaluated the completely uncharacterized role of ADP-ribosylation in atherogenesis. We hypothesized that ADP-ribosylated proteins circulate from liver, accumulate in aorta and promote atherogenesis. Methods and Results: We harvested the aorta, liver, and plasma of LDL receptor-deficient ( Ldlr -/- ) mice that were on a regular chow or high-fat diet for 3 or 6 months (n=40/condition). To increase ADP-ribosyl peptide signals in the aorta, we applied our novel recently optimized ion mobility mass spectrometry strategy to generate ADP-ribosylation proteomics data. We analyzed 160 mice aortas and identified 3 APOA1 and 3 APOE ADP-ribosylated peptides in both the aorta and the liver (Fig. A). In addition, these peptides were differentially abundant in the aorta of HFD-fed mice, compared to controls (i.e, APOA1 ARPALEDLR peptide relative abundance (Fig. B)). Using the same mouse plasma, we then validated the presence of ADP-ribosylated APOA1 and ADP-ribosylated APOE in HDL and chylomicron/VLDL/LDL fractions (Western blot), respectively. This finding indicates that classical apolipoproteins circulate as ADP-ribosylated forms, representing a completely novel class of modified apolipoproteins. Immunohistochemistry confirmed the enrichment of aortic lesions in macrophages and ADP-ribosylation signal (5-fold increase, p=0.0006). Conclusions: This work provides the first in vivo evidence that ADP-ribosylation occurs in atherosclerotic lesions, which may originate from the liver via circulating blood (Fig. C). Future studies will examine whether ADP-ribosylation of apolipoproteins, specifically APOA1, alters anti-atherogenic functions of HDL.

Ion Mobility-Mass Spectrometry Captures the Structural Consequences of Lipid Nanoparticle Encapsulation on Ribonucleic Acid Cargo.

Ribonucleic acids (RNAs) are becoming increasingly significant in our search for improved biotherapeutics. RNA-based treatments offer high specificity, targeted delivery, and potentially lower-cost options for various debilitating human diseases. Despite these benefits, there are still relatively few FDA-approved RNA-based therapies, with the notable exceptions being the mRNA (mRNA) COVID-19 vaccines, which are delivered using lipid nanoparticle (LNP) systems. LNPs are distinctive drug delivery systems (DDSs) because of their ability to target specific cells, their biocompatibility, and their efficiency in merging with cellular membranes to enhance treatment effectiveness. While the biophysical landscapes of RNA structures in solution are relatively well understood, the impact of the LNP environment on RNA remains less clear. This study uses native ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) techniques to investigate how LNP encapsulation affects RNA structure and stability. We examine how various factors, such as ionization polarity, cofactor binding, lipid types, and lipid ratios, influence LNP-released RNA cargo. Our findings reveal that LNP DDSs induce significant changes in the structures and stabilities of their RNA cargo. However, the extent of these changes strongly depends on the type and composition of the lipids used. We conclude by discussing how IM-MS and CIU can aid in the continued development of more efficient LNP DDSs and improve DDS selection methodologies overall.

Exploring the structural heterogeneity of IgG1s: A comprehensive analysis of glycosylated and deglycosylated forms using TIMS-TOF-MS technique

Denaturation of IgG is a complex and multistep process that involves various structural changes in the protein as it loses its native conformation. IgG denaturation is crucial in therapeutic antibody development, manufacturing, and storage. Glycosylated IgG is generally more resistant to denaturing conditions due to the stabilizing effect of its glycans. In contrast, deglycosylated IgG is more vulnerable to structural disruption. In this study, we examined the intact level and large fragments of IgG glycoforms. Our investigation focused on changes in structural heterogeneity through glycosylation under denaturing conditions. Collision cross-sections (CCS) of intact level and large fragments of IgG1 molecules have been determined using reversed phase-high performance liquid chromatography-electrospray ionization-trapped ion mobility-time-of-flight mass spectrometry (RP-HPLC-ESI-TIMS-TOF-MS) technique. At the intact level, deglycosylated bevacizumab (IgG1B) and trastuzumab (IgG1T) could be differentiated at specific charge states and CCS ranges. The deglycosylated IgG1B molecule showed a broader CCS range than the IgG1T molecule. This result demonstrated that the deglycosylated IgG1T was altered from deglycosylated IgG1B in the gas phase under denaturing conditions. The lowering in the collision cross-section distribution (CCSD) values indicates a decrease in the structural heterogeneity of the intact IgG after deglycosylation. The ion mobility-mass spectrometry (IM-MS) analyses performed in this study showed that the glycans on the Fc region of IgG1s can trigger structural changes in the molecule under denaturing conditions. Depending on whether they are glycosylated or deglycosylated, various conformations of IgG1 molecules could be distinguished in RP-HPLC-TIMS-TOF-MS analysis.

Elucidating Protein Structures in the Gas Phase: Traversing Configuration Space with Biasing Methods.

Achieving accurate characterization of protein structures in the gas phase continues to be a formidable challenge. To tackle this issue, the present study employs Molecular Dynamics (MD) simulations in tandem with enhanced sampling techniques (methods designed to efficiently explore protein conformations). The objective is to identify suitable structures of proteins by contrasting their calculated Collision Cross-Section (CCS) with those observed experimentally. Significant discrepancies were observed between the initial MD-simulated and experimentally measured CCS values through Ion Mobility-Mass Spectrometry (IMS-MS). To bridge this gap, we employed two distinct enhanced sampling methods, Harmonic Biasing Potential and Adaptive Biasing Force, which help the proteins overcome energy barriers to adopt more compact configurations. These techniques leverage the radius of gyration as a reaction coordinate (guiding parameter), guiding the system toward compressed states that potentially match experimental configurations more closely. The guiding forces are only employed to overcome existing barriers and are removed to allow the protein to naturally arrive at a potential gas phase configuration. The results demonstrated close alignment (within ∼4%) between simulated and experimental CCS values despite using different strengths and/or methods, validating their efficacy. This work lays the groundwork for future studies aimed at optimizing biasing methods and expanding the collective variables used for more accurate gas-phase structural predictions.

Simultaneous Determination of the Position and Cis-Trans Configuration of Lipid C═C Bonds via Asymmetric Derivatization and Ion Mobility-Mass Spectrometry.

The position and cis-trans configuration of C═C bonds in unsaturated lipids significantly affect their biological activities. Simultaneous identification of the position and cis-trans configuration of C═C bonds in unsaturated lipids is important; nonetheless, it still remains a challenging task. Herein, a stereoselective asymmetric reaction was used to recognize cis-trans isomers of the C═C bonds, and the derivatized precursor ions and product ions were subjected to tandem ion mobility-mass spectrometry (IM-MS) analysis. The theoretical calculation revealed that the formation of intramolecular hydrogen bonds after the cyclization reaction amplified the structural difference between diastereomers and increased the separation efficiency in IM. Consequently, a simple, sensitive, and highly selective platform for simultaneous determination of the position and cis-trans configuration of various C═C bonds in unsaturated lipids was established. It was then successfully applied to pinpoint the cis-trans geometry conversion of the located C═C bonds in lipids of the bacterial membrane under environmental stress and track the heterogeneous distribution of unsaturated lipids in rats after spinal cord injury. The present study also offers new insights into the application of IM-MS technology in resolving molecular structures and demonstrates the potential as a platform for a broad range of applications.

Eliminating the Deadwood: A Machine Learning Model for CCS Knowledge-Based Conformational Focusing for Lipids.

Accurate elucidation of gas-phase chemical structures using collision cross section (CCS) values obtained from ion-mobility mass spectrometry benefits from a synergism between experimental and in silico results. We have shown in recent work that for a molecule of modest size with a proscribed conformational space we can successfully capture a conformation(s) that can match experimental CCS values. However, for flexible systems such as fatty acids that have many rotatable bonds and multiple intramolecular London dispersion interactions, it becomes necessary to sample a much greater conformational space. Sampling more conformers, however, accrues significant computational cost downstream in optimization steps involving quantum mechanics. To reduce this computational expense for lipids, we have developed a novel machine learning (ML) model to facilitate conformer filtering according to the estimated gas-phase CCS values. Herein we report that the implementation of our CCS knowledge-based approach for conformational sampling resulted in improved structure prediction agreement with experiment by achieving favorable average CCS prediction errors of ∼2% for lipid systems in both the validation set and the test set. Moreover, most of the gas-phase candidate conformations obtained by using CCS focusing achieved lower energy-minimum geometries than the candidate conformations without focusing. Altogether, the implementation of this ML model into our modeling workflow has proven to be beneficial for both the quality of the results and the turnaround time. Finally, while our approach is limited to lipids, it can be readily extended to other molecules of interest.

Rapid separation of bile acid isomers via ion mobility mass spectrometry by complexing with spiramycin.

Bile acid (BA) is one of the main active components of bile and has multiple isomers, the structure or content of its isomers often changes due to diseases and other health problems; thus, the accurate detection of BA isomers is very important. In this study, two groups of BA isomers of glycine-conjugated BAs and taurine-conjugated BAs were simultaneously separated and quantitatively analyzed by ion mobility mass spectrometry (IM-MS). Especially, baseline mobility separation between the isomers was achieved by the formation of binary complexes via simple interaction with spiramycin (SPM), for which a separation resolution (Rp-p) of 1.96was reached. Moreover, BA isomers were quantitatively analyzed, and the limit of detection (LOD) of absolute quantification for TCDCA/TUDCA and GUDCA/GCDCA/GHDCA was 0.514 and 0.611ng∙mL-1, respectively; the LODs for molar ratio ranges of relative quantification for TCDCA/TUDCA, GUDCA/GHDCA, and GCDCA/GHDCA were 1:18-30:1, 1:18-21:1, and 1:19-21:1, respectively. Additionally, BA isomers analyzed in pig bile powder and bear bile powder were measured, which were in good consistency with those labeled, revealing the differences in BA composition and content between the two powders. Finally, BA detection and recovery analyses were performed on serum samples, with a recovery rate of ≥73.69%, RSD of ≤6.8%, and SR (standard deviation of recoveries, the degree of difference between measured values and average recovery) of ≤1.27. Due to the simple, rapid, and lack of need for complex sample preparation and chromatographic separation, the proposed method can be an effective method for BA detection in practical samples.

B-282 Cost and Benefits of Utilization of Ion Mobility Mass Spectrometry Testing for Drugs of Abuse Compared to Immunoassay Screening in a Large Urban Hospital

Abstract Background Urine drugs of abuse testing (DOAU) in US hospitals are mainly orders from the emergency department performed by immunoassay screening. Testing can be relatively expensive, and results may suffer from non-idealities in sensitivity, specificity, and from panel limitations. A potential approach to these issues for immunoassays is instead to employ mass spectrometry (MS), where a large and flexible analyte list can be evaluated providing needed specificity and reducing send out costs for confirmatory testing. We compared results and cost impacts of a DOAU assay on a prototype, ion mobility mass spectrometry system to those obtained from an array of immunoassays offered as routine tests at a large urban hospital. Methods Immunoassay screen-positive and screen-negative urine samples were isolated from among those submitted for a DOAU panel (amphetamines, barbiturates, benzodiazepines, cannabis, cocaine, opiates, and methadone) or for additional single drug tests (fentanyl, oxycodone, phencyclidine, buprenorphine, propoxyphene). Sample aliquots (3 mL) were retrieved after not more than 5 days of refrigerated storage of the original sample and were subsequently frozen. These samples were then subjected to MS analysis using the prototype system where identification of 62 compounds was based on mass and ion mobility-derived Collision Cross Section (CCS). An established library of m/z, SLIMCCSHe, peak intensity, and peak width for each analyte was used, with acceptable method performance characteristics as based on applicable CLIA validation guidelines. The MS list included compounds covered by the immunoassay classes as well as additional drugs not expected to be positive among the immunoassays test results but of value for routine toxicological assessments. Results comparisons, utility of results, sample-to-result turnaround times, and overall financial implications were compared between the two workflows. Results Among immunoassay screen-negative samples, MS analysis showed additional positive results for metabolites of parent drugs as well as novel psychoactive substances and known adulterants in >10% of cases. Among the immunoassay screen-positive results, MS analysis confirmed immunoassay results (>90%) but picked up an additional positive component in over 15% of cases. Cost per complete patient result for the immunoassay was approximately $2.80 in comparison to an estimated cost of $5 for the MS method. Confirmation testing for positive immunoassay screen results (16% of all samples) would cost an additional $70 per sample, whereas the need for confirmation testing is eliminated for the MS method. Thus, use of the MS testing workflow would be superior to an immunoassay with reflex testing workflow while at the same time providing a clinically superior service of more diverse positive test results and elimination of often overlooked false negatives. Turnaround time between immunoassay (1h) and MS (50m) were judged to be similar. Conclusions The MS approach confirmed >90% of all immunoassay positive screen results. In addition, the MS method identified additional positive results not detected by the immunoassay despite an overall similar estimated time for sample to patient. Need for development of a user friendly, MS analyzer for use in hospitals is supported based on cost-effective and analytically superior tests for DOAU compared to screens performed using immunoassay panels.

Recent additions and access to a multidimensional lipidomic database containing liquid chromatography, ion mobility spectrometry, and tandem mass spectrometry information.

The importance of lipids in biology continues to grow with their recent linkages to more diseases and conditions, microbiome fluctuations, and environmental exposures. These associations have motivated researchers to evaluate lipidomic changes in numerous matrices and studies. Lipidomic analyses, however, present numerous challenges as lipid species have broad chemistries that require different extraction methods and instrumental analyses to evaluate and separate their many isomers and isobars. Increasing knowledge about different lipid characteristics is therefore crucial for improving their separation and identification. Here, we present a multidimensional database for lipids analyzed on a platform combining reversed-phase liquid chromatography, drift tube ion mobility spectrometry, collision-induced dissociation, and mass spectrometry (RPLC-DTIMS-CID-MS). This platform and the different separation characteristics it provides enables more confident lipid annotations when compared to traditional tandem mass spectrometry platforms, especially when analyzing highly isomeric molecules such as lipids. This database expands on our previous publication containing only human plasma and bronchoalveolar lavage fluid lipids and provides experimental RPLC retention times, IMS collision cross section (CCS) values, and m/z information for 877 unique lipids from additional biofluids and tissues. Specifically, the database contains 1504 precursor [M + H]+, [M + NH4]+, [M + Na]+, [M-H]-, [M-2H]2-, [M + HCOO]-, and [M + CH3COO]- ion species and their associated CID fragments which are commonly targeted in clinical and environmental studies, in addition to being present in the chloroform layer of Folch extractions. Furthermore, this multidimensional RPLC-DTIMS-CID-MS database spans 5 lipid categories (fatty acids, sterols, sphingolipids, glycerolipids, and glycerophospholipids) and 24 lipid classes. We have also created a webpage (tarheels.live/bakerlab/databases/) to enhance the accessibility of this resource which will be populated regularly with new lipids as we identify additional species and integrate novel standards.

From Microsize Chromatographic Manufacturing for Fast Desalting to Its Characterization.

Microfluidic devices are becoming increasingly popular in protein analysis due to their ability to reduce sample and buffer volumes. However, there is a research gap concerning the coupling of this technology with ion mobility and mass spectrometry (IM-MS). This study aims to fill this void by introducing the manufacture and the characterization of a microsize exclusion chromatography (μSEC) module for fast desalting and its integration into microfluidics, along with its coupling to electrospray ionization and ion mobility mass spectrometry (ESI-IM-MS). To assess the feasibility of this approach, the desalting of α-synuclein (αS) was investigated using Bio Spin P6 gel as a stationary phase in the manufacture of a microfluidic device. αS detection by MS gives insight into the sample purity, while IM combined with MS provides information about protein structure. IM allowed both the recording of qualitative and quantitative information. The qualitative data provided a map of the conformers in equilibrium, while the calculation of the respective abundances (quantitative profile) of the conformers afforded the opportunity to describe the dynamics of the system. Our experiments, serving as proof-of-concept, demonstrate αS desalting, exchange buffer efficiency, and reduced solvent usage, without compromising the protein's structure.

In Vitro Inhibition of Enzymes and Antioxidant and Chemical Fingerprinting Characteristics of Azara serrata Ruiz & Pav. Fruits, an Endemic Plant of the Valdivian Forest of Chile

The World Health Organization has emphasized the importance of consuming small fruits for the prevention of chronic health problems, including diabetes, cardiovascular diseases, cancer, and obesity, which are named chronic non-communicable diseases (NCDs). Azara serrata Ruiz & Pav., commonly called “aroma de Castilla”, is a shrub endemic to Chile from the Salicaceae family that produces an underutilized blue-grey berry that grows wild in southern Chile. The species is widely used as a medicinal plant by the Andean communities of southern Chile. In this work, a high-resolution mass spectrometric analysis of the methanolic extract revealed several phenolic compounds for the first time in the edible berry of this endemic species. Furthermore, several glycosylated anthocyanins were detected and quantified using UHPLC coupled with UV/Vis detection and trapped ion mobility mass spectrometry (UHPLC-DAD-TIMS-TOF) for the anthocyanin-rich extract, which was prepared using an optimized anthocyanin extraction protocol. The extract proved to be active in the inhibition of several enzymes linked to NCDs, such as acetylcholinesterase, tyrosinase, amylase, lipase, and glucosidase (IC50 = 3.92 ± 0.23, 12.24 ± 0.03, 11.12 ± 0.10, 32.43 ± 0.0, and 371.6 ± 0.0 μg/mL, respectively). Furthermore, the extract concentrated in anthocyanins showed good antioxidant activity evidenced by the bleaching of the radicals DPPH and ABTS, ferric-reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC). The results show that these neglected endemic small berries can be a source of healthy phytochemicals. These Chilean berries can be used as functional food and their extracts are candidates for use as functional ingredients in naturally healthy products.

Investigating Anion Effects on Metal Ion Binding Interactions With Amyloid β Peptide by Ion Mobility Mass Spectrometry.

The study of metal ion's role in the biological processes of Alzheimer's disease has spurred investigations into the coordination chemistry of amyloid beta peptide and its fragments. Nano-electrospray ionization mass spectrometry (nESI-MS) has been utilized to examine the stabilization of bound anions on multiprotein complexes without bulk solvent. However, the effects of anions on metal ion binding interactions with amyloid beta peptide have not been explored. This study directly examined metal-peptide complexes using nESI-MS and investigated the effects of various anions on the binding ratio and stability of these complexes from ammonium salt solutions. The results indicate that different anions have distinct effects on the binding ratio and stability of various metal-peptide complexes. Of these, the bicarbonate ion exhibits the highest binding ratios for metal-peptide complexes, while binding ratios for these complexes in phosphate are comparatively low. Our results suggest that acetate, formate, bicarbonate, and phosphate have weak affinities and act as weak stabilizers of the metal-peptide complex structure in the gas phase. Intriguingly, chloride and sulfate act as stabilizers of the metal-peptide complex in the gas phase. The rank order determined from these data is substantially different from the Hofmeister salt series in solution. Although this outcome was anticipated due to the reduced influence of anions and water solvation, our findings correlate well with expected anion binding in solution and emphasize the importance of both hydration layer and anion-metal-peptide binding effects for Hofmeister-type stabilization in solution. This approach proved useful in examining the interactions between metal ions and amyloid beta peptide, which are relevant to Alzheimer's disease, using direct ESI-MS.

Characterization of Anticancer Drug Protomers Using Electrospray Ionization and Ion Mobility Spectrometry-Mass Spectrometry.

We used electrospray ionization and ion mobility spectrometry-mass spectrometry to detect and characterize the three anticancer drugs palbociclib, copanlisib, and olaparib. Ion mobility-mass spectrometry and density functional theory revealed that these compounds generate isomers during ionization (protomers) due to the presence of multiple protonation sites within their chemical structures. Our work has implications for understanding the solution- and gas-phase chemistry of these molecules during spray-based ionization processes.