Transaminase-Catalyzed Synthesis of ß-Branched Noncanonical Amino Acids Driven by a Lysine Amine Donor.

Transaminases are choice biocatalysts for the synthesis of chiral primary amines, including amino acids bearing contiguous stereocenters. In this study, we employ lysine as a "smart" amine donor in transaminase-catalyzed dynamic kinetic resolution reactions to access ß-branched noncanonical arylalanines. Our mechanistic investigation demonstrates that, upon transamination, the lysine-derived ketone byproduct readily cyclizes to a six-membered imine, driving the equilibrium in the desired direction and thus alleviating the need to load superstoichiometric quantities of the amine donor or deploy a multienzyme cascade. Lysine also shows good overall compatibility with a panel of wild-type transaminases, a promising hint of its application as a smart donor more broadly. Indeed, by this approach, we furnished a broad scope of ß-branched arylalanines, including some bearing hitherto intractable cyclopropyl and isopropyl substituents, with high yields and excellent selectivities.

Improved assay development of pharmaceutical modalities using feedback-controlled liquid chromatography optimization.

Development of meaningful and reliable analytical assays in the (bio)pharmaceutical industry can often be challenging, involving tedious trial and error experimentation. In this work, an automated analytical workflow using an AI-based algorithm for streamlined method development and optimization is presented. Chromatographic methods are developed and optimized from start to finish by a feedback-controlled modeling approach using readily available LC instrumentation and software technologies, bypassing manual user intervention. With the use of such tools, the time requirement of the analyst is drastically minimized in the development of a method. Herein key insights on chromatography system control, automatic optimization of mobile phase conditions, and final separation landscape for challenging multicomponent mixtures are presented (e.g., small molecules drug, peptides, proteins, and vaccine products) showcased by a detailed comparison of a chiral method development process. The work presented here illustrates the power of modern chromatography instrumentation and AI-based software to accelerate the development and deployment of new separation assays across (bio)pharmaceutical modalities while yielding substantial cost-savings, method robustness, and fast analytical turnaround.

Two-Dimensional SEC-SEC-UV-MALS-dRI Workflow for Streamlined Analysis and Characterization of Biopharmaceuticals.

The emergence of complex biological modalities in the biopharmaceutical industry entails a significant expansion of the current analytical toolbox to address the need to deploy meaningful and reliable assays at an unprecedented pace. Size exclusion chromatography (SEC) is an industry standard technique for protein separation and analysis. Some constraints of traditional SEC stem from its restricted ability to resolve complex mixtures and notoriously long run times while also requiring multiple offline separation conditions on different pore size columns to cover a wider molecular size distribution. Two-dimensional liquid chromatography (2D-LC) is becoming an important tool not only to increase peak capacity but also to tune selectivity in a single online method. Herein, an online 2D-LC framework in which both dimensions utilize SEC columns with different pore sizes is introduced with a goal to increase throughput for biomolecule separation and characterization. In addition to improving the separation of closely related species, this online 2D SEC-SEC approach also facilitated the rapid analysis of protein-based mixtures of a wide molecular size range in a single online experimental run bypassing time-consuming deployment of different offline SEC methods. By coupling the second dimension with multiangle light scattering (MALS) and differential refractive index (dRI) detectors, absolute molecular weights of the separated species were obtained without the use of calibration curves. As illustrated in this report for protein mixtures and vaccine processes, this workflow can be used in scenarios where rapid development and deployment of SEC assays are warranted, enabling bioprocess monitoring, purity assessment, and characterization.

Computer-assisted multifactorial method development for the streamlined separation and analysis of multicomponent mixtures in (Bio)pharmaceutical settings.

The (bio)pharmaceutical industry is rapidly moving towards complex drug modalities that require a commensurate level of analytical enabling technologies that can be deployed at a fast pace. Unsystematic method development and unnecessary manual intervention remain a major barrier towards a more efficient deployment of meaningful analytical assay across emerging modalities. Digitalization and automation are key to streamline method development and enable rapid assay deployment. This review discusses the use of computer-assisted multifactorial chromatographic method development strategies for fast-paced downstream characterization and purification of biopharmaceuticals. Various chromatographic techniques such as reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and supercritical fluid chromatography (SFC) are addressed and critically reviewed. The most significant parameters for retention mechanism modelling, as well as mapping the separation landscape for optimal chromatographic selectivity and resolution are also discussed. Furthermore, several computer-assisted approaches for optimization and development of chromatographic methods of therapeutics, including linear, nonlinear, and multifactorial modelling are outlined. Finally, the potential of the chromatographic modelling and computer-assisted optimization strategies are also illustrated, highlighting substantial productivity improvements, and cost savings while accelerating method development, deployment and transfer processes for therapeutic analysis in industrial settings.

A Liquid Chromatography High-Resolution Tandem Mass Spectrometry Method to Quantify QS-21 Adjuvant and Its Degradation Products in Liposomal Drug Formulations.

Identification and quantification of an active adjuvant and its degradation product/s in drug formulations are important to ensure drug product safety and efficacy. QS-21 is a potent adjuvant that is currently involved in several clinical vaccine trials and a constituent of licensed vaccines against malaria and shingles. In an aqueous milieu, QS-21 undergoes pH- and temperature-dependent hydrolytic degradation to form a QS-21 HP derivative that may occur during manufacturing and/or long-term storage. Intact QS-21 and deacylated QS-21 HP elicit different immune response profiles; thus, it is imperative to monitor QS-21 degradation in vaccine adjuvant formulation. To date, a suitable quantitative analytical method for QS-21 and its degradation product in drug formulations is not available in the literature. In view of this, a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and qualified to accurately quantify the active adjuvant QS-21 and its degradation product (QS-21 HP) in liposomal drug formulations. The method was qualified according to the FDA Guidance for Industry: Q2(R1). Study results showed that the described method presents good specificity for QS-21 and QS-21 HP detection in a liposomal matrix, good sensitivity characterized by the limit of detection (LOD)/limit of quantitation (LOQ) in the nanomolar range, linear regressions with correlation coefficients, R2 > 0.999, recoveries in the range of 80-120%, and precise detection and quantification with % relative standard deviation (RSD) < 6% for QS-21 and < 9% for the QS-21 HP impurity assay. The described method was successfully used to accurately evaluate in-process and product release samples of the Army Liposome Formulation containing QS-21 (ALFQ).

Automated Hydrophobic Interaction Chromatography Screening Combined with In Silico Optimization as a Framework for Nondenaturing Analysis and Purification of Biopharmaceuticals.

The mounting complexity of new modalities in the biopharmaceutical industry entails a commensurate level of analytical innovations to enable the rapid discovery and development of novel therapeutics and vaccines. Hydrophobic interaction chromatography (HIC) has become one of the widely preferred separation techniques for the analysis and purification of biopharmaceuticals under nondenaturing conditions. Inarguably, HIC method development remains very challenging and labor-intensive owing to the numerous factors that are typically optimized by a "hit-or-miss" strategy (e.g., the nature of the salt, stationary phase chemistry, temperature, mobile phase additive, and ionic strength). Herein, we introduce a new HIC method development framework composed of a fully automated multicolumn and multieluent platform coupled with in silico multifactorial simulation and integrated fraction collection for streamlined method screening, optimization, and analytical-scale purification of biopharmaceutical targets. The power and versatility of this workflow are showcased by a wide range of applications including trivial proteins, monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), oxidation variants, and denatured proteins. We also illustrate convenient and rapid HIC method development outcomes from the effective combination of this screening setup with computer-assisted simulations. HIC retention models were built using readily available LC simulator software outlining less than a 5% difference between experimental and simulated retention times with a correlation coefficient of >0.99 for pharmaceutically relevant multicomponent mixtures. In addition, we demonstrate how this approach paves the path for a straightforward identification of first-dimension HIC conditions that are combined with mass spectrometry (MS)-friendly reversed-phase liquid chromatography (RPLC) detection in the second dimension (heart-cutting two-dimensional (2D)-HIC-RPLC-diode array detector (DAD)-MS), enabling the analysis and purification of biopharmaceutical targets.

Dual-Gradient Unified Chromatography: A New Paradigm for Versatility in Simultaneous Multicomponent Analysis.

Generality in analytical chemistry can be manifested in impactful platforms that can streamline modern organic synthesis and biopharmaceutical processes. We herein introduce a hybrid separation technique named Dual-Gradient Unified Chromatography (DGUC), which is built upon an automated dynamic modulation of CO2 , organic modifier, and water blends with various buffers. This concept enables simultaneous multicomponent analysis of both small and large molecules across a wide polarity range in single experimental runs. After a careful investigation of its fundamental aspects, a DGUC-DAD-MS screening workflow that combines multiple orthogonal column and mobile phase choices across a far-reaching universal elution profile is also reported. The power of this framework is demonstrated with new analytical applications guiding academic and industrial laboratories in the development of new (bio)pharmaceutical targets (e.g. synthetic intermediates, nucleosides, cyclic and linear peptides, proteins, antibody drug conjugates).

A Rapid Method for Direct Quantification of Antibody Binding-Site Concentration in Serum.

The quantitation of the available antibody binding-site concentration of polyclonal antibodies in serum is critical in defining the efficacy of vaccines against substances of abuse. We have conceptualized an equilibrium dialysis (ED)-based approach coupled with fluorimetry (ED-fluorimetry) to measure the antibody binding-site concentration to the ligand in an aqueous environment. The measured binding-site concentrations in monoclonal antibody (mAb) and sera samples from TT-6-AmHap-immunized rats by ED-fluorimetry are in agreement with those determined by a more established equilibrium dialysis coupled with ultraperformance liquid chromatography tandem mass spectrometry (ED-UPLC-MS/MS). Importantly, we have shown that the measured antibody binding-site concentrations to the ligand by ED-fluorimetry were not influenced by the sample serum matrix; thus, this method is valid for determining the binding-site concentration of polyclonal antibodies in sera samples. Further, we have demonstrated that under appropriate analytical conditions, this method resolved the total binding-site concentrations on a nanomolar scale with good accuracy and repeatability within the microliter sample volumes. This simple, rapid, and sample preparation-free approach has the potential to reliably perform quantitative antibody binding-site screening in serum and other more complex biological fluids.

Design, Synthesis, and In Vivo Evaluation of C1-Linked 4,5-Epoxymorphinan Haptens for Heroin Vaccines.

In our continuing effort to develop effective anti-heroin vaccines as potential medications for the treatment of opioid use disorder, herein we present the design and synthesis of the haptens: 1-AmidoMorHap (1), 1-AmidoMorHap epimer (2), 1 Amido-DihydroMorHap (3), and 1 Amido-DihydroMorHap epimer (4). This is the first report of hydrolytically stable haptenic surrogates of heroin with the attachment site at the C1 position in the 4,5-epoxymorophinan nucleus. We prepared respective tetanus toxoid (TT)-hapten conjugates as heroin vaccine immunogens and evaluated their efficacy in vivo. We showed that all TT-hapten conjugates induced high antibody endpoint titers against the targets but only haptens 2 and 3 can induce protective effects against heroin in vivo. The epimeric analogues of these haptens, 1 and 4, failed to protect mice from the effects of heroin. We also showed that the in vivo efficacy is consistent with the results of the in vitro drug sequestration assay. Attachment of the linker at the C1 position induced antibodies with weak binding to the target drugs. Only TT-2 and TT-3 yielded antibodies that bound heroin and 6-acetyl morphine. None of the TT-hapten conjugates induced antibodies that cross-reacted with morphine, methadone, naloxone, or naltrexone, and only TT-3 interacted weakly with buprenorphine, and that subtle structural difference, especially at the C6 position, can vastly alter the specificity of the induced antibodies. This study is an important contribution in the field of vaccine development against small-molecule targets, providing proof that the chirality at C6 in these epoxymorphinans is a vital key to their effectiveness.

In Silico Method Development of Achiral and Chiral Tandem Column Reversed-phase Liquid Chromatography for Multicomponent Pharmaceutical Mixtures.

Tandem column liquid chromatography (LC) is a convenient, cost-effective approach to resolve multicomponent mixtures by serially coupling columns on readily available one-dimensional separation systems without specialized user training. Yet, adoption of this technique remains limited, mainly due to the difficulty in identifying optimal selectivity out of many possible tandem column combinations. At this point, method development and optimization require laborious "hit-or-miss" experimentation and "blind" screening when investigating different column selectivity without standard analytes. As a result, many chromatography practitioners end up combining two columns of similar selectivity, limiting the scope and potential of tandem column LC as a mainstay for industrial applications. To circumvent this challenge, we herein introduce a straightforward in silico multifactorial approach as a framework to expediently map the separation landscape across multiple tandem columns (achiral and chiral) and eluent combinations (isocratic and gradient elution) under reversed-phase LC conditions. Retention models were built using commercially available LC simulator software showcasing less than 2% difference between experimental and simulated retention times for analytes of interest in multicomponent pharmaceutical mixtures (e.g., metabolites and cyclic peptides).

Novel chimeric monoclonal antibodies that block fentanyl effects and alter fentanyl biodistribution in mice.

The prevalence and societal impact of opioid use disorder (OUD) is an acknowledged public health crisis that is further aggravated by the current pandemic. One of the devastating consequences of OUD is opioid overdose deaths. While multiple medications are now available to treat OUD, given the prevalence and societal burden, additional well-tolerated and effective therapies are still needed. To this point, we have developed chimeric monoclonal antibodies (mAb) that will specifically complex with fentanyl and its analogs in the periphery, thereby preventing them from reaching the central nervous system. Additionally, mAb-based passive immunotherapy offers a high degree of specificity to drugs of abuse and does not interfere with an individual's ability to use any of the medications used to treat OUD. We hypothesized that sequestering fentanyl and its analogs in the periphery will mitigate their negative effects on the brain and peripheral organs. This study is the first report of chimeric mAb against fentanyl and its analogs. We have discovered, engineered the chimeric versions, and identified the selectivity of these antibodies, through in vitro characterization and in vivo animal challenge studies. Two mAb candidates with very high (0.1-1.3 nM) binding affinities to fentanyl and its analogs were found to be effective in engaging fentanyl in the periphery and blocking its effects in challenged animals. Results presented in this work constitute a major contribution in the field of novel therapeutics targeting OUD.

Synthesis and immunological effects of C14-linked 4,5-epoxymorphinan analogues as novel heroin vaccine haptens.

Active immunization is being explored as a potential therapeutic to combat accidental overdose and to mitigate the abuse potential of opioids. Hapten design is one of the crucial factors that determines the efficacy of a candidate vaccine to substance abuse and remains one of the most active areas of research in vaccine development. Herein we report for the first time the synthesis of three novel opiate surrogates with the linker attachment site at C14, 1 (6,14-AmidoHap), 2 (14-AmidoMorHap), and 3 (14-AmidoHerHap) as novel heroin haptens. The compounds 1, 2, and 3 are analogues with different substituents at C6: an acetamide, a hydroxyl moiety, and an acetate, respectively. All three haptens had a phenolic hydroxyl group at C3. The haptens were conjugated to the tetanus toxoid carrier protein, adjuvanted with liposomal monophosphoryl lipid A/aluminum hydroxide and were tested in mice in terms of immunogenicity and efficacy. Immunization of mice resulted in antibody endpoint titers of >105 against all the haptens. Neither of the conjugates of 1, 2, and 3 had induced antibodies with selectivity broad enough to recognize and bind heroin, 6-AM, and morphine resulting in little to no protection against the antinociceptive effects of heroin in vivo. Only the mice immunized with conjugate 3 were partially protected against heroin-induced antinociception. These results contribute to the growing body of knowledge that the linker position and the subtle structural differences in the hapten scaffold impact the selectivity of the induced antibodies. Together, these highlight the importance of rational hapten design for heroin vaccine development.

Bivalent Conjugate Vaccine Induces Dual Immunogenic Response That Attenuates Heroin and Fentanyl Effects in Mice.

Opioid use disorders and fatal overdose due to consumption of fentanyl-laced heroin remain a major public health menace in the United States. Vaccination may serve as a promising potential remedy to combat accidental overdose and to mitigate the abuse potential of opioids. We previously reported the heroin and fentanyl monovalent vaccines carrying, respectively, a heroin hapten, 6-AmHap, and a fentanyl hapten, para-AmFenHap, conjugated to tetanus toxoid (TT). Herein, we describe the mixing of these antigens to formulate a bivalent vaccine adjuvanted with liposomes containing monophosphoryl lipid A (MPLA) adsorbed on aluminum hydroxide. Immunization of mice with the bivalent vaccine resulted in IgG titers of >105 against both haptens. The polyclonal sera bound heroin, 6-acetylmorphine, morphine, and fentanyl with dissociation constants (Kd) of 0.25 to 0.50 nM. Mice were protected from the anti-nociceptive effects of heroin, fentanyl, and heroin +9% (w/w) fentanyl. No cross-reactivity to methadone and buprenorphine was observed in vivo. Naloxone remained efficacious in immunized mice. These results highlighted the potential of combining TT-6-AmHap and TT-para-AmFenHap to yield an efficacious bivalent vaccine that could ablate heroin and fentanyl effects. This vaccine warrants further testing to establish its potential translatability to humans.

Synthesis of a deuterated 6-AmHap internal standard for the determination of hapten density in a heroin vaccine drug product.

A deuterated hapten was designed and synthesized that will be essential for a future study of residual hapten and stability of a hapten-protein conjugate. This hapten, 6-AmHap, was chosen for a heroin vaccine that is now slated for a Phase 1 clinical trial. A maleimide-thiol bioconjugation strategy was successfully applied to our heroin vaccine to connect the hapten 6-AmHap with an immunogenic carrier protein (tetanus toxoid, TT) through a trityl-protected 3-mercaptopropanamide linker. The antibodies induced by the vaccine have been found to have activity against several opioids, including heroin and its metabolites, and, importantly, leave alternate pain treatment medications such as methadone untouched. To the best of our knowledge, no other hapten for a heroin vaccine has been deuterated, yet this tool may prove to be of great importance in the study of residual hapten during product release and the long-term stability program of a hapten-protein conjugate as part of FDA regulatory requirements. Hydrocodone was the starting material for the synthesis of the deuterated 6-AmHap, with a stable amide at C6 and a 3-mercaptopropanamide linker attached at C3. The desired deuterated product was prepared as the disulfide, 3,3'-disulfanediylbis(N-((7S,7aR,12bS)-7-acetamido-3-[2 H3 ]methyl)-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)propanamide), that could be easily reduced to form the needed hapten, N-((4aR,7S,7aR,12bS)-7-acetamido-3-[2 H3 ]methyl]-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)-3-mercaptopropanamide.

Recent advances in the mass spectrometric analysis of glycosphingolipidome - A review.

Aberrant expression of glycosphingolipids has been implicated in a myriad of diseases, but our understanding of the strucural diversity, spatial distribution, and biological function of this class of biomolecules remains limited. These challenges partly stem from a lack of sensitive tools that can detect, identify, and quantify glycosphingolipids at the molecular level. Mass spectrometry has emerged as a powerful tool poised to address most of these challenges. Here, we review the recent developments in analytical glycosphingolipidomics with an emphasis on sample preparation, mass spectrometry and tandem mass spectrometry-based structural characterization, label-free and labeling-based quantification. We also discuss the nomenclature of glycosphingolipids, and emerging technologies like ion mobility spectrometry in differentiation of glycosphingolipid isomers. The intrinsic advantages and shortcomings of each method are carefully critiqued in line with an individual's research goals. Finally, future perspectives on analytical sphingolipidomics are stated, including a need for novel and more sensive methods in isomer separation, low abundance species detection, and profiling the spatial distribution of glycosphingolipid molecular species in cells and tissues using imaging mass spectrometry.

Novel Vaccine That Blunts Fentanyl Effects and Sequesters Ultrapotent Fentanyl Analogues.

Active immunization is an emerging potential modality to combat fatal overdose amid the opioid epidemic. In this study, we described the design, synthesis, formulation, and animal testing of an efficacious vaccine against fentanyl. The vaccine formulation is composed of a novel fentanyl hapten conjugated to tetanus toxoid (TT) and adjuvanted with liposomes containing monophosphoryl lipid A adsorbed on aluminum hydroxide. The linker and hapten N-phenyl-N-(1-(4-(3-(tritylthio)propanamido)phenethyl)piperidin-4-yl)propionamide were conjugated sequentially to TT using amine-N-hydroxysuccinimide-ester and thiol-maleimide reaction chemistries, respectively. Conjugation was facile, efficient, and reproducible with a protein recovery of >98% and a hapten density of 30-35 per carrier protein molecule. In mice, immunization induced high and robust antibody endpoint titers in the order of >106 against the hapten. The antisera bound fentanyl, carfentanil, cyclopropyl fentanyl, para-fluorofentanyl, and furanyl fentanyl in vitro with antibody-drug dissociation constants in the range of 0.36-4.66 nM. No cross-reactivity to naloxone, naltrexone, methadone, or buprenorphine was observed. In vivo, immunization shifted the antinociceptive dose-response curve of fentanyl to higher doses. Collectively, these preclinical results showcased the desired traits of a potential vaccine against fentanyl and demonstrated the feasibility of immunization to combat fentanyl-induced effects.

Differential Isotope Labeling by Permethylation and Reversed-Phase Liquid Chromatography-Mass Spectrometry for Relative Quantification of Intact Neutral Glycolipids in Mammalian Cells.

Probing the role of glycolipids in health and disease warrants development of practical strategies to determine these molecules at the intact structural level, namely to simultaneously characterize and quantify the glycan and lipid moieties without breaking the linkage between them. Herein we present such an approach utilizing differential isotope labeling and reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) for structural characterization and relative quantification of intact neutral glycolipids. In this approach, each individual sample and a pooled aliquot of each sample were permethylated using 12CH3I and 13CH3I, respectively, with the latter one serving as internal reference standard. The individual 12C-permethylated samples were spiked with equal amounts of the 13C-permethylated pooled sample and analyzed by RPLC-MS/MS. Permethylation not only increased the ionization efficiency of glycolipids but also facilitated structural characterization of both moieties. The ratio of the peak areas between the 12C- and 13C-labeled glycolipids served as surrogate measure of their relative concentrations. The coefficient of variation of the method was <6% measured across four representative glycolipids in five different ratios and triplicate experiments, after correction of natural isotopic distribution. When analyzing the low abundant glycolipids in total lipid extract, permethylation can dramatically reduce the analytical background by depleting most of the highly abundant ester-linked lipids. Application to conduritol B epoxide-, a ß-glucocerebrosidase inhibitor, treated RAW 264.7 cells demonstrated the practical utility of this method in profiling the temporal accumulation of different glycolipids. Overall, this methodology offers a practical LC-MS based identification and quantification strategy to advance intact glycolipids analysis in mammalian cells.

Fragmentation Behavior and Gas-Phase Structures of Cationized Glycosphingolipids in Ozone-Induced Dissociation Mass Spectrometry.

The role of cationization in the fragmentation behavior of glycoconjugates is amply documented in collisional activation techniques but remains less explored in ozone-induced dissociation mass spectrometry (OzID-MS). OzID-MS has been used to elucidate the location of carbon-carbon double bonds in unsaturated lipids. Previously, we demonstrated the structural analysis of unsaturated glycosphingolipids using OzID-MS by mass-selecting the [M+Na]+ adduct for fragmentation. In this work, we aimed to examine the effect of different adducts, namely [M+Na]+, [M+Li]+, and [M+H]+ on the OzID-MS fragmentation behavior of a representative unsaturated glycosphingolipid, LacCer d18:1/18:1(9Z). Our data show that [M+H]+ primarily undergoes dehydration followed by collision-induced dissociation-like loss of the headgroup, while [M+Li]+ and [M+Na]+ dissociate at the double bonds albeit with slightly different intensities of the resulting fragments. Using molecular mechanics and theoretical calculations at the semiempirical level, we report for the first time the gas-phase structure of cationized glycosphingolipids, which helps rationalize the observed bond cleavage. Our findings highlight that the type of adducts can influence gas-phase ion structure of glycosphingolipids and subsequently affect their fragmentation in OzID-MS. This study contributes to the growing body of knowledge on OzID-MS and gas-phase structures of ionized lipids and the findings have the potential to be extended to other more complex glycoconjugates.

Isobaric Labeling of Intact Gangliosides toward Multiplexed LC-MS/MS-Based Quantitative Analysis.

Gangliosides are sialic acid-containing glycosphingolipids recognized to play essential role in biological processes. Both the glycan and lipid structures influence their biological function and thus necessitate their determination as intact molecular species. To our knowledge, no multiplexed method for intact gangliosides currently exists. In this paper, we aimed to demonstrate an approach for isobaric labeling of intact gangliosides. Specifically, we carried out the rapid, chemoselective oxidation of sialic acid side chain in common ganglioside core structures using NaIO4 followed by ligation with a carbonyl-reactive isobaric tandem mass tag (TMT) reagent and subsequent RPLC-MS/MS analysis. Attachment of the isobaric label was observed to improve the ionization efficiency of complex gangliosides using electrospray ionization. Fragmentation of the resulting [M + 2H]2+ ions of TMT-labeled gangliosides provided information-rich spectra containing fragments from the glycan, lipid, and TMT reporter ions. This facile approach enabled simultaneous quantification of up to six samples as well as identification of glycan and lipid compositions in a single injection. As a proof-of-concept, using porcine brain total ganglioside extracts pooled at known ratios, we obtained overall sample-to-sample precision of <12% RSD and mean error of <10%. This showcased the great promise and feasibility of this strategy for high-throughput analysis of intact gangliosides in biological extracts.

Structural Analysis of Unsaturated Glycosphingolipids Using Shotgun Ozone-Induced Dissociation Mass Spectrometry.

Glycosphingolipids are essential biomolecules widely distributed across biological kingdoms yet remain relatively underexplored owing to both compositional and structural complexity. While the glycan head group has been the subject of most studies, there is paucity of reports on the lipid moiety, particularly the location of unsaturation. In this paper, ozone-induced dissociation mass spectrometry (OzID-MS) implemented in a traveling wave-based quadrupole time-of-flight (Q-ToF) mass spectrometer was applied to study unsaturated glycosphingolipids using shotgun approach. Resulting high resolution mass spectra facilitated the unambiguous identification of diagnostic OzID product ions. Using [M+Na]+ adducts of authentic standards, we observed that the long chain base and fatty acyl unsaturation had distinct reactivity with ozone. The reactivity of unsaturation in the fatty acyl chain was about 8-fold higher than that in the long chain base, which enables their straightforward differentiation. Influence of the head group, fatty acyl hydroxylation, and length of fatty acyl chain on the oxidative cleavage of double bonds was also observed. Application of this technique to bovine brain galactocerebrosides revealed co-isolated isobaric and regioisomeric species, which otherwise would be incompletely identified using contemporary collision-induced dissociation (CID) alone. These results highlight the potential of OzID-MS in glycosphingolipids research, which not only provides complementary structural information to existing CID technique but also facilitates de novo structural determination of these complex biomolecules. Graphical Abstract ᅟ.