Innovative methods to advance the analysis of intact glycolipids by mass spectrometry

The common etiological feature among the majority, if not all, of human diseases, is the impairment of crucial cellular events stemming from complex reactions at the molecular level. Precise understanding of these processes, such as structure-function relationships between biomolecules, could help guide the design of successful clinical interventions to prevent or halt diseases. Consequently, it is important to measure and characterize biomolecules at the finest possible granularity. Glycolipids, biomolecules comprised of a sugar (glycan) head and a lipid tail, serve as one of the key components of the cell membrane. Aberrant glycolipid metabolism is a hallmark of many pathologies, which accentuates their importance in disease pathogenesis. Despite their vital role, our present understanding of glycolipid biology remains incomplete, partly because of the dearth of technologies available to study them. Mass spectrometry (MS) has inarguably fueled much of our current knowledge on glycolipids. But, contemporary methods using this platform are not powerful enough to either detect low abundant glycolipids or elucidate subtle structural details that could facilitate uncovering of their precise biological functions. Often, the glycan and lipid components are analyzed separately, making correlation between structure and biological function impossible. Recent evidences have showcased that both glycan and lipid moieties dictate the overall function of glycolipids, which underlines the importance of structural analysis at the intact molecule level. In this respect, this dissertation was conceived to address the following aims, 1) To develop innovative MS methodologies to measure and characterize glycolipids in its intact, native form; and 2) To apply the developed methods in reasonably complex biological mixtures. Each method developed here is in-line with the following objectives: 1) to enhance detectability of glycolipids by increasing ionization efficiency, 2) to facilitate structural analysis, and 3) to improve quantification of glycolipids with potential for high-throughput applications. Both aims were addressed in four standalone projects that employed both commercially available standard glycolipids and complex biological samples. The first two projects sought to establish a method to determine double bond locations in unsaturated glycolipids. We exploited ozone-induced dissociation MS (OzID-MS), a relatively new fragmentation technique that uses ozone gas inside the collision cell of MS in lieu of conventional inert gas. Ozone reacts selectively to carbon-carbon double bonds, as such, the products of the ozonolysis in situ could be detected by MS and being used to locate the double bonds. Using complex bovine brain sample, this method revealed low abundant glycolipids, mostly isomers and isobars, that are otherwise non-detectable and non-distinguishable when conventional methods are used. We also discovered that the use of different adducts, such as [M+Na]+, [M+Li]+, and [M+H]+, could provide distinct OzID-MS patterns. Thus, to rationalize the observed OzID-MS data, theoretical calculations were performed to establish the structures of ionized glycolipids in the gas-phase. The in silico generated models were consistent with the experimentally observed fragmentation patterns. Overall, these projects emphasized that innovative approaches like OzID-MS could uncover previously unknown molecular species in a complex sample and the use of different adducts could provide distinct levels of structural detail. The third project aimed to establish a method that exploits a relatively new approach to quantitation called isobaric labeling, a technique that has been extensively used in proteomics and glycomics fields as a multiplexed analytical tool. It involves the covalent attachment of a molecular tag to an analyte that generates reporter ions when fragmented in MS. The intensities of the reporter ions serve as surrogate measures of their relative concentration in the sample. Because this tag only reacts with a reactive aldehyde or ketone, which is absent in native glycolipids structures, we first employed a chemoselective oxidation approach to introduce a reactive site in the intact glycolipids, using sialic acid-containing glycolipids called gangliosides as a model. When applied to complex porcine brain total lipids extract, this method not only enabled multiplexed analysis of up to six independent samples and improved sensitivity of gangliosides by two orders, but also provided rich spectra that facilitated the structural analysis of both the sugar head group and lipid backbone. The fourth project addressed the increasing demand for heavy isotope labeled internal standards, which are currently limited and costly, while improving the detectability, facilitating structural characterization, and enabling multiplexed analysis. In this project, we employed permethylation, a reaction that converts active protons in the molecule to a methyl group using methyl iodide. In this so-called differential isotope labeling approach, using methyl iodide with either light (12C) or heavy (13C) carbon isotopes, two different samples were separately labeled, one of which is a pooled aliquot of individual samples, then mixed and analyzed by LC-MS. The pooled sample, labeled with heavy carbon isotope served as a universal internal standard which was spiked to individual samples at constant amount. Samples were analyzed using reversedphase liquid chromatography mass spectrometry (RPLC-MS), and the resulting peak areas were used to calculate the 12C/13C ratio as a surrogate measure of the relative concentration of analytes in each sample. Using an in vitro model of Gaucher’s disease, characterized by accumulation of neutral glycolipids, temporal changes in glycolipid profile were measured and each glycolipid was annotated using retention time and MS/MS fragmentation. [This abstract has been edited to remove characters that will not display in this system. Please see the PDF for the full abstract.]]]> 2019 Glycolipids Gangliosides Mass spectrometry English http://libres.uncg.edu/ir/uncg/f/Barrientos_uncg_0154D_12835.pdf oai:libres.uncg.edu/26787 2019-08-19T15:07:35Z UNCG The Triumvirate: Effective Communication Across Publisher, Library, and Discovery Channels Bernhardt, Beth NC DOCKS at The University of North Carolina at Greensboro <![CDATA[In examining data transfer challenges from both librarian and publisher perspectives, this article based on a NC Serials Conference presentation calls for proactive industry communication across publisher, library, and discovery service channels. The authors discuss the importance of collaborative communication across the industry

Development and optimization of a MALDI-QTOF MS Method for the Analysis of Glucans from Daedalea quercina

This study demonstrated the applicability of Maltooligosaccharides (MOS) from a commercial beer as cheap and readily-available calibrant and model compounds for the optimization of MALDI-MS and MS/MS analysis of glycans in a Quadrupole Time-of-Flight (Q-TOF) MS platform. Important parameters such as homogeneity of matrix, in-source fragmentation and collision energy were investigated. The optimized methodology was used to characterize the glycan from the bioactive fraction of Daedalea quercina. In this study, we report for the first time the extraction, isolation, and the proposed structure of a polysaccharide from the fruiting bodies of D. quercina. The monosaccharide composition was mainly glucose as identified using GC-MS of the isolate. FTIR-ATR spectroscopy showed that DQW1Pa1 has a β configuration. The average molecular weight of this β-glucan obtained using size exclusion chromatography was 1.6 x 104 Da, consistent with glucans derived from mushrooms of the order Polyporaceae. MALDI-QTOF MS/MS was carried out to identify the linkage and connectivity of the glucose units. Collision Induced Dissociation (CID) of selected parent ions of different oligosaccharide lengths showed the presence of characteristic glycosidic bond cleavages Bn/Cn and the linear backbone by 1-6 linkage, cross-ring fragment, 0,3An. Presence of branching unit was identified from high intensity 0,3A4 fragment and verified from diagnostic ion of [D] and [D-H2O] types. To confirm the linkage assignment obtained using the developed and optimized MALDI-QTOF MS/MS method, DQW1Pa1 was subjected to methylation analysis. Results showed the presence of 1-3, 1-6, 1- and 1-3-6 linked glucose in the order of decreasing abundance, respectively. The repeating unit of isolate DQW1Pa1 was deduced as 1-3 linked linear glucose backbone with branches composed of three 1-3 linked glucose units linked to backbone by 1-6 linkage of approximately 7%

Extraction, isolation and MALDI-QTOF MS/MS analysis of β-D-Glucan from the fruiting bodies of Daedalea quercina

© 2016 Elsevier B.V. We report for the first time the extraction, isolation, and the proposed structure of a polysaccharide from the fruiting bodies of Daedalea quercina. The monosaccharide composition of D. quercina isolate (DQW1Pa1) was mainly glucose as identified using GC–MS. FTIR-ATR spectroscopy and absolute configuration studies showed that this polysaccharide is a β-D-glucan. Its average molecular weight obtained using size exclusion chromatography was 1.6 × 104 Da, consistent with glucans derived from the order Polyporaceae. MALDI-QTOF MS/MS was carried out to identify the linkage and connectivity of the glucose units. Collision Induced Dissociation (CID) of selected parent ions of different oligosaccharide lengths showed the presence of characteristic glycosidic bond cleavages Bn/Cn, the linear backbone by 1-6 linkage, and the cross-ring fragment, 0,3An. Presence of branching unit was identified from high intensity 0,3A4 fragment and verified from diagnostic ion of [D] and [D-H2O] types. To confirm the linkage assignment obtained using MALDI-QTOF MS/MS, DQW1Pa1 was subjected to methylation analysis. Results showed the presence of 1-3, 1-6, 1- and 1-3-6 linked glucose in the order of decreasing abundance, respectively. The repeating unit of isolate DQW1Pa1 was deduced as 1-3 linked linear glucose backbone with branches composed of three 1-3 linked glucose units connected to backbone by 1-6 linkage

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 &gt;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.</p

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