356 research outputs found
Fig. 2. Observed HMBC correlations for 1–2 in Biofilm blocking sesquiterpenes from Teucrium polium
No full textFig. 2. Observed HMBC correlations for 1–2 as indicated by the arrows.Published as part of <i>Elmasri, Wael A., Hegazy, Mohamed-Elamir F., Aziz, Mina, Koksal, Ekrem, Amor, Wail, Mechref, Yehia, Hamood, Abdul N., Cordes, David B. & Paré, Paul W., 2014, Biofilm blocking sesquiterpenes from Teucrium polium, pp. 107-113 in Phytochemistry 103</i> on page 109, DOI: 10.1016/j.phytochem.2014.03.029, <a href="http://zenodo.org/record/10489548">http://zenodo.org/record/10489548</a>
Analysis of mucosal mucins separated by SDS-urea agarose polyacrylamide composite gel electrophoresis
No full textEfficient separation of mucins (200?kDa2?MDa) was demonstrated using gradient SDS agarose/polyacrylamide composite gel electrophoresis (SDS-AgPAGE). Inclusion of urea (SDS-UAgPAGE) in the gels casting were shown to have no effect on the migration of mucins in the gel and allowed casting of gel at room temperature. This simplified the procedure for multiple casting of agarose polyacrylamide gradients and increased reproducibility of these gels. Hence, the implementation of urea makes the technique applicable for high throughput isolation and screening of mucin oligosaccharides by LC-MS after releasing the oligosaccharides from isolated, blotted mucin subpopulations. It was also shown that the urea addition had no effect on other supporting applications such as western and lectin blotting. In addition, identification of the mucin protein after tryptic digestion and LC-MS was possible and no protein carbamylation due to the presence of urea in the gel was detected. LC-MS software developed for metabolomic analysis was used for O-linked oligosaccharide detection and differential display of various mucin samples. Using this method, heterogeneous glycosylation of mucins and mucin-type molecules isolated by SDS-AgPAGE and SDS-UAgPAGE was shown to consist of more than 80 different components in a single band, and in the extreme cases, up to 300500 components (MUC5B/AC from saliva and sputum and). Metabolomic software was also used to show that the migration of mucin isoforms within the gel is due to heterogeneous size distribution of the oligosaccharides, with the slower migrating bands enriched in high-molecular-weight oligosaccharides
High-throughput lectin magnetic bead array-coupled tandem mass spectrometry for glycoprotein biomarker discovery
No full textAlterations in protein glycosylation occur during development and progression of many diseases, hence glycomics and glycoproteomics have emerged as important tools in glycobiomarker discovery. High-throughput glycan profiling can now be achieved with the recent developments in MS-based techniques. To enable identification and rapid monitoring of glycosylation changes in serum proteins, we developed a semi-automated high-throughput glycoprotein biomarker discovery platform termed lectin magnetic bead array-coupled tandem mass spectrometry (LeMBA-MS) which includes (i) effective single-step serum glycoprotein isolation using a panel of 20 individual lectin-coated magnetic beads in microplate format, (ii) on-bead trypsin digestion, and (iii) nanoLC-MS/MS with lectin exclusion list. With use of appropriate sequence databases, LeMBA-MS can detect glycosylation changes regardless of the species. By spiking known amounts of titrated ovalbumin to a serum sample, we report nanomolar sensitivity, and linearity of response of LeMBA-MS using concanavalin A-coupled beads. Neuraminidase treatment led to reduction of binding to sialic acid-binding lectins. Interestingly, we found that desialylation caused increased binding of haptoglobin and hemopexin to mannose-specific lectins, pointing to the importance of identifying a signature of lectin-binding. High-throughput LeMBA-MS to generate glycosylation signatures will facilitate glycobiomarker discovery. LeMBA can be coupled to down-stream detection platforms for validation, making it a truly versatile platform
Method development for characterization and quantification of glycomics, proteomics, and glycoproteomics by LC-MS/MS
No full textProtein glycosylation is one of the most prevalent and crucial post-translational modifications (PTMs). It has been reported that over 50% of mammalian proteins undergo glycosylation. This modification plays a pivotal role in various biological functions, including cell-cell signaling, protein degradation, and immune response. Despite its significance, studying glycosylation poses challenges due to factors such as low abundance, complex microheterogeneity of glycosylation sites, and the low ionization efficiency of glycopeptides.
To overcome the challenges in glycosylation studies, recent advances in sensitive techniques have been developed. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) stands out as a powerful approach widely employed for the identification, characterization, and quantification of protein glycosylation. This technique facilitates in-depth analysis and provides valuable insights into the intricate world of glycosylation.
In this dissertation, a metabolic isotopic labeling method was designed to simultaneously label both glycan moiety and peptide backbone, called GlyProSILC. It enables quantification of glycomics, proteomics, and glycoproteomics by using the same batch of cell lines. Through the employment of GlyProSILC and differential labeling of glycan and peptide, a multiplex glycoproteomic approach was developed to facilitate direct comparison of glycoproteomic quantitation and enhance throughput. The analysis of sialylated glycopeptides, especially sialic acid linkage isomers, remains challenging due to low abundance and isomer similarity. Here, a derivatization method was illustrated to efficiently separate sialylated glycopeptide isomers by utilizing regular reverse phase column and gradient time. A glycopeptide capture-and-release method was introduced for the selective enrichment of sialic acid-containing glycopeptides. Different MS acquisition methods for enhancing the quantification of glycopeptides were also investigated and compared.Embargo status: Restricted until 06/2030. To request the author grant access, click on the PDF link to the left
Exploring the Molecularity of Olfactory and Gustatory Perceptions
No full textThe sense of smell is crucial for a species' fight or flight response. It ensures that an
animal can survive and feed itself. Smell and taste are strongly connected. The sense of
smell contributes to 75-95% of our overall perceived flavor. Humans have just four
tastes: sweet, sour, salty, bitter, and in some populations, umami, yet we can taste
multiple different flavors because smell and taste combine.
The approach we use for this study explores the intermolecular contributions to the
perception of specific odors and tastes. This approach goes beyond associating specific
chemosensory perceptions with a molecule’s gross structural features such as aromatic
rings, aliphatic rings or the chain lengths of these compounds. Our approach goes a step
further, focusing on specific atom pairs, whether bonded or non-bonded (remote) within
compounds. We identified structural-electronic signatures of these atom pairs, which we
associate with specific odors and tastes. For an atom pair, the structure is the interatomic
distance. The electronic features are determined by Nuclear Magnetic Resonance (NMR)
chemical shifts that indicate the atom’s electronic environment. This thesis explores
how atom pairs with the same structure-electronic signatures are reproducible in
molecules that exhibit the same odor and taste responses.
We identified 19 molecules associated with the smell brown and 18 molecules associated
with the taste brown–the determinations for these having been made by expert smellers.
These “Brown” odorants and tastants were associated with additional smells and tastes,
which we categorized into cooked, vegetable and sweet. We extended the exploration of
these signatures from the linear (atom pairs) to the planar (atom triads) by calculating
reproducible angles.
This work explores the molecularity of odorants and tastants for smell, taste, for five
molecules that are common for brown taste and smell, and for molecules that were
exclusively identified as being associated with brown taste and smell. This notion allows
us to explore the how taste is influenced by smell at the molecular level.
Our future plans involve extending these signatures in the third dimension by identifying
reproducible four- atom tetrads by calculating dihedral angles between these atoms.
This work also adds a unique perspective of how smell and taste combine not only with
visual perception but are the result of experiential memory.Embargo status: Restricted until 06/2025. To request the author grant access, click on the PDF link to the left
Development of quantitative analytical methods for the reliable assessment of the roles of glycans in the development and progression of breast cancer
No full textThough there are many post translational modifications made to proteins, glycosylation is one of the most common. Glycans play important biological roles and have key functions in processes like cell signaling and cell adhesion. The aberrant expression of glycans has been linked to diseases such as various types of cancer. Differences in glycan expression are key to discovering possible biomarkers for the diseases and the variations. While there are many ways in which glycans can be analyzed, liquid chromatography mass spectrometry is method that will be the main focus for the applications in this work.
As ionization energy can fluctuate between sample runs and there is a need for high throughput methods of glycomic analysis, multiplexing is becoming a promising methodology for running glycomic analysis. There are many multiplexing strategies but many face complex issues like low reporter ion yield and labeling overlap while few stabilize the labile moieties in glycans. An 8-plex method using CH3I, CH2DI, CHD2I, CD3I, 13CH3I, 13CH2DI, 13CHD2I, 13CD3I that was previously published by our group avoids these problems and herein this work the method has been expanded to 16-plex through the use of 18O labeling. The 16-plex method was tested on standard glycoproteins through the use of the C18 liquid chromatography column the original 8-plex method was designed for. However, this work also tests to see if the 16-plex method can work on micro pillar array columns.
As mentioned, aberrant glycosylation has been linked to cancer. This work specifically looks into the significant expression changes between the four major subtypes of breast cancer: luminal A, luminal B, HER2 positive, and triple negative. One hundred and twenty breast cancer samples were tested, thirty each coming from ten patients of each cancer subtype. The significant glycan expression differences between the subtypes were explored and possible candidates for glycan based biomarkers were found with which the cancer subtypes may be able to be differentiated.
This work also outlines the future work needed for the projects discussed herein.Restricted until 06/2027. To request the author grant access, click on the PDF link to the left
Belgian Mosque Recognition Data, Anonimyzed
No full textThis dataset was originally used to study the recognition rates of mosque-communities in Belgium. Each row represents a unique Belgian mosque, which has been anonymized. For each unit, I include a regional indicator, ethnicity indicator, network indicator, and recognition status indicator. All variables from the original dataset that could potentially be used to de-anonymize a mosque have been excluded, but may be available upon request from the author. The data made available has been sourced from publicly available reports, such as those produced by the Executive for Muslims in Belgium
Sensitive glycopeptide analysis by enrichment and LC-MS/MS
No full textGlycosylation plays an important role in many biological processes, and aberrant glycosylation has been found to play a critical role in many diseases and cancer progression. Studies of glycoproteins take two approaches for full characterization in that both the peptide backbones and glycan structures must be obtained during analysis. Changes in glycosylation during disease progression are key to discovering biomarkers for diagnosis. Glycoproteomic strategies for identification and quantification are monitored through liquid chromatography separation interfaced with tandem mass spectrometry in this work. Glycopeptides, however, are abundantly low and express poor ionization efficiencies. Enrichment techniques are needed to capture glycopeptides and remove interfering species.
Three HILIC enrichment parameters were tested on cell line HTB-131 and compared to the optimized 5mg Cotton HILIC protocol for blood serum sample analysis. Tests were conducted with various cotton amounts, buffer solutions, and incubation times. For sufficient glycopeptide capturing and peptide removal, it was found that 5 mg of cotton, a washing buffer consisting of 90% ACN /0.1% FA, and one hour incubation time provided the best results. Under these conditions, 715 peptides were identified while 206 glycosylation sites were identified. Some sample loss was observed, but an approximate four times increase in signal was observed utilizing these enrichment conditions.
In breast cancer metastasis to the brain the blood brain barrier, a region of the brain that regulates the entrance of ions, diseases, toxins, etc., fails to block breast cancer cells from crossing. Here, we present a study of identifying and quantifying the glycosylation of six breast and brain cancer cell lines using hydrophilic interaction liquid chromatography (HILIC) and electrostatic repulsion liquid chromatography (ERLIC) enrichments and LC-MS/MS analysis. Qualitative and quantitative analyses of N-linked glycosylation were performed by both enrichment techniques for individual and complementary comparison. Cancer glycopeptide biomarkers were identified and confirmed by chemometric and statistical evaluations. A total of 497 glycopeptides were characterized with 401 common glycopeptides (80.6% overlap) determined from both enrichment techniques. HILIC enrichment yielded 320 significant glycopeptides out of 494 unique glycopeptides, and sequential HILIC-ERLIC enrichment yielded 212 significant glycopeptides out of 404 unique glycopeptides. The results provide the first comprehensive glycopeptide listing for these six cell lines.
Methods for surface analysis of biological samples include Secondary Ion Mass Spectrometry (SIMS), Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI MS), and Liquid Extraction Surface Analysis (LESA). LESA techniques were utilized in a previous study published from our lab. In this study on-tissue digestion was conducted for glycomic profiling of mouse brain tissue sections. Manual deposition of PNGase F enzyme was spotted on to regions of the tissue. A collection of the released glycans and analysis by LC-MS resulted in the identification of 43 glycan structures. The success of this experimental design led to the development of an automated instrument for enzyme deposition termed ‘Microliter Deposition Device.’
Different materials were tested for the creation of masks. These masks functioned as stencil-like templates for enzyme deposition by creating wells/holes of fixed diameters. Polyetheretherketone (PEEK), polyoxymethylene (POM; Delrin), and polytetrafluoroethylene (PTFE, Teflon) were the materials tested. It was found that the Teflon mask with the circular holes packed in a hexagonal pattern provided the most efficient sealing. The second generation microliter deposition device was then constructed to achieve automated on-tissue digestion. The new device had three step motors for the movement in the x, y, and z directions. It included fixed locations for the mask and well plate positioning. The device is controlled by a LabVIEW VI of 30 stacked sequences. The capabilities of this new device, include deposition and withdrawing control, syringe pump control, temperature control, increased precision, and increased spatial resolution. The goal of this project is to implement the device for on-tissue digestion and analysis eventually for imaging mass spectrometry (IMS)
Flowing atmospheric pressure afterglow drift tube ion mobility spectrometry evidence discrimination
No full textDrift Tube Ion Mobility Spectrometry (DTIMS) is a gas-phase analytical separation method allows separation of chemical compounds based on the individual mobilities as they travel through a uniform electric field and collide with neutral gas molecules. DTIMS features advantages including excellent detection limits (ppb), fast response, simplicity in design, and portability. It has been widely used for detection of chemical warfare agents, narcotics, environmental contaminations, quality control, and biological applications.
Ionization source is one of the main sections in DTIMS which ionizes neutral analyte molecules prior to any separation. Ionization sources such as Corona Discharge (CD), Electrospray (ESI), Radioactive (63Ni) been coupled to DTIMS; however, ionization of all forms of compounds (solid, liquid, gas) are not possible with the aforementioned ionization sources. Plasma-based Ambient Desorption ionization sources (ADI) like Low-Temperature Plasma (LTP) and Direct Analysis In Real Time (DART), permits direct desorption/ionization of compounds with no to minimal sample preparation. However, obtained plasma gas temperature for desorption and abundance of charged species are not sufficient for a wide range of compounds with low volatility. Flowing Atmospheric-Pressure Afterglow Discharge (FAPA) is another plasma-based ADI source not only provides plasma gas temperatures higher than LTP and DART but also wider ranges of charged species are generated through more available ionization mechanism.
In chapter one, different aspects of DTIMS including theories, experimental part, and preliminary results were discussed. DTIMS was designed and constructed, then optimization of several parameters including drift gas flow rate and BNG injection times was done. Initial IMS spectra were obtained and improved in terms of long background observed in the peaks.
In chapter two, the flowing atmospheric-pressure afterglow (FAPA) ambient desorption/ionization source has been coupled with stand-alone Drift Tube Ion Mobility Spectrometry (DTIMS) for the first time. A tip repeller electrode, modified to allow higher bias potential still below the Townsend’s breakdown, was implemented at the FAPA/DTIMS interface to overcome the opposing potentials and facilitate ion transmission. The effect of the lab-built DTIMS and FAPA’s operating conditions (such as plasma voltage, current, gas flow rate, repeller’s potential and positioning, FAPA orientation, etc.) on the signal of selected analytes was studied, for both gas-phase injection and desorption. The FAPA reactant ion peak (RIP) reduced mobility coefficient (K0) corresponds to protonated water clusters (H2O)nH+. The FAPA-DTIMS spectra of several selected compounds showed that their K0 agrees with literature values. Moreover, quantitative characterization of acetaminophen and 2,6-di-tert-butylpyridine (2,6-DTBP) based on desorption or gas-phase injection yield limits of detection (LODs) of 0.03 μg and 18 ppb, respectively.
In chapter three, a field-switching method obviates the needs of Bradbury-Nielsen Gate (BNG) was proposed for FAPA-DTIMS. In this geometry, packet of ions are formed with applying pulses to the repeller. In this work, all repeller and FAPA’s operating conditions were optimized. A qualitative analysis of selected compounds with a wide range of mobilities were investigated to assess the capability of the proposed geometry for identification and determination of chemical compounds. A quantitative analysis of two compounds including 2,6-Di-Tert-Butylpyridine (2,6-DTBP) and acetaminophen was done, where around 6 and 10 times better limit of detections (LODs) were obtained, respectively. The Gate Depletion Effect (GDE) discriminates against the low mobility ions was shown. Field-switching repeller FAPA-DTIMS, improves the obtained peak intensities for lower mobility ions, then reduces the GDE due to lack of blocking field in BNG
Sensitive glycomics for understanding biological attributes of glycans associated with proteins
No full textGlycosylation of proteins, an important posttranslational modification (PTM) of proteins, provides extreme diversity of proteins, which have a great number of functions in biological and physiological process of eukaryotic cells. Moreover, it is of no surprise that aberrant glycosylation of proteins has been associated with all kinds of human disease, especially cancers. So it is of great pathological and therapeutic significance to accurately and comprehensively profile glycans of glycoproteins, on which my research is focused. A prototype of the microfluidic device is developed for sensitive glycomics, and the device is called Glycan Chip, which is composed of an immobilized enzyme reactor (IMER) and a porous graphitized carbon (PGC) column.
The first part of my job was to optimize the solid matrix of enzyme reactors. The solid matrix is made of poly (glycidylmetharylate-co-ethylene dimethacrylate) [poly (GMA-co-EDMA). The polymerization of the matrix, traditionally initiated by water bath incubation, was activated by microwave and the reaction time was reduced from 24h to 20min. The resultant polymeric monolith shows similar morphology with that made from the traditional method. Moreover, the reproducibility of microwave activation for the reaction proved ideal by fabrication of 5 replicates of polymeric monolith.
The second part of my research was to fabricate IMER in capillaries to test the activity of immobilized enzymes. Model glycoproteins, such as ribonuclease B, fetuin, and porcine thyroglobulin were digested by IMERs, and it shows that fast and efficient release of glycans can be achieved in about 5 min. Combined with MALDI-MS, which is capable of fast first screening of glycans, IMERs can provide glycomic profiling with high turnover. This is of great advantage in the case of a large number of samples.
The last part of my work focused on the development of Glycan Chip, a prototype of a microfluidic device. The integrated PGC column can purify the released glycans efficiently, which in turn facilitates the sensitive detection. Interfaced with ESI-MS, which is more sensitive than MALDI-MS, IMER has the capability of accurately profiling glycans of complex samples. In addition, the portable chips are convenient for liquid control with the aid of check valves and have potential in field application
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