Absolute Affinities from Quantitative Shotgun Glycomics Using Concentration-Independent (COIN) Native Mass Spectrometry.

Native mass spectrometry (nMS) screening of natural glycan libraries against glycan-binding proteins (GBPs) is a powerful tool for ligand discovery. However, as the glycan concentrations are unknown, affinities cannot be measured directly from natural libraries. Here, we introduce ncentration-dependent (COIN)-nMS, which enables quantitative screening of natural glycan libraries by exploiting slow mixing of solutions inside a nanoflow electrospray ionization emitter.

Comparative anti-herpes simplex virus type 1 and chemical profiling of and collected from North Africa.

Herpes simplex virus (HSV) can infect millions of people worldwide causing mild to life-threating infections. The current study demonstrates the first comparative anti-HSV type 1 activity and phytochemical investigation of and collected from Egypt and Libya. Liquid chromatography/mass spectrometry (LC/MS) analysis allowed the identification of 56 and 38 compounds in the Egyptian and Libyan ethanolic extracts, respectively, in addition to 46 and 50 compounds in the Egyptian and Libyan ethanolic extracts, respectively.

Fucosylated Human Milk Oligosaccharide Foraging within the Species Bifidobacterium pseudocatenulatum Is Driven by Glycosyl Hydrolase Content and Specificity.

Human milk enriches members of the genus in the infant gut. One species, Bifidobacterium pseudocatenulatum, is found in the gastrointestinal tracts of adults and breastfed infants. In this study, B.

In vitro biological activities and in vivo hepatoprotective role of brown algae-isolated fucoidans.

Brown seaweeds are rich in polysaccharides, such as fucoidan (FUC) which has shown beneficial effects in several medical conditions. The aim of the present study was to assess the antioxidant, anti-inflammatory, and hepatoprotective properties of Colpomenia sinuosa- and Sargassum prismaticum-isolated FUC in vitro and in vivo. The hot acid extraction method was used to isolate FUC from C.

Enhancement of phenolic content, antioxidant and cytotoxic activities of leaf and seed by suspension culture.

family is well known for a wide range of biological activities and a complex phytochemical composition. The current study investigates tissue culture protocols for leaves and seeds. For static culture initiation, Murashige and skooge (MS) as a basal medium with hormonal supply of (0-10 µM) of 2,4-dicholorophenoxy acetic acid and 6-benzyl aminopurine for seeds and leaves was employed.

Antibacterial activity of Centaurea pumilio L. root and aerial part extracts against some multidrug resistant bacteria.

Background: In the context of searching for potent, safe, natural antimicrobial agents to combate the global antimicrobial resistance (AMR) phenomenon, the current study evaluates for the first time ever, the broad-spectrum antimicrobial activity of essential oil (EO) and extracts from the rare wild plant Centaurea pumilio L.. It has tremendous ethnomedicinal values; its dried root is used as a fattening agent, a treatment for bad breath and diabetes, and screened for schistosomicidal activity.

Chemical constituents, Antibacterial and Acetylcholine esterase inhibitory activity of leaves.

The chemical constituents of were investigated. A new neolignan glycoside (1) in addition to nine known compounds were isolated. The acetylcholinesterase (AChE) inhibitory activity and antibacterial activity against methicillin-resistant (MRSA) of different fractions and isolates of were evaluated.

A novel purified Lactobacillus acidophilus 20079 exopolysaccharide, LA-EPS-20079, molecularly regulates both apoptotic and NF-κB inflammatory pathways in human colon cancer.

Background: The direct link between inflammatory bowel diseases and colorectal cancer is well documented. Previous studies have reported that some lactic acid bacterial strains could inhibit colon cancer progression however; the exact molecules involved have not yet been identified. So, in the current study, we illustrated the tumor suppressive effects of the newly identified Lactobacillus acidophilus DSMZ 20079 cell-free pentasaccharide against colon cancer cells.

Screening natural libraries of human milk oligosaccharides against lectins using CaR-ESI-MS.

Human milk oligosaccharides (HMOs) afford many health benefits to breast-fed infants, such as protection against infection and regulation of the immune system, through the formation of non-covalent interactions with protein receptors. However, the molecular details of these interactions are poorly understood. Here, we describe the application of catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) for screening natural libraries of HMOs against lectins.

High-Throughput Label- and Immobilization-Free Screening of Human Milk Oligosaccharides Against Lectins.

The intense interest in the mechanisms responsible for the beneficial effects of breast-feeding on infant health has created a significant need for analytical methods capable of rapidly identifying interactions between human milk oligosaccharides (HMOs) and their protein receptors. Currently, there are no established, high-throughput assays for the screening libraries of free (unmodified) HMOs against lectins. The present work describes a rapid and label- and immobilization-free assay, based on catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS), capable of simultaneously screening mixtures of free HMOs of known concentration for binding to lectins in vitro.

Novel Chromatographic Methods for Simultaneous Quantification of Fish and Wheat Germ Oils Mixture in Pharmaceutical Dosage Forms.

Two simple, direct and environment-friendly chromatographic methods, high-performance liquid chromatography (HPLC) and high-performance thin layer chromatographic (HPTLC), were developed for the determination of a binary mixture of fish oil (FO) and wheat germ oil (WGO), for the first time, in their pharmaceutical dosage forms with no need for any sample pretreatment. The HPLC separation was carried out using C-18 stationary phase with mobile phase of 15% formic acid (pH 6), methanol and acetonitrile through gradient-elution, 1.5 mL min-1 flow-rate and detection at 215 nm for FO and 280 nm for WGO.

Recognition of human milk oligosaccharides by bacterial exotoxins.

The affinities of the most abundant oligosaccharides found in human milk for four bacterial exotoxins (from Vibrio cholerae and pathogenic Escherichia coli) were quantified for the first time. Association constants (Ka) for a library of 20 human milk oligosaccharides (HMOs) binding to Shiga toxin type 2 holotoxin (Stx2) and the B subunit homopentamers of cholera toxin, heat-labile toxin and Shiga toxin type 1 (CTB5, HLTB5 and Stx1B5) were measured at 25°C and pH 7 using the direct electrospray ionization mass spectrometry assay. Notably, all four bacterial toxins bind to a majority of the HMOs tested and five of the HMOs (2'-fucosyllactose, lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II and lacto-N-fucopentaose III) are ligands for all four toxins.

Evaluation of a focused virtual library of heterobifunctional ligands for Clostridium difficile toxins.

A focused library of virtual heterobifunctional ligands was generated in silico and a set of ligands with recombined fragments was synthesized and evaluated for binding to Clostridium difficile toxins. The position of the trisaccharide fragment was used as a reference for filtering docked poses during virtual screening to match the trisaccharide ligand in a crystal structure. The peptoid, a diversity fragment probing the protein surface area adjacent to a known binding site, was generated by a multi-component Ugi reaction.

Screening carbohydrate libraries for protein interactions using the direct ESI-MS assay. Applications to libraries of unknown concentration.

A semiquantitative electrospray ionization mass spectrometry (ESI-MS) binding assay suitable for analyzing mixtures of oligosaccharides, at unknown concentrations, for interactions with target proteins is described. The assay relies on the differences in the ratio of the relative abundances of the ligand-bound and free protein ions measured by ESI-MS at two or more initial protein concentrations to distinguish low affinity (≤10(3) M(-1)) ligands from moderate and high affinity (>10(5) M(-1)) ligands present in the library and to rank their affinities. Control experiments were performed on solutions of a single chain antibody and a mixture of synthetic oligosaccharides, with known affinities, in the absence and presence of a 40-component carbohydrate library to demonstrate the implementation and reliability of the assay.

Mycobacteriophage cell binding proteins for the capture of mycobacteria.

Slow growing subsp. (MAP) causes a deadly condition in cattle known as Johne's disease where asymptomatic carriers are the major source of disease transmission. MAP was also shown to be associated with chronic Crohn's disease in humans.

Quantifying protein interactions with isomeric carbohydrate ligands using a catch and release electrospray ionization-mass spectrometry assay.

The application of a catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to quantify interactions between proteins and isomeric carbohydrate ligands is described. Absolute affinities for each ligand are determined from the abundance ratio of ligand-bound to free protein measured directly by ESI-MS and the relative abundances of the individual isomeric ligands, which are established by releasing the ligands, in their deprotonated form, from the protein using collision-induced dissociation (CID) and subjecting them to ion mobility separation (IMS) or another stage of CID to fragment the ions. Using Gaussian functions to represent the contributions of individual ligands to the arrival time distributions (ATDs) measured by IMS, the relative abundance of each ligand bound to the protein can be established.

Quantifying carbohydrate-protein interactions by electrospray ionization mass spectrometry analysis.

The development of analytical methods capable of characterizing carbohydrate-protein interactions, which are critical for many biological processes, represents an active area of research. Recently, the direct electrospray ionization mass spectrometry (ESI-MS) assay has emerged as a valuable tool for identifying and quantifying carbohydrate-protein complexes in vitro. The assay boasts a number of strengths, including its simplicity, speed, low level of sample consumption, and the unique ability to directly probe binding stoichiometry and to measure multiple binding equilibria simultaneously.

Quantifying ligand binding to large protein complexes using electrospray ionization mass spectrometry.

An electrospray ionization mass spectrometry (ESI-MS) method for quantifying protein-ligand complexes that cannot be directly detected by ESI-MS is described. The proxy protein ESI-MS method combines direct ESI-MS binding measurements with competitive protein-ligand binding. To implement the method, a proxy protein (P(proxy)), which interacts specifically with the ligand of interest with known affinity and can be detected directly by ESI-MS, is used to quantitatively monitor the extent of ligand binding to the protein of interest.

Reliable determinations of protein-ligand interactions by direct ESI-MS measurements. Are we there yet?

The association-dissociation of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small molecules, are critical events in many biological processes. The discovery and characterization of these interactions is essential to a complete understanding of biochemical reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein-ligand interactions in vitro.

Applications of a catch and release electrospray ionization mass spectrometry assay for carbohydrate library screening.

Applications of a catch and release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against target proteins are described. Direct ESI-MS measurements were performed on solutions containing a target protein (a single chain antibody, an antigen binding fragment, or a fragment of a bacterial toxin) and a library of carbohydrates containing multiple specific ligands with affinities in the 10(3) to 10(6) M(-1) range. Ligands with moderate affinity (10(4) to 10(6) M(-1)) were successfully detected from mixtures containing >200 carbohydrates (at concentrations as low as 0.

Binding of Clostridium difficile toxins to human milk oligosaccharides.

The binding of recombinant fragments of the C-terminal cell-binding domains of the two large exotoxins, toxin A (TcdA) and toxin B (TcdB), expressed by Clostridium difficile and a library consisting of the most abundant neutral and acidic human milk oligosaccharides (HMOs) was examined quantitatively at 25°C and pH 7 using the direct electrospray ionization mass spectrometry (ES-MS) assay. The results of the ES-MS measurements indicate that both toxin fragments investigated, TcdB-B1 and TcdA-A2, which possess one and two carbohydrate binding sites, respectively, bind specifically to HMOs ranging in size from tri- to heptasaccharides. Notably, five of the HMOs tested bind to both toxins: Fuc(α1-2)Gal(β1-4)Glc, Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc, Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)Gal(β1-4)Glc and Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc.

Quantifying labile protein-ligand interactions using electrospray ionization mass spectrometry.

A new electrospray ionization mass spectrometry (ES-MS) approach for quantifying protein-ligand complexes that are prone to in-source (gas-phase) dissociation is described. The method, referred to here as the reference ligand ES-MS method, is based on the direct ES-MS assay and competitive ligand binding. A reference ligand (L(ref)), which binds specifically to the protein (P), at the same binding site as the ligand (L) of interest, with known affinity and forms a stable protein-ligand complex in the gas phase, is added to the solution.

Gas phase stabilization of noncovalent protein complexes formed by electrospray ionization.

The use of gas phase additives to stabilize noncovalent protein complexes in electrospray ionization mass spectrometry (ES-MS) is demonstrated for two protein-ligand interactions, an enzyme-small molecule inhibitor complex, and a protein-disaccharide complex. It is shown that the introduction of gas phase imidazole into the ES ion source effectively protects gas phase protein-ligand complexes against in-source dissociation. The stabilizing effect of imidazole vapor is comparable to that observed upon addition of imidazole to the ES solution.