Gas Chromatography-Mass Spectrometry (GC-MS) Metabolite Profiling of Citrus limon (L.) Osbeck Juice Extract Evaluated for its Antimicrobial Activity Against Streptococcus mutans.

Purpose The present study aimed to determine the antimicrobial nature of juice extract against and to identify its metabolic profile by gas chromatography-mass spectrometry (GC-MS) technique. Materials and methods The cariogenic bacteria were procured from Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India, and revived on brain heart infusion (BHI) agar. The (L.

Metabolite Profiling of Bark Using Advanced LC/MS and GC/Q-TOFTechnology.

There is an urge for traditional herbal remedies as an alternative to modern medicine in treating several ailments. is one such plant, used traditionally to treat several diseases. In several reports, there are findings related to the use of this plant extract that demonstrate its therapeutic value.

Qualitative assessment of extractables from single-use components and the impact of reference standard selection.

The advent of single-use bioprocess systems used for the delivery, storage or manufacture of biopharmaceuticals has introduced a new potential source for extractables and leachables (E&L) as these systems are comprised of polymeric materials. Several industry working groups, the FDA and USP have issued guidance and draft guidance on E&L analyses for a variety of applications. These documents typically indicate that mass spectrometry should be applied for discovery of E&L's but provide little guidance as to the exact analytical methodology which should be used.

LC-MS-based serum metabolomic analysis reveals dysregulation of phosphatidylcholines in esophageal squamous cell carcinoma.

Unlabelled: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers with poor prognosis. Here, we carried out liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS)-based untargeted metabolomic analysis of ESCC serum samples. Statistical analysis resulted in the identification of 652 significantly dysregulated molecular features in serum from ESCC patients as compared to the healthy subjects.

Production of antipeptide antibodies.

Peptides (8-20 residues) are as effective as proteins in raising antibodies, both polyclonal and monoclonal with a titer above 20,000 easily achievable. A successful antipeptide antibody production depends on several factors such as peptide sequence selection, peptide synthesis, peptide-carrier protein conjugation, the choice of the host animal, and antibody purification. Peptide sequence selection is likely the most difficult and critical step in the development of antipeptide antibodies.

Matrix metalloprotease selective peptide substrates cleavage within hydrogel matrices for cancer chemotherapy activation.

To utilize biologic mechanisms to elicit controlled release in response to disease, protease-sensitive devices have been created. Hydrogels were created with pendant peptide-drug complexes. For the matrix metalloproteases (MMPs) examined, a length of six amino acids greatly improved the specificity of the peptide (k(cat)/K(m) approximately 2.

Capturing biotinylated proteins and peptides by avidin functional affinity electrophoresis.

Avidin functional affinity electrophoresis (AFAEP) is a variational method of affinity electrophoresis. In this technique, avidin is immobilized within a small area of the gel matrix by interaction with acrylamide and/or polyacrylamide either directly or through bifunctional linker glutaraldehyde during polymerization. Analytes can be heated with Tris-glycine sodium dodecyl sulfate (SDS) sample buffer so that biotinylated peptides/proteins are negatively charged and migrate electrophoretically towards the cathode through the avidin zone regardless of their isoelectric point (pI) values.

An improved protocol for coupling synthetic peptides to carrier proteins for antibody production using DMF to solubilize peptides.

We present an improved protocol for coupling synthetic peptides to carrier proteins. In this protocol, dimethyl-formamide is used as the solvent to solubilize peptides instead of phosphate-buffered saline (PBS) or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7).

G-protein-coupled receptor 1, G-protein Galpha-subunit 1, and prephenate dehydratase 1 are required for blue light-induced production of phenylalanine in etiolated Arabidopsis.

Different classes of plant hormones and different wavelengths of light act through specific signal transduction mechanisms to coordinate higher plant development. A specific prephenate dehydratase protein (PD1) was discovered to have a strong interaction with the sole canonical G-protein Galpha-subunit (GPA1) in Arabidopsis (Arabidopsis thaliana). PD1 is a protein located in the cytosol, present in etiolated seedlings, with a specific role in blue light-mediated synthesis of phenylpyruvate and subsequently of phenylalanine (Phe).

Characterization of oligosaccharide moieties of intact glycoproteins by microwave-assisted partial acid hydrolysis and mass spectrometry.

A method, which utilizes microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), to elucidate oligosaccharide composition of intact glycoproteins is presented here. Glycoproteins, such as ribonuclease B, avidin, alpha1-acid glycoprotein, and fetuin, are used as model systems to demonstrate this technique. Partial cleavage of oligosaccharides from whole intact glycoproteins with trifluoroacetic acid was observed after a short exposure to microwaves.

Oligosaccharide analyses of glycopeptides of horseradish peroxidase by thermal-assisted partial acid hydrolysis and mass spectrometry.

Thermal-assisted partial acid hydrolysis of the carbohydrate moieties of N-glycosylated peptides of horseradish peroxidase (HRP) is used to generate oligosaccharide cleavage ladders. These ladders allow direct reading of components of the oligosaccharides by mass spectrometry. Acid hydrolysis performed with 1.

Characterization of oligosaccharide moieties of glycopeptides by microwave-assisted partial acid hydrolysis and mass spectrometry.

Microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used to study oligosaccharide structures of glycopeptides. Tryptic N-glycosylated peptides of horseradish peroxidase, with MH+ ions at m/z 2533, 2612, 3355, 3673, and 5647, were used as test cases. Within a microwave exposure with trifluoroacetic acid of 2 min, partial cleavages of the oligosaccharides of these tryptic N-glycosylated peptides were observed.

Mass spectrometric detection of biotinylated peptides captured by avidin functional affinity electrophoresis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to detect biotinylated peptides captured by avidin functional affinity electrophoresis (AFAEP). Peptide samples loaded onto AFAEP were heated with sodium dodecyl sulfate to ensure that the peptides are negatively charged, and thus migrate electrophoretically toward the cathode through the embedded avidin zone in the middle of the gel. To detect the biotinylated peptides, the band containing the avidin-biotinylated peptide complexes was excised from a 7.

Capturing sodium dodecyl sulfate-treated protein antigens by antibody affinity electrophoresis.

Modifications to antibody affinity electrophoresis for improved detection of proteins have been developed. The bifunctional linker glutaraldehyde is added to the polyacrylamide gel solution for better incorporation of the bait antibody into a distinct region of a 10% w/v polyacrylamide gel. The addition of glutaraldehyde alleviates the need of an electrophoresis buffer with a specific pH.

Three-dimensional chemical structures by protein functionalized micron-sized beads bound to polylysine-coated silicone surfaces.

A novel method is described here that allows three-dimensional (3D) control of both chemistry and morphology by a series of wet chemical steps: the attachment of protein functionalized micron-sized beads onto a flat silicone surface that has been functionalized with a distinct chemical modification. Bovine serum albumin (BSA), laminin, or polylysine is covalently bound to 6.5-microm-diameter spherical beads.

Capturing SDS-treated biotinylated protein and peptide by avidin functional affinity electrophoresis with or without SDS in the gel running buffer.

Avidin functional affinity electrophoresis (AFAEP) is a new method of affinity electrophoresis. In this technique, bifunctional linker glutaraldehyde is added to the polyacrylamide gel solution to embed avidin within the gel matrix by interaction with the amino/amide groups. Samples are heated with triglycine sodium dodecyl sulfate (SDS) sample buffer to ensure that biotinylated proteins biotinylated peptides are negatively charged and migrate electrophoretically toward the cathode through the avidin zone regardless of their pI values.

Catching protein antigens by antibody affinity electrophoresis.

A new kind of affinity electrophoresis called antibody affinity electrophoresis is a technique used to capture protein antigens based on their interactions with specific monoclonal or polyclonal antibodies incorporated in the polyacrylamide gel. Polyclonal anti-glutathione-S-transferase (anti-GST), monoclonal anti-bovine serum albumin (anti-BSA), and polyclonal anti-human alpha-lactalbumin are embedded in distinct areas of a 7.5% native polyacrylamide gel.

Catching and separating protein ligands by functional affinity electrophoresis.

A new kind of affinity electrophoresis called functional affinity electrophoresis (FAEP) is a technique used to separate and/or capture proteins according to their functions in a native polyacrylamide gel. Protein A:immunoglobulin G, avidin:biotin, antibody:antigen, and concanavalin A:glycoprotein interactions are used to demonstrate this technique. Protein A, avidin, monoclonal anti-bovine serum albumin (BSA) antibody, and concanavalin A are embedded in distinct regions of a 7.

RGD and YIGSR synthetic peptides facilitate cellular adhesion identical to that of laminin and fibronectin but alter the physiology of neonatal cardiac myocytes.

In the mammalian heart, the extracellular matrix plays an important role in regulating cell behavior and adaptation to mechanical stress. In cell culture, a significant number of cells detach in response to mechanical stimulation, limiting the scope of such studies. We describe a method to adhere the synthetic peptides RGD (fibronectin) and YIGSR (laminin) onto silicone for culturing primary cardiac cells and studying responses to mechanical stimulation.

Derivatization of surface-bound peptides for mass spectrometric detection via threshold single photon ionization.

Chemical derivatization of peptides allows efficient F2 laser single photon ionization (SPI) of Fmoc-derivatized peptides covalently bound to surfaces. Laser desorption photoionization mass spectrometry using 337-nm pulses for desorption and 157.6-nm pulses for threshold SPI forms large ions identified as common peptide fragments bound to either Fmoc or the surface linker.

GRGDSP peptide-bound silicone membranes withstand mechanical flexing in vitro and display enhanced fibroblast adhesion.

Mechanobiological studies of cardiac tissue require devices that allow forces to be exerted on cells in vitro. Silicone elastomer is often used in these devices because it is flexible and transparent, permitting optical imaging of the cells. However, native untreated silicone is hydrophobic and is unsuitable for cell culture.