Metabolite Profiling of Alangium salviifolium 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. Alangium salviifolium 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. However, very few attempts have been made to identify the extensive metabolite composition of this plant. Here, we performed metabolite profiling and identification from the bark of A. salviifolium by extracting the sample in organic and aqueous solvents. The organic and aqueous extracts were fraction-collected using the Agilent 1260 Analytical Scale Fraction Collection System. Each of the fractions was analyzed on Liquid Chromatogaphy/Quadrupole Time-of-Flight LC/Q-TOF and Gas Chromatography/Quadrupole Time-of-Flight GC/instruments. The Liquid Chromatography/Mass Spectrometry (LC/MS) analyses were performed using Hydrophilic Ineraction Liquid Chromatography (HILIC), as well as reversed-phase chromatography using three separate, orthogonal reverse phase columns. Samples were analyzed using an Agilent Jet Stream (AJS) source in both positive and negative ionization modes. The compounds found were flavonoids, fatty acids, sugars, and terpenes. Eighty-one secondary metabolites were identified as having therapeutic potential. The data produced was against the METLIN database using accurate mass and/or MS/MS library matching. Compounds from Alangium that could not be identified by database or library matching were subsequently searched against the ChemSpider) database of over 30 million structures using MSMS data and Agilent MSC software.In order to identify compounds generated by GC/MS, the data were searched against the AgilentFiehn GCMS Metabolomics Library as well as the Wiley/NIST libraries.

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. Alternatively, if the behavior of the biotinylated analytes is known, the SDS treatment is not required. The polarity of the electrodes is set such that biotinylated analytes migrate electrophoretically through the avidin zone. This technique can work with or without SDS in gel running buffer. The AFAEP method allows the capture and concentration of biotinylated peptides/proteins. The values of this technique stem from a combination of merits of polyacrylamide gel electrophoresis and affinity technology.

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. We investigated the extractable profiles of a model single-use bioprocessing system consisting of a single-use bioprocess bag, connector tubing, and a hydrophilic disk filter including filter housing. Extractions were performed in water, ethanol, ethanol/water (50:50) and saline solutions. Extracts were analyzed using a stepwise analytical methodology including a variety of screening and mass spectrometry methods We then used this model system to demonstrate the use of recursive feature finding to automatically detect unique extractables followed by statistical filtering to focus on differentially present extractables which were above the analytical evaluation threshold (AET). We further show the significant affects of standard selection on the number of compounds determined to be above AET when reducing liquid chromatography-mass spectrometry (LC/MS) data. A relative response factor database consisting of 14 structurally diverse commercially available polymer additives was used to arrive at an LC/MS identification threshold. The results of this study demonstrate that significant care should be taken when selecting standards for LC/MS analysis to avoid under reporting of extractables and leachables.

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). Additionally, the last desalting or dialyzing step to remove uncoupled peptides as in the traditional method is eliminated. Finally, 3 ml of 0.1 M ammonium bicarbonate is added to the carrier protein conjugated peptide solution to help the lyophilization process. Coupling of Cys-containing synthetic peptides to keyhole limpet hemocyanin or bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester are used as the test cases. This method produces high-quality antipeptide antibodies. Also, compared to the traditional method, this procedure is simpler and useful for peptides with solubility problems in PBS or 6 M guanidine-HCl.

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 1.4, 3.1, 4.5, or 6.7M trifluoroacetic acid at 37, 65, or 95 degrees C for 30min to 24h hydrolyzed mainly the oligosaccharide units of glycopeptides with least peptide bond or amino acid side chain hydrolysis. Tryptic N-glycosylated peptides from HRP with molecular weights of 2533, 2612, 3355, 3673, and 5647Da were used as test systems in these experiments. Data showed that the most labile group of oligosaccharides is the fucose (Fuc) and the majority of the end cleavage products are peptides with one or no N-acetylglucosamine (GlcNAc) residue linked to Asparagine (Asn). Additionally, the data agree with previous reports that glycopeptides 3355 and 3673Da carry an oligosaccharide (Xyl)Man3(Fuc)GlcNAc2, glycopeptide 5647Da carries two oligosaccharides (Xyl)Man3(Fuc)GlcNAc2, and glycopeptides 2612 and 2533Da carry (Xyl)Man3GlcNAc2 and (Fuc)GlcNAc, respectively. However, the glycosylation site of the 2612Da peptide at Asn286 is partially occupied. This method is particularly useful in identifying glycopeptides and obtaining monosaccharide compositions of glycopeptides.

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

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. Phosphatidylcholines were identified as a major class of dysregulated metabolites in this study suggesting potential perturbation of phosphocholine metabolism in ESCC. By using a targeted MS/MS approach both in positive and negative mode, we were able to characterize and confirm the structure of seven metabolites. Our study describes a quantitative LC-MS approach for characterizing dysregulated lipid metabolism in ESCC. BIOLOGICAL SIGNIFICANCE: Altered metabolism is a hallmark of cancer. We carried out (LC-MS)-based untargeted metabolomic profiling of serum from esophageal squamous cell carcinoma (ESCC) patients to characterize dysregulated metabolites. Phosphatidylcholine metabolism was found to be significantly altered in ESCC. Our study illustrates the use of mass spectrometry-based metabolomic analysis to characterize molecular alterations associated with ESCC. This article is part of a Special Issue entitled: Proteomics in India.

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). Insertion mutagenesis confirms that GPA1 and the sole canonical G-protein-coupled receptor (GCR1) in Arabidopsis also have a role in this blue light-mediated event. In vitro analyses indicate that the increase in PD1 activity is the direct and specific consequence of its interaction with activated GPA1. Because of their shared role in the light-mediated synthesis of phenylpyruvate and Phe, because they are iteratively interactive, and because activated GPA1 is directly responsible for the activation of PD1; GCR1, GPA1, and PD1 form all of or part of a signal transduction mechanism responsible for the light-mediated synthesis of phenylpyruvate, Phe, and those metabolites that derive from that Phe. Data are also presented to confirm that abscisic acid can act through the same pathway. An additional outcome of the work is the confirmation that phenylpyruvate acts as the intermediate in the synthesis of Phe in etiolated plants, as it commonly does in bacteria and fungi.

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 7.5% w/v native polyacrylamide gel, and biotinylated peptides were extracted with aqueous 95% v/v formamide (pH 8.2), aqueous 6 M guanidine HCl (pH 1.5), or water, at temperatures from 4 to 95 degrees C for periods from 5 min to 24 h. It was observed that all three solvents are capable of extracting biotinylated peptides and avidin from the gel, but the best results were obtained with aqueous 95% v/v formamide (pH 8.2) at 65 degrees C for 20 min. However, some AFAEP-captured biotinylated peptides are not stable and are extensively modified by formamide during extraction at too high a temperature or too long an extraction time.

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. The protein sample to be analyzed is treated with 2% w/v sodium dodecyl sulfate (SDS) to ensure that they carry a negative charge. The negative charge will allow the proteins to migrate towards the cathode and hence pass through the area embedded with the bait antibody. It is observed that electrophoretic migration of bovine serum albumin (BSA) or protein G ceases upon encounter with anti-BSA whereas proteins ovalbumin, beta-lactoglobulin A, and myoglobin migrate freely. However, the addition of 0.1% w/v SDS in the native gel running buffer disrupts the antibody-antigen bond and neither BSA nor protein G can be captured by anti-BSA.

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. Due to the high-resolution mass spectra obtained by MALDI-TOFMS from glycoproteins with molecular weights less than 20 kDa, the compositions of oligosaccharides are readily derived for ribonuclease B and avidin. The data agree with the proposed oligosaccharide structures of ribonuclease B (five glycoforms) and avidin (eight glycoforms). Larger glycoproteins such as alpha1-acid glycoprotein (many glycoforms) and fetuin (many glycoforms) exhibited only broad peaks with no glycoform resolution. Nevertheless, this method can be used successfully for analysis of glycoproteins with molecular weights greater than 20 kDa to determine the presence or absence of glycosylation.

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. The AFAEP method allows the capture and concentration of biotinylated proteins or biotinylated peptides irrespective of the use of SDS in both the sample buffer and the gel running buffer.

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. The data showed that the most labile group within the oligosaccharides is the fucose (Fuc) residue, and that a majority of the end cleavage products are peptides with one N-acetylglucosamine (GlcNAc) residue linked to asparagine (Asn). In addition, the glycopeptides with m/z 3355 and 3673 carry an oligosaccharide (Xyl)Man3(Fuc)GlcNAc2, the glycopeptide at m/z 5647 carries two oligosaccharides (Xyl)Man3(Fuc)GlcNAc2, and the glycopeptides at m/z 2612 and 2533 carry (Xyl)Man3GlcNAc2 and (Fuc)GlcNAc, respectively. However, the glycosylation site of the m/z 2612 peptide at Asn286 is partially occupied. This simple and rapid method is particularly useful in identifying glycopeptides and obtaining monosaccharide compositions of glycopeptides.

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 7.5% native polyacrylamide gel. Some of each of the embedded proteins get covalently and/or noncovalently incorporated into the gel matrix network. Under electrophoresis conditions, these proteins do not show significant electrophoretic mobility or they migrate in a direction opposite to the protein analytes, as in avidin. We clearly observe that polyclonal anti-human myoglobin antibody, biotinylated insulin, BSA, and ovalbumin (glycoprotein) are captured and separated in distinct regions of a FAEP gel by protein A, avidin, monoclonal anti-BSA antibody, and concanavalin A, respectively.