Identification, Chemical Synthesis, and Sweetness Evaluation of Rhamnose or Xylose Containing Steviol Glycosides of Stevia () Leaves.

Steviol glycosides obtained from leaves are increasingly used in the food industry as natural low-calorie sweeteners. Among them, the sweetness of major glycosides composed of glucose residues (e.g.

Diaminoanthraquinone Enhances Alkaloid Ionization in MALDI-MS.

The alkaloids epinastine, 3-methylxanthine and camptothecin were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The ionization efficiencies of epinastine and 3-methylxanthine were improved upon the addition of 1,5-diaminoanthraquinone (DAAQ). DAAQ did not show ultraviolet absorbance peaks at wavelengths around 337 nm and 355 nm that are used in conventional MALDI-MS instruments.

Multimatrix Variation Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry as a Tool for Determining the Bonding of Nitrogen Atoms in Alkaloids.

The reactivity of alkaloids in dehydrogenation was investigated using multimatrix variation matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of over 20 different alkaloids with six matrices. The dehydrogenated molecular ions [M - H] generated by in-source decay were detected in the MALDI mass spectra of some types of alkaloids such as reserpine. The dehydrogenation proceeded at the cyclic tertiary amine rather than double-bonded nitrogen atoms and indole rings involved in the electron-delocalized systems.

Fragmentation and Ionization Efficiency of Positional and Functional Isomers of Paeoniflorin Derivatives in Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Paeoniflorin and albiflorin, which are functional isomers, are the major constituents of an herbal medicine derived from . Those functional isomers and their galloylated derivatives, which are positional isomers, were studied by matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS). The resulting mass spectra are discussed based on the fragmentation patterns of the sodium adducts.

Distinguishing between isomeric neoxanthin and violaxanthin esters in yellow flower petals using liquid chromatography/photodiode array atmospheric pressure chemical ionization mass spectrometry and tandem mass spectrometry.

Rationale: Liquid chromatography/photodiode array atmospheric pressure chemical ionization mass spectrometry (LC/PDA-APCI-MS) is used for the analysis of various carotenoid pigments in plants. Among them, it is difficult to distinguish between the isomeric violaxanthin/neoxanthin esters.

Methods: The yellow pigments of tomato petals were extracted with acetone, and the extracts were kept at -30°C to allow the contaminating triacylglycerols to settle out physically.

Amidation/non-amidation top-down analysis of endogenous neuropeptide Y in brain tissue by nano flow liquid chromatography orbitrap Fourier transform mass spectrometry.

Neuropeptide Y (NPY) is a transmitter molecule in nerve system, and it was an over 4-kDa large peptide with the C-terminal end amidation. NPY is biosynthesized through many maturation processes from a large pre-pro-peptide with peptide-cleavages and amidation that is important to study the biosynthesis regulation. Previously, it was reported that cathepsin L participates in the production of NPY and that cathepsin L generates both of amidated and non-amidated NPYs.

Practical Protocol for Making Calibration Curves for Direct and Sensitive Quantitative LC Orbitrap-MS of Large Neuropeptides.

Peptides larger than 3-4 kDa, such as neuropeptide Y (NPY), orexin-B, and alpha-MSH, have practical issues that arise when conducting direct and sensitive quantitative liquid chromatography (LC) orbitrap-FT mass spectrometry (MS) due to their adsorption and low ionization efficiency, especially in standard solutions. A mixing solvent consisting of 0.5% trifluoroacetic acid (TFA) and 35-50% aq.

Practical optimization of liquid chromatography/mass spectrometry conditions and pretreatment methods toward the sensitive quantification of auxin in plants.

Rationale: The plant hormone auxin, indole-3-acetic acid, regulates many aspects of plant growth and development. Auxin quantification should offer broad insights into its mechanistic action in plants. However, limited auxin content in plant tissues hampers the establishment of quantification methods without the highest graded instruments or deeply specialized experimental techniques.

Troubleshooting Carry-Over in the LC-MS Analysis of Biomolecules: The Case of Neuropeptide Y.

We describe systematic troubleshooting of the carry-over of neuropeptide Y (NPY) in LC-MS analysis. The objective was to remove candidate parts of the LC-MS system that are responsible for carry over one-by-one. The findings indicate that the carry-over of NPY occurs on the column, particularly in the guard column and at the consumable seals of the sample-needle and high-pressure valves.

Development of Software for the In-Depth Analysis of Protein Dynamics as Determined by MALDI Mass Spectrometry-Based Hydrogen/Deuterium Exchange.

Hydrogen/deuterium exchange (HDX) coupled with pepsin digestion is useful for rapidly analyzing the kinetic properties of small amounts of protein. However, the analysis of HDX by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is time-consuming due to a lack of dedicated software. Currently available software programs mainly calculate average mass shifts, even though the isotopic distribution width contains information regarding multiple protein conformations.

Imaging Mass Spectrometry Analysis of Flavonoids in Blue Viola Petals and Their Enclosure Effects on Violanin during Color Expression.

The color expression of anthocyanin pigments in blue flowers is precisely controlled by their chemical and physical properties such as pH and the presence of metal ions or colorless copigments. Despite the large number of known blue flowers, their coloration mechanisms have not been examined in sufficient detail. In this work, the blue coloration of Viola cornuta petals was expressed via the copigmentation of various flavonol 3- O-glycosides.

Fragmentation of Oligosaccharides from Sodium Adduct Molecules Depends on the Position of -Acetyl Hexosamine Residue in Their Sequences in Mass Spectrometry.

Six different sequences of hexasaccharides, pyridylaminated malto-hexaoses containing one -acetyl hexosamine (HexNAc) residue, were analyzed using matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF) mass spectrometry (MS). Based on the product ion spectra of sodium adducts [M+Na], the chemical species of the observed product ions contained a HexNAc residue and had high ion abundance, indicating that the HexNAc residue had a higher affinity to sodium atom than glucopyranose. The acetamide group coordinated easily to sodium atom.

Probing the catalytic site of rabbit muscle glycogen phosphorylase using a series of specifically modified maltohexaose derivatives.

Glycogen phosphorylase (GP) is an allosteric enzyme whose catalytic site comprises six subsites (SG, SG, SG, SG, SG, and SP) that are complementary to tandem five glucose residues and one inorganic phosphate molecule, respectively. In the catalysis of GP, the nonreducing-end glucose (Glc) of the maltooligosaccharide substrate binds to SG and is then phosphorolyzed to yield glucose 1-phosphate. In this study, we probed the catalytic site of rabbit muscle GP using pyridylaminated-maltohexaose (Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA, where GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol]; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (Glc -AltNAc-Glc -GlcPA, where m + n = 4 and AltNAc is 3-acetoamido-3-deoxy-D-altrose).

Mechanism for odd-electron anion generation of dihydroxybenzoic acid isomers in matrix-assisted laser desorption/ionization mass spectrometry with density functional theory calculations.

Rationale: Proton and radical are transferred between matrices and matrix and analyte in matrix-assisted laser desorption/ionization (MALDI) and these transfers drive ionization of analytes. The odd-electron anion [M-2H] was generated in dihydroxybenzoic acids (DHBs) and the ion abundance of the 2,5-DHB was the highest among six DHB isomers. We were interested in the mechanism of the ion generation of the odd-electron anion.

High-resolution mass spectrometry for detecting Acetylcholine in Arabidopsis.

Acetylcholine (ACh) was first identified a century ago, and has long been known as a neurotransmitter in animals. However, it has been shown recently that the occurrence of ACh is widespread among various non-animal species including higher plants. Although previous reports suggest that various plant species are capable of responding to exogenously applied ACh, the molecular basis for ACh biosynthesis and regulatory mechanisms mediated by endogenous ACh are largely unclear.

Cyclodepsipeptides produced by actinomycetes inhibit cyclic-peptide-mediated quorum sensing in Gram-positive bacteria.

Cyclic peptides are commonly used as quorum-sensing autoinducers in Gram-positive Firmicutes bacteria. Well-studied examples of such molecules are thiolactone and lactone, used to regulate the expression of a series of virulence genes in the agr system of Staphylococcus aureus and the fsr system of Enterococcus faecalis, respectively. Three cyclodepsipeptides WS9326A, WS9326B and cochinmicin II/III were identified as a result of screening actinomycetes culture extracts for activity against the agr/fsr system.

Non-neuronal acetylcholine as an endogenous regulator of proliferation and differentiation of Lgr5-positive stem cells in mice.

Non-neuronal acetylcholine (ACh) is predicted to function as a local cell signaling molecule. However, the physiological significance of the synthesis of non-neuronal ACh in the intestine remains unclear. Here, experiments using crypt-villus organoids that lack nerve and immune cells in culture led us to suggest that endogenous ACh is synthesized in the intestinal epithelium to evoke growth and differentiation of the organoids through activation of muscarinic ACh receptors (mAChRs).

Laser-induced hydrogen radical removal in UV MALDI-MS allows for the differentiation of flavonoid monoglycoside isomers.

Negative-ion matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra and tandem mass spectra of flavonoid mono-O-glycosides showed the irregular signals that were 1 and/or 2 Da smaller than the parent deprotonated molecules ([M - H](-)) and the sugar-unit lost fragment ions ([M - Sugar - H](-)). The 1 and/or 2 Da mass shifts are generated with the removing of a neutral hydrogen radical (H*), and/or with the homolytic cleavage of the glycosidic bond, such as [M - H* - H](-), [M - Sugar - H* - H](-), and [M - Sugar - 2H* - H](-). It was revealed that the hydrogen radical removes from the phenolic hydroxy groups on the flavonoids, not from the sugar moiety, because the flavonoid backbones themselves absorb the laser.

Amino and acetamide functional group effects on the ionization and fragmentation of sugar chains in positive-ion mass spectrometry.

To elucidate the influence of amino (-NH2) and acetamide (-NHCOCH3, -NAc) groups in sugar chains on their ionization and fragmentation, cycloamyloses (cyclodextrins, CyDs) and lacto-oligosaccharide are analyzed by MALDI TOF/TOF and ESI Q-TOF mass spectrometry. CyD derivatives substituted by amino or acetamide groups are ideal analytes to extract the function group effects, which are amino-CyD with one hexosamine (HexNH2) and acetamide-CyD with one N-acetyl hexosamine (HexNAc). Interestingly, the relative ion intensities and isotope-like patterns in their product ion spectra depend on the functional groups and ion forms of sugar chains.

MPIase is a glycolipozyme essential for membrane protein integration.

Protein integration into biological membranes is a vital cellular event for all organisms. We previously reported an integration factor in the inner membrane of Escherichia coli, named MPIase (membrane protein integrase). Here we show that in contrast to previously identified integration factors that are proteins, MPIase is a glycolipid composed of diacylglycerol and a glycan chain of three acetylated aminosugars linked through pyrophosphate.

Solution NMR analysis of the binding mechanism of DIVS6 model peptides of voltage-gated sodium channels and the lipid soluble alkaloid veratridine.

Voltage-gated sodium channels (VGSCs) are responsible for generating action potentials in nervous systems. Veratridine (VTD), a lipid soluble alkaloid isolated from sabadilla lily seed, is believed to bind to segment 6 of VGSCs and act as a partial agonist. However, high resolution structural interaction mechanism between VGSCs and VTD is difficult to elucidate because of the large size and membrane localization of VGSCs.

Time-course changes in fungal elicitor-induced lignan synthesis and expression of the relevant genes in cell cultures of Linum album.

Linum album has been shown to accumulate anti-tumor podophyllotoxin (PTOX) and its related lignans. In the present study, we examined the effects of five fungal extracts on the production of lignans in L. album cell cultures.