Structural Categorization of Adenine, Guanine, and Xanthine Derivatives Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry with 5-Nitrosalicylic Acid and 1,5-Diaminonaphtalene.

In this study, we analyzed purine derivatives using multimatrix variation matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) with α-cyano-4-hydroxycinnamic acid (CHCA), 1,5-diaminonaphtalene (DAN), 5-formylsalicylic acid (FSA), and 5-nitrosalicylic acid (NSA) as matrices. Further, we focused on the abstraction/attachment of hydrogen from/to analytes and detected [M - H], [M + 2H] and/or [M + 3H] in MALDI MS spectra of compounds containing nitrogen and/or carbonyl oxygen. Although [M - H] generation of purine compounds in MALDI MS with conventional matrices was challenging, NSA-MALDI MS effectively yielded the [M - H]species of purine derivatives compared with CHCA, FSA, and DAN, and the [M - H]/[M + H] ratios reflected their structures, such as the substituting groups and positions.

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.

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.

Correction to: Cell-free synthesis of functionally active HSPB5.

In the original publication, the given name of the last author was incorrectly displayed as the name must read: Katsuyoshi Masuda.

Cell-free synthesis of functionally active HSPB5.

Human αB-crystallin (HSPB5) is frequently modified post-translationally by UV radiation, oxidation, and age-associated processes, which complicates functional analyses of the protein using natural sources. Thus, determining the biological function of HSPB5 at the molecular structure level requires unmodified protein. Here, we employed an Escherichia coli cell-free protein synthesis system to prepare unmodified, functionally active human HSPB5.

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.

Amino acid residues of bitter taste receptor TAS2R16 that determine sensitivity in primates to β-glycosides.

In mammals, bitter taste is mediated by TAS2Rs, which belong to the family of seven transmembrane G protein-coupled receptors. Since TAS2Rs are directly involved in the interaction between mammals and their dietary sources, it is likely that these genes evolved to reflect species-specific diets during mammalian evolution. Here, we analyzed the amino acids responsible for the difference in sensitivities of TAS2R16s of various primates using a cultured cell expression system.

Characterization of GnRH-like peptides from the nerve ganglia of Yesso scallop, Patinopecten yessoensis.

There is yet no firm experimental evidence that the evolutionary ancient gonadotropin-releasing hormone GnRH (i.e., GnRH1) also acts in invertebrate gametogenesis.

Continuous flow atmospheric pressure laser desorption/ionization using a 6-7-µm-band mid-infrared tunable laser for biomolecular mass spectrometry.

A continuous flow atmospheric pressure laser desorption/ionization technique using a porous stainless steel probe and a 6-7-µm-band mid-infrared tunable laser was developed. This ion source is capable of direct ionization from a continuous flow with a high temporal stability. The 6-7-µm wavelength region corresponds to the characteristic absorption bands of various molecular vibration modes, including O-H, C=O, CH3 and C-N bonds.

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.

Localization and enzymatic activity profiles of the proteases responsible for tachykinin-directed oocyte growth in the protochordate, Ciona intestinalis.

We previously substantiated that Ci-TK, a tachykinin of the protochordate, Ciona intestinalis (Ci), triggered oocyte growth from the vitellogenic stage (stage II) to the post-vitellogenic stage (stage III) via up-regulation of the gene expression and enzymatic activity of the proteases: cathepsin D, carboxypeptidase B1, and chymotrypsin. In the present study, we have elucidated the localization, gene expression and activation profile of these proteases. In situ hybridization showed that the Ci-cathepsin D mRNA was present exclusively in test cells of the stage II oocytes, whereas the Ci-carboxypeptidase B1 and Ci-chymotrypsin mRNAs were detected in follicular cells of the stage II and stage III oocytes.

Peptidomic analysis of the central nervous system of the protochordate, Ciona intestinalis: homologs and prototypes of vertebrate peptides and novel peptides.

The phylogenetic position of ascidians as the chordate invertebrates closest to vertebrates suggests that they might possess homologs and/or prototypes of vertebrate peptide hormones and neuropeptides as well as ascidian-specific peptides. However, only a small number of peptides have so far been identified in ascidians. In the present study, we have identified various peptides in the ascidian, Ciona intestinalis.

Oxytocin/vasopressin and gonadotropin-releasing hormone from cephalopods to vertebrates.

Recent advances in peptide search methods have revealed two peptide systems that have been conserved through metazoan evolution. Members of the oxytocin/vasopressin-superfamily have been identified from protostomian and deuterostomian animals, indicating that the oxytocin/vasopressin hormonal system represents one of the most ancient systems. In most protostomian animals, a single member of the superfamily shares oxytocin-like and vasopressin-like actions.

Mutational deglycosylation of the Fc portion of immunoglobulin G causes O-sulfation of tyrosine adjacently preceding the originally glycosylated site.

Mutagenesis directed to a specific glycosylation site has been widely used to examine biological roles of individual glycans. However, occurrence of any post-translational modification on such deglycosylated mutants has not yet been well characterized. Here we performed mass spectrometric analyses of the Fc fragment of an unglycosylated mutant of mouse immunoglobulin G2b, whose conserved N-glycosylation site, i.

Key structures of bacterial peptidoglycan and lipopolysaccharide triggering the innate immune system of higher animals: Chemical synthesis and functional studies".

Chemistry-based investigation is reviewed which led to identification of the active entities responsible for the immunostimulating potencies of peptidoglycan and lipopolysaccharide. Though these glycoconjugates which ubiquitously occur in wide range of bacteria as the essential components of their cell envelopes have long been known to enhance the immunological responses of higher animals, neither the precise chemical structures required nor the mechanism of their action had been elucidated until early 1970s. Chemical synthesis of partial structures of peptidoglycan proved N-acetylmuramyl-L-alanyl-D-isoglutamine to be the minimum structure responsible for the activity and led to later identification of its receptor protein Nod2 present in animal cells.

Key structures of bacterial peptidoglycan and lipopolysaccharide triggering the innate immune system of higher animals: chemical synthesis and functional studies.

Chemistry-based investigation is reviewed which led to identification of the active entities responsible for the immunostimulating potencies of peptidoglycan and lipopolysaccharide. Though these glycoconjugates which ubiquitously occur in wide range of bacteria as the essential components of their cell envelopes have long been known to enhance the immunological responses of higher animals, neither the precise chemical structures required nor the mechanism of their action had been [corrected] elucidated until early 1970s. Chemical synthesis of partial structures of peptidoglycan proved N-acetylmuramyl-L-alanyl-D-isoglutamine to be the minimum structure responsible for the activity and led to later identification of its receptor protein Nod2 present in animal cells.

Lipopolysaccharide induces raft domain expansion in membrane composed of a phospholipid-cholesterol-sphingomyelin ternary system.

The molecular behavior and interaction of Re-type lipopolysaccharide (ReLPS) and phospholipids were investigated in two different types of model membrane systems, a pure phospholipid membrane consisting of 1,2-dielaidoyl-snglycero-3-phosphoethanolamine (DEPE) and a raft-forming membrane composed of equimolar DEPE, sphingomyelin (SM), and cholesterol (Chol) by solid-state NMR spectroscopy. A remarkable influence of ReLPS on the property of lipid bilayer was found by analyzing the (13)C-NMR spectra. Namely, while both liquid-ordered (L(o)) and liquid-disordered (L(d)) phases co-exist in DEPE/SM/Chol, only the L(o) phase is present in DEPE/SM/Chol/ReLPS.

Comparative overview of toll-like receptors in lower animals.

Toll-like receptors (TLRs) have been shown to play a crucial role in host defense against pathogenic microbes in innate immunity in mammals. Recent genome-wide analyses have suggested that TLRs or related genes are conserved in the genome of non-mammalian organisms such as fishes, cyclostomes, ascidians, cephalochordates, sea urchins, and hydras. However, neither active forms nor functions of authentic invertebrate TLRs had been elucidated.

Neuropeptides, hormone peptides, and their receptors in Ciona intestinalis: an update.

The critical phylogenetic position of ascidians leads to the presumption that neuropeptides and hormones in vertebrates are highly likely to be evolutionarily conserved in ascidians, and the cosmopolitan species Ciona intestinalis is expected to be an excellent deuterostome Invertebrate model for studies on neuropeptides and hormones. Nevertheless, molecular and functional characterization of Ciona neuropeptides and hormone peptides was restricted to a few peptides such as a cholecystokinin (CCK)/gastrin peptide, cionin, and gonadotropin-releasing hormones (GnRHs). In the past few years, mass spectrometric analyses and database searches have detected Ciona orthologs or prototypes of vertebrate peptides and their receptors, including tachykinin, insulin/relaxin, calcitonin, and vasopressin.

Photoactive ligands probing the sweet taste receptor. Design and synthesis of highly potent diazirinyl D-phenylalanine derivatives.

Some D-amino acids such as d-tryptophan and D-phenylalanine are well known as naturally-occurring sweeteners. Photoreactive D-phenylalanine derivatives containing trifluoromethyldiazirinyl moiety at 3- or 4-position of phenylalanine, were designed as sweeteners for functional analysis with photoaffinity labeling. The trifluoromethyldiazirinyl D-phenylalanine derivatives were prepared effectively with chemo-enzymatic methods using L-amino acid oxidase and were found to have potent activity toward the human sweet taste receptor.

Functional diversity of signaling pathways through G protein-coupled receptor heterodimerization with a species-specific orphan receptor subtype.

Gonadotropin-releasing hormones (GnRHs) play pivotal roles in control of reproduction via a hypothalamic-pituitary-periphery endocrine system and nervous systems of not only vertebrates but also invertebrates. GnRHs trigger several signal transduction cascades via GnRH receptors (GnRHRs), members of the G protein-coupled receptor (GPCR) family. Recently, six GnRHs (tunicate GnRH [tGnRH]-3 to tGnRH-8) and four GnRHRs (Ciona intestinalis [Ci]-GnRHR1 to GnRHR-4), including a species-specific paralog, Ci-GnRHR4 (R4) regarded as an orphan receptor or nonfunctional receptor, were identified in the protochordate, C.