Proteomic analysis of human dental cementum and alveolar bone.

Unlabelled: Dental cementum (DC) is a bone-like tissue covering the tooth root and responsible for attaching the tooth to the alveolar bone (AB) via the periodontal ligament (PDL). Studies have unsuccessfully tried to identify factors specific to DC versus AB, in an effort to better understand DC development and regeneration. The present study aimed to use matched human DC and AB samples (n=7) to generate their proteomes for comparative analysis.

Rapid assessment of RNAi-mediated protein depletion by selected reaction monitoring mass spectrometry.

We describe the use of a targeted proteomics approach, selected reaction monitoring (SRM) mass spectrometry, to detect and assess RNAi-mediated depletion or "knockdown" of specific proteins from human cells and from Drosophila flies. This label-free approach does not require any specific reagents to confirm the depletion of RNAi target protein(s) in unfractionated cell or whole organism extracts. The protocol described here is general, can be developed rapidly, and can be multiplexed to detect and measure multiple proteins at once.

The development of selected reaction monitoring methods for targeted proteomics via empirical refinement.

Software advancements in the last several years have had a significant impact on proteomics from method development to data analysis. Herein, we detail a method, which uses our in-house developed software tool termed Skyline, for empirical refinement of candidate peptides from targeted proteins. The method consists of four main steps from generation of a testable hypothesis, method development, peptide refinement, to peptide validation.

A method to determine the kinetics of multiple proteins in human infants with respiratory distress syndrome.

We report a method to measure in vivo turnover of four proteins from sequential tracheal aspirates obtained from human newborn infants with respiratory distress syndrome using targeted proteomics. We detected enrichment for all targeted proteins approximately 3 h from the start of infusion of [5,5,5-(2)H(3)] leucine, secretion times that varied from 1.2 to 2.

Effect of collision energy optimization on the measurement of peptides by selected reaction monitoring (SRM) mass spectrometry.

Proteomics experiments based on Selected Reaction Monitoring (SRM, also referred to as Multiple Reaction Monitoring or MRM) are being used to target large numbers of protein candidates in complex mixtures. At present, instrument parameters are often optimized for each peptide, a time and resource intensive process. Large SRM experiments are greatly facilitated by having the ability to predict MS instrument parameters that work well with the broad diversity of peptides they target.

Measurement of human surfactant protein-B turnover in vivo from tracheal aspirates using targeted proteomics.

We describe a method to measure protein synthesis and catabolism in humans without prior purification and use the method to measure the turnover of surfactant protein-B (SP-B). SP-B, a lung-specific, hydrophobic protein essential for fetal-neonatal respiratory transition, is present in only picomolar quantities in tracheal aspirate samples and difficult to isolate for dynamic turnover studies using traditional in vivo tracer techniques. Using infusion of [5,5,5-(2)H(3)] leucine and a targeted proteomics method, we measured both the quantity and kinetics of SP-B tryptic peptides in tracheal aspirate samples of symptomatic newborn infants.

Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.

Summary: Skyline is a Windows client application for targeted proteomics method creation and quantitative data analysis. It is open source and freely available for academic and commercial use. The Skyline user interface simplifies the development of mass spectrometer methods and the analysis of data from targeted proteomics experiments performed using selected reaction monitoring (SRM).

Overexpression of catalase targeted to mitochondria attenuates murine cardiac aging.

Background: Age is a major risk for cardiovascular diseases. Although mitochondrial reactive oxygen species have been proposed as one of the causes of aging, their role in cardiac aging remains unclear. We have previously shown that overexpression of catalase targeted to mitochondria (mCAT) prolongs murine median lifespan by 17% to 21%.

Expediting the development of targeted SRM assays: using data from shotgun proteomics to automate method development.

Selected reaction monitoring (SRM) is a powerful tandem mass spectrometry method that can be used to monitor target peptides within a complex protein digest. The specificity and sensitivity of the approach, as well as its capability to multiplex the measurement of many analytes in parallel, has made it a technology of particular promise for hypothesis driven proteomics. An underappreciated step in the development of an assay to measure many peptides in parallel is the time and effort necessary to establish a usable assay.

Distonoid ions.

By Yates, Bouma, and Radom's definition, distonic radical ions are those formally arising by ionization of diradicals or zwitterionic molecules (including ylides). These ions differ, therefore, from conventional radical ions by displaying the charge site and unpaired electron site (spin) localized mandatorily on separate atoms or group of atoms; that is, these sites are separated in all of their major resonance forms. Many conventional radical ions with a major resonance form in which charge and spin sites reside formally on the same atom or group of atoms display, however, high degree of discretionary (non-mandatory) charge-spin separation.

Cyclization reactions of acylium and thioacylium ions with isocyanates and isothiocyanates: gas phase synthesis of 3,4-dihydro-2,4-dioxo-2H-1,3,5-oxadiazinium ions.

Gas-phase reactions of several acylium and thioacylium ions, that is H2C=N-C+=O, H2C=N-C+=S, O=C=N-C+=O, S=C=N-C+=O, H3C-C+=O, and (CH3)2N-C+=O, with both a model isocyanate and isothiocyanate, that is, C2H5-N=C=O and C2H5-N=C=S, were investigated using tandem-in-space pentaquadrupole mass spectrometry. In these reactions, the formation of mono- and double-addition products is observed concurrently with proton transfer products. The double-addition products are far more favored in reactions with ethyl isocyanate, whereas the reactions with ethyl isothiocyanate form, preferentially, either the mono-addition product or proton transfer products, or both.

Isolation and structure determination of bioactive isoflavones from callus culture of Dipteryx odorata.

Crude extracts of callus and roots of Dipteryx odorata were analyzed by HPLC to detect and quantify isoflavone contents. Based on spectroscopic and X-ray crystallography data the structures of two isoflavones were elucidated as 7-hydroxy-4',6-dimethoxyisoflavone and 3',7-dihydroxy-4',6-dimethoxyisoflavone. The production of dry biomass of 7-hydroxy-4',6-dimethoxyisoflavone in cultured callus was 4.

Electrospray ionization mass spectrometry fingerprinting of beer.

After just simple degassing, dilution, pH adjustment and direct flow injection, characteristic fingerprint spectra of beer samples have been obtained by fast (few seconds) electrospray ionization mass spectrometry (ESI-MS) analysis in both the negative and positive ion modes. A total of 29 samples belonging to the two main beer types (lagers and ales) and several beer subtypes from USA, Europe and Brazil could be clearly divided into three groups both by simple visual inspection of their ESI(+)-MS and ESI(-)-MS fingerprints as well as by chemometric treatment of the MS data. Diagnostic ions with contrasting relative abundances in both the positive and negative ion modes allow classification of beers into three major types: P = pale (light) colored (pilsener, pale ale), D = dark colored (bock, stout, porter, mild ale) and M = malt beer.

Electrospray mass and tandem mass spectrometry of homologous and isomeric singly, doubly, triply and quadruply charged cationic ruthenated meso-(phenyl)m-(meta- and para-pyridyl)n (m + n = 4) macrocyclic porphyrin complexes.

Ten homologous or isomeric singly, doubly, triply and quadruply charged cationic macrocyclic complexes I-Va, bn+ (n = 1-4) formed by the coordination of [Ru(bipy)2Cl]+ to the pyridyl N-atoms of a series of meso-(phenyl)m-(meta or para-pyridyl)n-porphyrins (m + n = 4) were transferred to the gas phase and structurally characterized by electrospray ionization (ESI) mass (MS) and tandem mass (MS/MS) spectrometry. Previously known to be stable in solution and in the solid state, I-Va, bn+ are found to constitute also a new class of stable, long-lived multiply charged gas-phase ions with spatially separated charge sites. Increasing intramolecular electrostatic repulsion from Ia, b+ to IVa, b3+ facilitates in-source and tandem collision-induced dissociation (CID).

Electrospray ionization mass spectrometry fingerprinting of propolis.

Crude ethanolic extracts of propolis, a natural resin, have been directly analysed using electrospray ionization mass (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) in the negative ion mode. European, North American and African samples have been analyzed, but emphasis has been given to Brazilian propolis which displays diverse and region-dependent chemical composition. ESI-MS provides characteristic fingerprint mass spectra, with propolis samples being divided into well-defined groups directly related to their geographical origins.

(trans-1,4-bis[(4-pyridyl)ethenyl]benzene)(2,2'-bipyridine)ruthenium(II) complexes and their supramolecular assemblies with beta-cyclodextrin.

Two novel ruthenium polypyridine complexes, [Ru(bpy)(2)Cl(BPEB)](PF(6)) and ([Ru(bpy)(2)Cl](2)(BPEB))(PF(6))(2) (BPEB = trans-1,4-bis[2-(4-pyridyl)ethenyl]benzene), were synthesized and their characterization carried out by means of elemental analysis, UV-visible spectroscopy, positive ion electrospray (ESI-MS), and tandem mass (ESI-MS/MS) spectrometry, as well as by NMR spectroscopy and cyclic voltammetry. Cyclic and differential pulse voltammetry for the mononuclear complex showed three set of waves around 1.2 V (Ru(2+/3+)), -1.

Mass spectrometric characterization of two novel inflammatory peptides from the venom of the social wasp Polybia paulista.

The social wasp P. paulista is relatively common in southeast Brazil causing many medically important stinging incidents. The seriousness of these incidents is dependent on the amount of venom inoculated by the wasps into the victims, and the characteristic envenomation symptoms are strongly dependent on the types of peptides present in the venom.

Tetrahydrofuran lignans from Nectandra megapotamica with trypanocidal activity.

The phytochemical investigation of the crude ethanol extract of the leaves of Nectandra megapotamica afforded three new (2d, 3b, and 3c) and eight known (1a-d, 2a-c, and 3a) tetrahydrofuran lignans. Regarding the in vitro trypanocidal activity, the lignan-rich fraction, at a concentration of 2.0 mg/mL, was 100% active.

New antioxidant C-glucosylxanthones from the stems of Arrabidaea samydoides.

Three new C-glucosylxanthones, 2-(2'-O-trans-caffeoyl)-C-beta-d-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (1), 2-(2'-O-trans-cinnamoyl)-C-beta-d-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (2), and 2-(2'-O-trans-coumaroyl)-C-beta-d-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (3), were isolated from the stems of Arrabidaea samydoides, in addition to three known C-glucosylxanthones, mangiferin (4), 2-(2'-O-benzoyl)-C-beta-d-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (5), and muraxanthone (6). Their chemical structures were assigned on the basis of MS and 1D and 2D NMR experiments. Xanthones 1-6 showed moderate free radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) as well as antioxidant activity evidenced by redox properties measured on ElCD-HPLC.

Turbinatine, a potential key intermediate in the biosynthesis of corynanthean-type indole alkaloids.

Extraction of the leaves of Chimarrhis turbinata has led to the isolation of turbinatine (1), a new corynanthean-type indole alkaloid, besides four known indole alkaloids, strictosidine, 5alpha-carboxystrictosidine, vallesiachotamine, and isovallesiachotamine. The structural determination of 1 was based on 1D and 2D spectroscopic data. An evaluation of the DNA-damaging activities of the isolates was performed by means of a bioassay using mutant strains of Saccharomyces cerevisiae, which indicated these compounds were weakly active.

Iridoid glucosides from Randia spinosa (Rubiaceae).

An iridoid glucoside: randinoside, along with five known iridoids: galioside, deacetylasperulosidic acid methyl ester, scandoside methyl ester, geniposide and gardenoside, were isolated from the stems of Randia spinosa. The structures were determined by spectroscopic analysis, including 2D NMR techniques.

Eudesmanolides from Dimerostemma vestitum.

A leaf rinse extract with dichloromethane from Dimerostemma vestitum afforded five new eudesmanolides (1-5) along with the known flavone eupafolin. Compounds 1-5 were isolated by chromatographic methods, and their structures were elucidated by spectral analyses. Eudesmanolides 1-4 show a functional group substitution pattern so far unreported in the subtribe Verbesininae (tribe Heliantheae) of the family Asteraceae.

Fiber introduction mass spectrometry: fully direct coupling of solid-phase microextraction with mass spectrometry.

This work describes the first fully direct coupling of solid-phase microextraction (SPME) with mass spectrometry. An inlet system using a septum as the only interface between the ambient and the high-vacuum mass spectrometer was constructed to allow the introduction of the SPME needle directly into the ionization region of a mass spectrometer. The PDMS-coated fiber was then placed and exposed exactly between the two ionization filaments.

Mannich-type reactions in the gas-phase: the addition of enol silanes to cyclic N-acyliminium ions.

The intrinsic gas-phase reactivity of cyclic N-acyliminium ions in Mannich-type reactions with the parent enol silane, vinyloxytrimethylsilane, has been investigated by double- and triple-stage pentaquadrupole mass spectrometric experiments. Remarkably distinct reactivities are observed for cyclic N-acyliminium ions bearing either endocyclic or exocyclic carbonyl groups. NH-Acyliminium ions with endocyclic carbonyl groups locked in s-trans forms participate in a novel tandem N-acyliminium ion reaction: the nascent adduct formed by simple addition is unstable and rearranges by intramolecular trimethylsilyl cation shift to the ring nitrogen, and an acetaldehyde enol molecule is eliminated.