RESUMO
The placenta is a crucial interface between the fetus and the maternal environment. It allows for nutrient absorption, thermal regulation, waste elimination, and gas exchange through the mother's blood supply. Furthermore, the placenta determines important adjustments and epigenetic modifications that can change the phenotypic expression of the individual even long after birth. Polyethylene glycol (PEG) is a polyether compound derived from petroleum with many applications, from medicine to industrial manufacturing. In this study, for the first time, an integration of ultra-high-performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS) was used to detect suites of PEG compounds in human placenta samples, collected from 12 placentas, originating from physiological pregnancy. In 10 placentas, we identified fragments of PEG in both chorioamniotic membranes and placental cotyledons, for a total of 36 samples.
Assuntos
Placenta , Espectrometria de Massas em Tandem , Humanos , Feminino , Gravidez , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Placenta/metabolismo , Plásticos/metabolismo , Polietilenoglicóis/metabolismoRESUMO
The present chapter describes methods for the separation and identification of photosynthetic proteins of thylakoid membranes present in chloroplasts by using different detergents, high-performance liquid chromatography and mass spectrometry. Thylakoid membranes represent a good model for setting up analytical methods suitable for membrane protein characterization.The first step in the procedure is the preparation of purified membrane fractions from plant tissues, followed by the fractionation of membrane proteins by differential solubilization using different detergents. Thus, several protein complexes can be isolated, collected, separated by ion-pair reversed-phase chromatography and detected online by UV-absorption and/or mass spectrometry. Finally, identification of the eluting proteins is accomplished by comparing the molecular mass determined in silico with the molecular mass measured by mass spectrometry.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Proteínas de Plantas/análise , Tilacoides/química , Fracionamento Químico , Detergentes/química , Dextranos/química , Digitonina/química , Hidroponia , Complexo de Proteína do Fotossistema I/análise , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/isolamento & purificação , Complexo de Proteína do Fotossistema II/análise , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Polietilenoglicóis/química , Spinacia oleracea/química , Spinacia oleracea/citologia , Spinacia oleracea/crescimento & desenvolvimentoRESUMO
The membrane protein components of photosystem I (PSI) and II (PSII) from different species were prefractionated by liquid extraction and sucrose gradient ultracentrifugation and subsequently analyzed by reversed-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) using poly-(styrene-divinylbenzene)-based monolithic capillary columns. The analytical method was shown to be very flexible and enabled the identification of antenna proteins as well as most of the proteins of the reaction center from PSI and PSII in various plant species with few RP-HPLC-ESI-MS analyses necessitating only minor adaptations in the gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid. The membrane proteins, ranging in molecular mass (Mr) from 4196 (I protein) to more than 80,000 (PSI A/B) as well as isoforms were identified on the basis of their intact Mr and comparison with Mr deduced from known DNA or protein sequences. High quality mass spectra enabled the identification and quantitation of the nonphosphorylated and phosphorylated reaction center subunits D1, D2, and CP43 of PSII, containing five to seven membrane-spanning alpha-helices. Because of its high flexibility and suitability for proteins having a very wide range of Mr and hydrophobicities, the method is generally applicable to the analysis of complex mixtures of membrane proteins.