RESUMEN
Enrichment of the plasma membrane represents one valuable method to characterize the surfaceome, along with other plasma membrane and structural proteins. Currently, the overlapping densities of many subcellular organelles hinder enrichment of the plasma membrane by centrifugation. However, external access to the plasma membrane of intact cells allows the attachment of a nanoparticle pellicle to enhance its density and facilitate enrichment. We describe the synthesis of iron oxide nanoparticles, attachment of the pellicle to suspended cells, and recovery of plasma membrane proteins for proteomic analysis.
Asunto(s)
Membrana Celular/química , Compuestos Férricos/química , Proteínas de la Membrana/aislamiento & purificación , Nanopartículas/química , Proteoma/química , Línea Celular Tumoral , Centrifugación/métodos , Humanos , Microscopía Electrónica de Transmisión , Proteómica , SuspensionesRESUMEN
In this laboratory module, introductory biochemistry students are exposed to two-dimensional (1) H-nuclear magnetic resonance of glycerophospholipids (GPLs). Working in groups of three, students enzymatically synthesized and purified a variety of 2-acyl lyso GPLs. The structure of the 2-acyl lyso GPL was verified using (1) H-correlation spectroscopy. Students scored significantly higher on an assessment of NMR knowledge after having participated in this lab module and in comparison to a similar cohort who did not participate. Inaddition, student confidence in their NMR knowledge and abilities increased 62% following the module and correlated with their ability to apply their NMR knowledge. Based on these results, the laboratory module was very effective at providing students with a more extensive understanding of the underlying concepts of NMR as a tool for structural determination.
Asunto(s)
Bioquímica/educación , Glicerofosfolípidos/química , Glicerofosfolípidos/síntesis química , Lisofosfolípidos/química , Lisofosfolípidos/síntesis química , Espectroscopía de Resonancia Magnética/métodos , Acilación , Cromatografía en Capa Delgada , Glicerofosfolípidos/aislamiento & purificación , Humanos , Lisofosfolípidos/aislamiento & purificación , ProtonesRESUMEN
Advances in mass spectrometry have facilitated the identification of novel lipid structures. In this work, we fractionated the lipids of Escherichia coli B and analyzed the fractions using negative-ion electrospray ionization mass spectrometry to reveal unknown lipid structures. Analysis of a fraction eluting with high salt from DEAE cellulose revealed a series of ions not corresponding to any of the known lipids of E. coli. The ions, with m/z 861.5, 875.5, 887.5, 889.5, and 915.5, were analyzed using collision-induced dissociation mass spectrometry (MS/MS) and yielded related fragmentation patterns consistent with a novel diacylated glycerophospholipid. Product ions arising by neutral loss of 216 mass units were observed with all of the unknowns. A corresponding negative product ion was also observed at m/z 215.0. Additional ions at m/z 197.0, 171.0, 146.0, and 128.0 were used to propose the novel structure phosphatidylserylglutamate (PSE). The hypothesized structure was confirmed by comparison with the MS/MS spectrum of a synthetic standard. Normal phase liquid chromatography-mass spectrometry analysis further showed that the endogenous PSE and synthetic PSE eluted with the same retention times. PSE was also observed in the equivalent anion exchange fractions of total lipids extracted from the wild-type E. coli K-12 strain MG1655.