RESUMO
Nontargeted single-cell proteomics analysis by mass spectrometry with sample multiplexing utilizing isobaric labeling is often performed using a carrier proteome. The presented protocol describes a targeted approach that replaces the carrier proteome with a set of synthetic peptides from selected proteins, which improves the identification and quantification of these proteins in single human cells.
Assuntos
Espectrometria de Massas , Proteoma , Proteômica , Análise de Célula Única , Proteômica/métodos , Análise de Célula Única/métodos , Humanos , Espectrometria de Massas/métodos , Proteoma/análise , Peptídeos/química , Peptídeos/metabolismo , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Recent studies have reported abundant presence of bacterial extracellular membrane vesicles in the marine environment. However, the ecological significance of these bacterial vesicles in the marine environment is only beginning to be explored. In present study, for the first time we report and characterize membrane vesicles secreted by a seaweed associated bacterium, Alteromonas macleodii KS62. Proteomics studies revealed that the vesicle proteome was rich in hydrolytic enzymes (30%) like glycoside hydrolases, proteases, sulphatases, lipases, nucleases and phosphatases. Zymography experiments and enzyme assays established that the vesicles carry active κ-carrageenan degrading enzymes. κ-carrageenan is a major polysaccharide of cell walls of certain red seaweeds like Kappaphycus. Purified membrane vesicles were successfully able to degrade Kappaphycus biomass. Based on these results, we discuss how the hydrolase-rich vesicles may play a role in red seaweed cell wall degradation so that the bacteria can invade and colonise the seaweed biomass establishing a saprophytic lifestyle. We also discuss the role of these vesicles in nutrient acquisition and their ecological significance in the marine environment.