Methods in enzymology
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Methods in enzymology · Jan 2017
Deep Profiling of Proteome and Phosphoproteome by Isobaric Labeling, Extensive Liquid Chromatography, and Mass Spectrometry.
Mass spectrometry-based proteomics has experienced an unprecedented advance in comprehensive analysis of proteins and posttranslational modifications, with particular technical progress in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an optimized LC-MS/MS platform to quantitate whole proteome and phosphoproteome. ⋯ Quality control steps are implemented for troubleshooting and evaluating experimental variation. Such a multiplexed robust method provides a powerful tool for dissecting proteomic signatures at the systems level in a variety of complex samples, ranging from cell culture, animal tissues to human clinical specimens.
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Methods in enzymology · Jan 2017
Cellular Assays for Evaluating Calcium-Dependent Translocation of cPLA2α to Membrane.
The group IVA phospholipase A2, commonly called cytosolic phospholipase A2α (cPLA2α), is a widely expressed enzyme that hydrolyzes membrane phospholipid to produce arachidonic acid and lysophospholipids, which are precursors for a number of bioactive lipid mediators. Arachidonic acid is metabolized through the cyclooxygenase and lipoxygenase pathways for production of prostaglandins and leukotrienes that regulate normal physiological processes and contribute to disease pathogenesis. cPLA2α is composed of an N-terminal C2 domain and a C-terminal catalytic domain that contains the Ser-Asp catalytic dyad. The catalytic domain contains phosphorylation sites and basic residues that regulate the catalytic activity of cPLA2α. ⋯ In resting cells, cPLA2α is localized in the cytosol but translocates to membrane including the Golgi, endoplasmic reticulum, and the peri-nuclear membrane in response to increases in intracellular calcium. Calcium binds to the C2 domain, which promotes the interaction of cPLA2α with membrane through hydrophobic interactions. In this chapter, we describe assays used to study the calcium-dependent translocation of cPLA2α to membrane, a regulatory step necessary for access to phospholipid and release of arachidonic acid.