Methods in molecular biology
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Allergic asthma is a chronic inflammatory lung disease mediated by type 2 cytokines produced by T helper 2 (Th2) cells as well as the recently discovered group 2 innate lymphoid cells (ILC2). Due to a lack of unique markers, the accurate phenotypic characterization and quantification of ILC2 requires a comprehensive panel of fluorescently labeled antibodies. ⋯ ILC2 are also activated in mouse models of allergic asthma based on the physiologically relevant house dust mite (HDM) allergen, which parallel eosinophilic airway inflammation observed in asthma patients. Here, we describe the analysis of ILC2 by flow cytometry in these two commonly used allergic airway inflammation models in the mouse.
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Post-translational modifications (PTMs) are an important source of protein regulation; they fine-tune the function, localization, and interaction with other molecules of the majority of proteins and are partially responsible for their multifunctionality. Usually, proteins have several potential modification sites, and their patterns of occupancy are associated with certain functional states. These patterns imply cross talk among PTMs within and between proteins, the majority of which are still to be discovered. Several methods detect associations between PTMs; these have recently combined into a global resource, the PTMcode database, which contains already known and predicted functional associations between pairs of PTMs from more than 45,000 proteins in 19 eukaryotic species.
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Glycosylation of membrane proteins plays a crucial role in various physiological events, including intercellular recognition and intermolecular interactions on the cell surface (Gornik et al., Biochim Biophys Acta 1820:1318-1326, 2012). To study composition and function of N-glycans on membrane proteins one has to have an efficient and reproducible analytical method, which includes protein extraction and analysis of glycans. In this chapter we provide an analytical approach that includes cloud-point extraction (CPE) of total membrane proteins with the non-ionic detergent Triton X-114 and subsequent analysis of their N-glycans using hydrophilic interaction liquid chromatography (HILIC)-UPLC/HPLC. The protocol presented here can be used for parallel analysis of both membrane and intracellular proteins.
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Phosphorylation is among the most important post-translational modifications of proteins and has numerous regulatory functions across all domains of life. However, phosphorylation is often substoichiometric, requiring selective and sensitive methods to enrich phosphorylated peptides from complex cellular digests. Various methods have been devised for this purpose and we have recently described a Fe-IMAC HPLC column chromatography setup which is capable of comprehensive, reproducible, and selective enrichment of phosphopeptides out of complex peptide mixtures. ⋯ Here, we provide a step-by-step protocol for the entire phosphopeptide enrichment procedure including sample preparation (lysis, digestion, desalting), Fe-IMAC column chromatography (column setup, operation, charging), measurement by LC-MS/MS (nHPLC gradient, MS parameters) and data analysis (MaxQuant). To increase throughput, we have optimized several key steps such as the gradient time of the Fe-IMAC separation (15 min per enrichment), the number of consecutive enrichments possible between two chargings (>20) and the column recharging itself (<1 h). We show that the application of this protocol enables the selective (>90 %) identification of more than 10,000 unique phosphopeptides from 1 mg of HeLa digest within 2 h of measurement time (Q Exactive Plus).
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High-throughput techniques are indispensable for aiding basic and translational research. Among them, recent advances in proteomics techniques have allowed biomedical researchers to characterize the proteome of multiple organisms. ⋯ This chapter provides an overview of computational strategies, methods, and techniques reported in this book for bioinformatics analysis of protein data. An outline of many bioinformatics tools, databases, and proteomic techniques described in each of the chapters is given here.