Methods in molecular biology
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Soybean Knowledge Base (SoyKB) is a comprehensive all-inclusive web resource for bridging the gap between soybean translational genomics and molecular breeding. It provides information for six entities including genes/proteins, microRNAs (miRNAs)/small interfering RNAs (sRNA), metabolites, single nucleotide polymorphisms (SNPs), and plant introduction lines and traits. It has a user-friendly web interface publicly available at http://soykb.org , which integrates and presents data in an intuitive manner to the soybean researchers, breeders, and consumers. It incorporates several informatics and analytical tools for integrating and merging various multi-omics datasets.
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Protein phosphorylation regulates brain development and neuronal activities; and dysregulation of phosphorylation contributes to neurobiological disorders. Phosphoproteomic analysis provides comprehensive modification maps for measuring protein activities in cellular pathways and biological processes. Here, we introduce a mass spectrometry (MS)-based protocol to quantitatively analyze the phosphoproteome of human postmortem brains of Alzheimer's disease. ⋯ To improve the coverage of phosphoproteome, the peptide mix is further fractionated by offline basic pH reversed-phase liquid chromatography (LC) with high-resolution power. Phosphopeptides in each fraction are then enriched by the titanium dioxide method and analyzed by online acidic pH reverse phase LC-MS/MS, leading to the analysis of tens of thousands of phosphorylation events. This protocol can also be adapted to profile phosphoproteome in other biological samples.
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DNA methylation is a major epigenetic modification that regulates gene expression, genome imprinting, and development and has a role in diseases including cancer. There are various methods for whole-genome methylation profiling that differ in cost and resolution. ⋯ In this chapter, we provide detailed protocols for whole-genome bisulfite sequencing (WGBS), which captures the complete methylome. Using WGBS, we are able to generate a reference DNA methylome for normal or malignant hematopoietic cells.
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Protein phosphorylation is one of the key events in the regulation of plant physiological responses to diverse environmental stimuli. As crucial regulators of phosphorylation, protein kinases have been linked to the control of seed germination, flowering, and stress responses. Identifying downstream substrates of kinases is important for dissecting kinase-substrate networks as well as delineating the underlying defense mechanisms in response to extracellular stimulation. ⋯ Moreover, it remains challenging to identify bona fide kinase substrates from proteome-wide analyses. Thus, developing methodologies with high sensitivity and specificity is imperative for understanding plant kinase-substrate cascades. Here, we describe a proteomic strategy termed kinase assay-linked phosphoproteomics (KALIP) approach for large-scale identification of the direct substrates of plant kinases with high sensitivity and a low false-positive rate.
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Network analysis methods are increasing in popularity. An approach commonly applied to analyze proteomics data involves the use of protein-protein interaction (PPI) networks to explore the systems-level cooperation between proteins identified in a study. ⋯ Here we describe a method for calculating robust empirical p-values for protein interaction networks. We also provide a worked example with python code demonstrating the implementation of this methodology.