Methods in molecular biology
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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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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.
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Post-translational modifications (PTMs) are one of the main contributors to the diversity of proteoforms in the proteomic landscape. In particular, protein phosphorylation represents an essential regulatory mechanism that plays a role in many biological processes. Protein kinases, the enzymes catalyzing this reaction, are key participants in metabolic and signaling pathways. ⋯ In this chapter, we demonstrate two text mining tools, RLIMS-P and eFIP, for the retrieval and extraction of kinase-substrate-site data and phosphorylation-dependent PPIs from the literature. These tools offer several advantages over a literature search in PubMed as their results are specific for phosphorylation. RLIMS-P and eFIP results can be sorted, organized, and viewed in multiple ways to answer relevant biological questions, and the protein mentions are linked to UniProt identifiers.
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Whole-genome bisulfite sequencing (WGBS) has become a powerful tool to dissect genome-wide methylation profiles at single-base resolution. In this chapter we describe in detail the bioinformatics pipeline used for the analysis of ARGONAUTE-dependent DNA methylation in Arabidopsis thaliana. We provide tools and command lines used for mapping bisulfite sequencing reads, for estimating methylation levels at individual cytosine sites, for identifying differentially methylated regions (DMRs), and for calculating methylation levels of DMRs.