Springer protocols feed by Plant Sciences 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Phosphorylation Stoichiometry Determination in Plant Photosynthetic Membranes
This chapter describes different strategies for the study of phosphorylation dynamics and stoichiometry in photosynthetic membranes. Detailed procedures for the detection, large-scale identification, and quantification of phosphorylated proteins optimized for plant thylakoid proteins are given. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
Absolute Quantitation of Protein Posttranslational Modification Isoform
Mass spectrometry has been widely applied in characterization and quantification of proteins from complex biological samples. Because the numbers of absolute amounts of proteins are needed in construction of mathematical models for molecular systems of various biological phenotypes and phenomena, a number of quantitative proteomic methods have been adopted to measure absolute quantities of proteins using mass spectrometry. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with internal peptide standards, i.e., the stable isotope-coded peptide dilution series, which was originated from the field of analy...
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
Kinase Activity and Specificity Assay Using Synthetic Peptides
Phosphorylation of substrate proteins by protein kinases can lead to activation or inactivation of signaling pathways or metabolic processes. Precise understanding of activity and specificity of protein kinases are important questions in characterization of kinase functions. Here, we describe a procedure to study kinase activity and specificity using kinase-GFP complexes purified from plant material and synthetic peptides as substrates. Magnetic GFP beads allow purifying receptor-like kinase-GFP complexes from microsomal fractions. Kinase-GFP complexes are then incubated with ATP and the synthetic peptides for kinase react...
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
Combining Metabolic 15N Labeling with Improved Tandem MOAC for Enhanced Probing of the Phosphoproteome
In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool for studying protein phosphorylation because it enables unbiased localization, and site-specific quantification of in vivo phosphorylation of hundreds of proteins in a single experiment. A common strategy for identifying phosphoproteins and their phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. However, despite high sensitivity of modern mass spectrometers ...
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
Phosphopeptide Profiling of Receptor Kinase Mutants
The family of transmembrane receptor kinase is the largest protein kinase family in Arabidopsis. However many of these kinases have yet uncharacterized functions and little is known about direct substrates of these kinases. Here, we present a large-scale phosphoproteomics method involving label-free quantitation-based comparative phosphopeptide profiling of knockout mutants in receptor-like kinases. This approach, among other physiological and cell biological experiments, is one step in understanding the functional roles of plant kinases in the context of their signaling networks. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
Screening of Kinase Substrates Using Kinase Knockout Mutants
Protein kinases are widely known to be major regulators of various signaling processes, particularly in eukaryotes, including plants. To understand their role in signal transduction pathways, it is necessary to determine which proteins are phosphorylated by these enzymes. Recent studies have applied a comparative phosphoproteomic approach to identify protein kinase substrates in plants. The results demonstrated that kinase knockout mutants are useful for screening protein kinase substrates via such a comparative analysis. Here some technical points are described for the experimental design and comparative analysis using ki...
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
Phosphoproteomics in Cereals
Cereals are the most important crop plant supplying staple food throughout the world. The economic importance and continued breeding of crop plants such as rice, maize, wheat, or barley require a detailed scientific understanding of adaptive and developmental processes. Protein phosphorylation is one of the most important regulatory posttranslational modifications and its analysis allows deriving functional and regulatory principles in plants. This minireview summarizes the current knowledge of phosphoproteomic studies in cereals. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
Phosphatases in Plants
Reversible protein phosphorylation is an essential posttranslational modification mechanism executed by opposing actions of protein phosphatases and protein kinases. About 1,000 predicted kinases in Arabidopsis thaliana kinome predominate the number of protein phosphatases, of which there are only ~150 members in Arabidopsis. Protein phosphatases were often referred to as “housekeeping” enzymes, which act to keep eukaryotic systems in balance by counteracting the activity of protein kinases. However, recent investigations reveal the crucial and specific regulatory functions of phosphatases in cell signaling. Ph...
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
The Plant Kinome
Plant kinases are one of the largest protein families in Arabidopsis. There are almost 600 membrane-located receptor kinases and almost 400 soluble kinases with distinct functions in signal transduction. In this minireview we discuss phylogeny and functional context of prominent members from major protein kinase subfamilies in plants. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
Phosphorylation Site Prediction in Plants
Protein phosphorylation events on serine, threonine, and tyrosine residues are the most pervasive protein covalent bond modifications in plant signaling. Both low and high throughput studies reveal the importance of phosphorylation in plant molecular biology. Although becoming more and more common, the proteome-wide screening on phosphorylation by experiments remains time consuming and costly. Therefore, in silico prediction methods are proposed as a complementary analysis tool to enhance the phosphorylation site identification, develop biological hypothesis, or help experimental design. These methods build statistical mod...
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
Databases for Plant Phosphoproteomics
Phosphorylation is the most studied posttranslational modification involved in signal transduction in stress responses, development, and growth. In the recent years large-scale phosphoproteomic studies were carried out using various model plants and several growth and stress conditions. Here we present an overview of online resources for plant phosphoproteomic databases: PhosPhAt as a resource for Arabidopsis phosphoproteins, P3DB as a resource expanding to crop plants, and Medicago PhosphoProtein Database as a resource for the model plant Medicago trunculata. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
Computational Identification of Protein Kinases and Kinase-Specific Substrates in Plants
The protein phosphorylation catalyzed by protein kinases (PKs) plays an essential role in almost all biological progresses in plants. Thus, the identification of PKs and kinase-specific substrates is fundamental for understanding the regulatory mechanisms of protein phosphorylation especially in controlling plant growth and development. In this chapter, we describe the computational methods and protocols for the identification of PKs and kinase-specific substrates in plants, by using Vitis vinifera as an example. First, the proteome sequences and experimentally identified phosphorylation sites (p-sites) in Vitis vinifera w...
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
Computational Phosphorylation Network Reconstruction: Methods and Resources
The succession of protein activation and deactivation mediated by phosphorylation and dephosphorylation events constitutes a key mechanism of molecular information transfer in cellular systems. To deduce the details of those molecular information cascades and networks has been a central goal pursued by both experimental and computational approaches. Many computational network reconstruction methods employing an array of different statistical learning methods have been developed to infer phosphorylation networks based on different types of molecular data sets such as protein sequence, protein structure, or phosphoproteomics...
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
Targeted Analysis of Protein Phosphorylation by 2D Electrophoresis
Two-dimensional (2D) gel electrophoresis combines isoelectric focusing in the first and SDS polyacrylamide gel electrophoresis in the second dimension to separate complex mixtures of proteins with unequalled resolution and sensitivity. It is well suited for the analysis of posttranslational protein modifications as most of them affect the isoelectric point and, therefore, the focusing behavior of the protein in the first dimension. It is particularly useful for low-abundance proteins, as it provides a first indication of PTMs, before establishing methods for protein isolation. For targeted proteomics of more abundant prote...
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
Plant Protein Kinase Substrates Identification Using Protein Microarrays
Protein kinases regulate signaling pathways by phosphorylating their targets. They play critical roles in plant signaling networks. Although many important protein kinases have been identified in plants, their substrates are largely unknown. We have developed and produced plant protein microarrays with more than 15,000 purified plant proteins. Here, we describe a detailed protocol to use these microarrays to identify plant protein kinase substrates via in vitro phosphorylation assays on these arrays. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - May 4, 2015 Category: Biology Source Type: news
