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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.Identification of Direct Kinase Substrates via Kinase Assay-Linked Phosphoproteomics
Protein phosphorylation plays an essential role in the regulation of various cellular functions. Dysregulation of phosphorylation is implicated in the pathogenesis of certain cancers, diabetes, cardiovascular diseases, and central nervous system disorders. As a result, protein kinases have become potential drug targets for treating a wide variety of diseases. Identification of kinase substrates is vital not only for dissecting signaling pathways, but also for understanding disease pathologies and identifying novel therapeutic targets. However, identification of bona fide kinase substrates has remained challenging, necessit...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Simple and Reproducible Sample Preparation for Single-Shot Phosphoproteomics with High Sensitivity
The traditional sample preparation workflow for mass spectrometry (MS)-based phosphoproteomics is time consuming and usually requires multiple steps, e.g., lysis, protein precipitation, reduction, alkylation, digestion, fractionation, and phosphopeptide enrichment. Each step can introduce chemical artifacts, in vitro protein and peptide modifications, and contaminations. Those often result in sample loss and affect the sensitivity, dynamic range and accuracy of the mass spectrometric analysis. Here we describe a simple and reproducible phosphoproteomics protocol, where lysis, denaturation, reduction, and alkylation are per...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Online LC-FAIMS-MS/MS for the Analysis of Phosphorylation in Proteins
High-field asymmetric waveform ion mobility spectrometry (FAIMS) is a gas-phase separation technique which, when coupled with liquid chromatography tandem mass spectrometry, offers benefits for analysis of complex proteomics samples such as those encountered in phosphoproteomics experiments. Results from LC-FAIMS-MS/MS are typically complementary, in terms of proteome coverage and isomer identification, to those obtained by use of solution-phase separation methods, such as prefractionation with strong cation-exchange chromatography. Here, we describe the protocol for large-scale phosphorylation analysis by LC-FAIMS-MS/MS. ...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Variable Digestion Strategies for Phosphoproteomics Analysis
In recent years, mass spectrometry-based phosphoproteomics has propelled our knowledge about the regulation of cellular pathways. Nevertheless, typically applied bottom-up strategies have several limitations. Trypsin, the preferentially used proteolytic enzyme shows impaired cleavage efficiency in the vicinity of phosphorylation sites. Moreover, depending on the frequency and distribution of tryptic cleavage sites (Arg/Lys), generated peptides can be either too short or too long for confident identification using standard LC-MS approaches. To overcome these limitations, we introduce an alternative and simple approach based...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Two Dimensional Gel Electrophoresis-Based Plant Phosphoproteomics
Phosphorylation is one of the most important reversible protein modifications and is involved in regulating signal transduction, subcellular localization and enzyme activity of target proteins. Phosphorylation or dephosphorylation of proteins is directly reflected in changed ratios of phosphoprotein abundance and total protein abundance. Two-dimensional gel electrophoresis (2-DE)-based proteomics allow quantification of both total protein abundance by Coomassie Brilliant Blue (CBB) staining and phosphoprotein abundance by fluorescence-based staining. Pro-Q diamond phosphoprotein stain (Pro-Q DPS) can bind to the phosphate ...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
The Use of Titanium Dioxide for Selective Enrichment of Phosphorylated Peptides
Titanium dioxide (TiO2) has very high affinity for phosphopeptides and in recent years it has become one of the most popular methods for phosphopeptide enrichment from complex biological samples. Peptide loading onto TiO2 resin in a highly acidic environment in the presence of 2,5-dihydroxybenzoic acid (DHB), phthalic acid, lactic acid, or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding of non-phosphorylated peptides. The phosphopeptides bound to the TiO2 are subsequently eluted from the chromatographic material using an alkaline buffer. TiO2 chromatography is extremely tole...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography
Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe3+, Ga3+, Al3+, Zr4+, and Ti4+ has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphory...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Enrichment Strategies in Phosphoproteomics
The comprehensive study of the phosphoproteome is heavily dependent on appropriate enrichment strategies that are most often, but not exclusively, carried out on the peptide level. In this chapter, I give an overview of the most widely used techniques. In addition to dedicated antibodies, phosphopeptides are enriched by their selective interaction with metals in the form of chelated metal ions or metal oxides. The negative charge of the phosphate group is also exploited in a variety of chromatographic fractionation methods that include different types of ion exchange chromatography, hydrophilic interaction chromatography (...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Phosphopeptide Enrichment Using Various Magnetic Nanocomposites: An Overview
Magnetic nanocomposites are hybrid structures consisting of an iron oxide (Fe3O4/γ-Fe2O3) superparamagnetic core and a coating shell which presents affinity for a specific target molecule. Within the scope of phosphopeptide enrichment, the magnetic core is usually first functionalized with an intermediate layer of silica or carbon to improve dispersibility and increase specific area, and then with an outer layer of a phosphate-affinity material. Fe3O4-coating materials include metal oxides, rare earth metal-based compounds, immobilized-metal ions, polymers, and many others. This chapter provides a generic overview of...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Offline High pH Reversed-Phase Peptide Fractionation for Deep Phosphoproteome Coverage
Protein phosphorylation, a process in which kinases modify serines, threonines, and tyrosines with phosphoryl groups is of major importance in eukaryotic biology. Protein phosphorylation events are key initiators of signaling responses which determine cellular outcomes after environmental and metabolic stimuli, and are thus highly regulated. Therefore, studying the mechanism of regulation by phosphorylation, and pinpointing the exact site of phosphorylation on proteins is of high importance. This protocol describes in detail a phosphoproteomics workflow for ultra-deep coverage by fractionating peptide mixtures based on hig...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation
Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with ...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Sequential Elution from IMAC (SIMAC): An Efficient Method for Enrichment and Separation of Mono- and Multi-phosphorylated Peptides
Phosphoproteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems, especially when using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., Immobilized Metal ion Affinity Chromatography and titanium dioxide chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. The number of multi-phosphorylated peptides identified in most published studies is rather low. Here we describe a protocol for a strategy that separates mono-phosphorylated peptides from multiply phosphorylated pept...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Quantitative Analysis of Tissue Samples by Combining iTRAQ Isobaric Labeling with Selected/Multiple Reaction Monitoring (SRM/MRM)
Mass spectrometry-based phosphoproteomics is an indispensible technique used in the discovery and quantification of phosphorylation events on proteins in biological samples. The application of this technique to tissue samples is especially useful for the discovery of biomarkers as well as biological studies. We herein describe the application of a large-scale phosphoproteome analysis and SRM/MRM-based quantitation to develop a strategy for the systematic discovery and validation of biomarkers using tissue samples. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Identification of Direct Kinase Substrates Using Analogue-Sensitive Alleles
Identifying the substrates of protein kinases remains a major obstacle in the elucidation of eukaryotic signaling pathways. Promiscuity among kinases and their substrates coupled with the extraordinary plasticity of phosphorylation networks renders traditional genetic approaches or small-molecule inhibitors problematic when trying to determine the direct substrates of an individual kinase. Here we describe methods to label, enrich, and identify the direct substrates of analogue-sensitive kinases by exploiting their steric complementarity to artificial ATP analogues. Using calcium-dependent protein kinases of Toxoplasma gon...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Peptide Labeling Using Isobaric Tagging Reagents for Quantitative Phosphoproteomics
Isobaric tagging reagents have become an invaluable tool for multiplexed quantitative proteomic analysis. These reagents can label multiple, distinct peptide samples from virtually any source material (e.g., tissue, cell line, purified proteins), allowing users the opportunity to assess changes in peptide abundances across many different time points or experimental conditions. Here, we describe the application of isobaric peptide labeling, specifically 8plex isobaric tags for relative and absolute quantitation (8plex iTRAQ), for quantitative phosphoproteomic analysis of cultured cells or tissue suspensions. For this partic...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
