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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.Using a Genetically Encoded FRET-Based Reporter to Visualize Calcineurin Phosphatase Activity in Living Cells
Calcineurin is an evolutionarily conserved, ubiquitously expressed protein phosphatase that serves as a major effector of Ca2+ signals, regulating diverse biological processes such as gene expression, tissue differentiation, immune responses, and neural plasticity. The following method describes how to monitor real-time calcineurin activity in cultured mammalian cells using a fluorescence resonance energy transfer (FRET)-based activity reporter. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
Imaging Kinase Activity at Protein Scaffolds
Kinase signaling is under tight spatiotemporal control, with signaling hubs within the cell often coordinated by protein scaffolds. Genetically encoded kinase activity reporters afford a unique tool to interrogate the rate, amplitude, and duration of kinase signaling at specific locations throughout the cell. This protocol describes how to assay kinase activity at a protein scaffold in live cells using a fluorescence resonance energy transfer (FRET)-based kinase activity sensor for protein kinase D (PKD) as an example. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
Computational Approaches to Selected Reaction Monitoring Assay Design
Selected reaction monitoring (SRM) is becoming the tool of choice for targeted quantitative proteomics, with applications as diverse as clinical diagnostics and systems biology. Assay design is critical to the success of every SRM experiment. For each protein of interest it is necessary to find a set of peptides that can be monitored as surrogates for that protein. These peptides must satisfy a number of criteria, including uniqueness in the proteome, detectability by mass spectrometry, and suitability of product ion series. Finding peptides that meet all these criteria is time consuming, especially when seeking to quantif...
Source: Springer protocols feed by Protein Science - May 16, 2013 Category: Biochemistry Source Type: news
Methods and Algorithms for Quantitative Proteomics by Mass Spectrometry
Protein quantitation by mass spectrometry (MS) is attractive since it is possible to obtain both identification and quantitative values of proteins and their posttranslational modifications in a single experiment. In contrast, protein arrays only provide quantitative values of targeted proteins and their modifications. There are an overwhelming number of quantitative MS methods for protein and peptide quantitation. The aim here is to provide an overview of the most common MS methods and algorithms used in quantitative proteomics and discuss the computational algorithms needed to reliably quantitate proteins, peptides, and ...
Source: Springer protocols feed by Protein Science - May 16, 2013 Category: Biochemistry Source Type: news
Improving Peptide Identification Using Empirical Scoring Systems
Peptides and proteins are routinely identified from peptide fragmentation spectra acquired in a mass spectrometer, analyzed by database search engines. The types of fragments that can be formed are known, and it is also well appreciated that certain fragment types are more common or more informative than others. However, most search engines do not use detailed knowledge of peptide fragmentation, but rather consider a limited range of fragments, giving each an equivalent weighting in their scoring system that decides which results are likely to be correct. This chapter discusses efforts to make use of information about the ...
Source: Springer protocols feed by Protein Science - May 16, 2013 Category: Biochemistry Source Type: news
Interpretation of Tandem Mass Spectra of Posttranslationally Modified Peptides
Tandem mass spectrometry provides a sensitive means of analyzing the amino acid sequence of peptides and modified peptides by providing accurate mass measurements of precursor and fragment ions. Modern mass spectrometry instrumentation is capable of rapidly generating many thousands of tandem mass spectra and protein database search engines have been developed to match the experimental data to peptide candidates. In most studies there is a schism between discarding perfectly valid data and including nonsensical peptide identifications—this is currently a major bottleneck in data-analysis and it calls for an understan...
Source: Springer protocols feed by Protein Science - May 16, 2013 Category: Biochemistry Source Type: news
Algorithms for Database-Dependent Search of MS/MS Data
The frequent used bottom-up strategy for identification of proteins and their associated modifications generate nowadays typically thousands of MS/MS spectra that normally are matched automatically against a protein sequence database. Search engines that take as input MS/MS spectra and a protein sequence database are referred as database-dependent search engines. Many programs both commercial and freely available exist for database-dependent search of MS/MS spectra and most of the programs have excellent user documentation. The aim here is therefore to outline the algorithm strategy behind different search engines rather t...
Source: Springer protocols feed by Protein Science - May 16, 2013 Category: Biochemistry Source Type: news
Retention Time Prediction and Protein Identification
In bottom-up proteomics, proteins are typically identified by enzymatic digestion into peptides, tandem mass spectrometry and comparison of the tandem mass spectra with those predicted from a sequence database for peptides within measurement uncertainty from the experimentally obtained mass. Although now decreasingly common, isolated proteins or simple protein mixtures can also be identified by measuring only the masses of the peptides resulting from the enzymatic digest, without any further fragmentation. Separation methods such as liquid chromatography and electrophoresis are often used to fractionate complex protein or ...
Source: Springer protocols feed by Protein Science - May 16, 2013 Category: Biochemistry Source Type: news
Isotopic Distributions
Isotopic information determined by mass spectrometry can be used in a wide variety of applications. Broadly speaking these could be classified as “passive” applications, meaning that they use naturally occurring isotopic information, and “active” applications, meaning that the isotopic distributions are manipulated in some way. The classic passive application is the determination of chemical composition by comparing observed isotopic patterns of molecules to theoretically calculated isotopic patterns. Active applications include isotope exchange experiments of a variety of types, as well as isotope ...
Source: Springer protocols feed by Protein Science - May 16, 2013 Category: Biochemistry Source Type: news
LC-MS Spectra Processing
Peak extraction from raw data is the first step in LC-MS data analysis. The quality of this procedure is important since it affects the quality and accuracy of all subsequent analysis such as database searches and peak quantitation. The most important and most accurately measured physical entity provided by mass spectrometers is m/z values which need to be extracted by state of art algorithms and scrutinized thoroughly. The aim of this chapter is to provide a discussion of peak processing methods and furthermore discuss some of the yet unresolved or neglected issues. A few novel concepts are also proposed for analysis and ...
Source: Springer protocols feed by Protein Science - May 16, 2013 Category: Biochemistry Source Type: news
Introduction to Mass Spectrometry-Based Proteomics
Mass spectrometry has been widely applied to study biomolecules and one rapidly developing field is the global analysis of proteins, proteomics. Understanding and handling mass spectrometry data is a multifaceted task that requires many decisions to be made to get the most comprehensive information from an experiment. Later chapters in this book deal in-depth with various aspects of the process and how different tools can be applied to the many analytical challenges. This introductory chapter is intended as a basic introduction to mass spectrometry (MS)-based proteomics to set the scene for newcomers and give pointers to r...
Source: Springer protocols feed by Protein Science - May 16, 2013 Category: Biochemistry Source Type: news
Simple Proteomics Data Analysis in the Object-Oriented PowerShell
Scripting languages such as Perl and Python are appreciated for solving simple, everyday tasks in bioinformatics. A more recent, object-oriented command shell and scripting language, Windows PowerShell, has many attractive features: an object-oriented interactive command line, fluent navigation and manipulation of XML files, ability to consume Web services from the command line, consistent syntax and grammar, rich regular expressions, and advanced output formatting. The key difference between classical command shells and scripting languages, such as bash, and object-oriented ones, such as PowerShell, is that in the latter ...
Source: Springer protocols feed by Protein Science - May 16, 2013 Category: Biochemistry Source Type: news
Proteomic Strategies to Characterize Signaling Pathways
Cells respond to external stimuli by transducing signals through a series of intracellular molecules and eliciting an appropriate response. The cascade of events through which the signals are transduced include post-translational modifications such as phosphorylation and ubiquitylation in addition to formation of multi-protein complexes. Improvements in biological mass spectrometry and protein/peptide microarray technology have tremendously improved our ability to probe proteins, protein complexes, and signaling pathways in a high-throughput fashion. Today, a single mass spectrometry-based investigation of a signaling path...
Source: Springer protocols feed by Protein Science - May 16, 2013 Category: Biochemistry Source Type: news
Tools for Protein Posttranslational Modifications Analysis: FAK, a Case Study
Recent advances in mass spectrometry have resulted in an exponential increase in annotation of posttranslational modifications (PTMs). Just in the Swiss-Prot Knowledgebase, there are 89,931 of a total of 27 characterized PTM types reported experimentally. A single protein can be dynamically modified during its lifetime for regulation of its function. Considering a PTM can occur at different levels and the number of different PTMs described, the number of possibilities for a single protein is unthinkable. Narrowing the study to a single PTM can be rather unmerited considering that most proteins are heavily modified. Current...
Source: Springer protocols feed by Protein Science - May 16, 2013 Category: Biochemistry Source Type: news
Proteomics Data Exchange and Storage: The Need for Common Standards and Public Repositories
Both the existence of data standards and public databases or repositories have been key factors behind the development of the existing “omics” approaches. In this book chapter we first review the main existing mass spectrometry (MS)-based proteomics resources: PRIDE, PeptideAtlas, GPMDB, and Tranche. Second, we report on the current status of the different proteomics data standards developed by the Proteomics Standards Initiative (PSI): the formats mzML, mzIdentML, mzQuantML, TraML, and PSI-MI XML are then reviewed. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - May 16, 2013 Category: Biochemistry Source Type: news
