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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.Spot Matching of 2-DE Images Using Distance, Intensity, and Pattern Information
We describe a simple and accurate method which allows to automatically and accurately match spots in 2-DE images. The method consists of simultaneously exploiting the distance between the spots, their intensity, and the pattern formed by their spatial configuration. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
Image Pretreatment Tools II: Normalization Techniques for 2-DE and 2-D DIGE
Gel electrophoresis is usually applied to identify different protein expression profiles in biological samples (e.g., control vs. pathological, control vs. treated). Information about the effect to be investigated (a pathology, a drug, a ripening effect, etc.) is however generally confounded with experimental variability that is quite large in 2-DE and may arise from small variations in the sample preparation, reagents, sample loading, electrophoretic conditions, staining and image acquisition. Obtaining valid quantitative estimates of protein abundances in each map, before the differential analysis, is therefore fund...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
Image Pretreatment Tools I: Algorithms for Map Denoising and Background Subtraction Methods
One of the critical steps in two-dimensional electrophoresis (2-DE) image pre-processing is the denoising, that might aggressively affect either spot detection or pixel-based methods. The Median Modified Wiener Filter (MMWF), a new nonlinear adaptive spatial filter, resulted to be a good denoising approach to use in practice with 2-DE. MMWF is suitable for global denoising, and contemporary for the removal of spikes and Gaussian noise, being its best setting invariant on the type of noise. The second critical step rises because of the fact that 2-DE gel images may contain high levels of background, generated...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
Comparative Evaluation of Software Features and Performances
Analysis of two-dimensional gel images is a crucial step for the determination of changes in the protein expression, but at present, it still represents one of the bottlenecks in 2-DE studies. Over the years, different commercial and academic software packages have been developed for the analysis of 2-DE images. Each of these shows different advantageous characteristics in terms of quality of analysis. In this chapter, the characteristics of the different commercial software packages are compared in order to evaluate their main features and performances. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
A Novel Gaussian Extrapolation Approach for 2-D Gel Electrophoresis Saturated Protein Spots
Analysis of images obtained from two-dimensional gel electrophoresis (2-D GE) is a topic of utmost importance in bioinformatics research, since commercial and academic software currently available have proven to be neither completely effective nor fully automatic, often requiring manual revision and refinement of computer generated matches. In this chapter, we present an effective technique for the detection and the reconstruction of over-saturated protein spots. Firstly, the algorithm reveals overexposed areas, where spots may be truncated, and plateau regions caused by smeared and overlapping spots. Next, it reconst...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
Detection and Quantification of Protein Spots by Pinnacle
Accurate spot detection and quantification is a challenging task that must be performed effectively in order to properly extract the proteomic information from two-dimensional (2-D) gel electrophoresis images. In Morris et al., Bioinformatics 24:529–536, 2008, we introduced Pinnacle, an automatic, fast, effective noncommercial package for spot detection and quantification for 2-D gel images, and subsequently we have developed a freely available gui-based interface for applying the method to a set of gels. In this chapter, we overview Pinnacle, and in a step-by-step manner we describe how to use the software to obtain...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
GENOCOP Algorithm and Hierarchical Grid Transformation for Image Warping of Two-Dimensional Gel Electrophoretic Maps
Hierarchical grid transformation is a powerful hierarchical approach to 2-D map warping, able to model both global and local deformations. The algorithm can be stopped when a desired degree of accuracy in the images alignment is obtained. The deformed image is warped and aligned to the target image using a grid where the number of nodes increases in each step of the algorithm. The numerical optimization of the position of the nodes of the grid can be efficiently solved by genetic algorithms, ensuring the achievement of the optimal position of the nodes with a low computational cost with respect to other methods. Here, the ...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
Two-Dimensional Gel Electrophoresis Image Analysis via Dedicated Software Packages
Analyzing two-dimensional gel electrophoretic images is supported by a number of freely and commercially available software. Although the respective program is highly specific, all the programs follow certain standardized algorithms. General steps are: (1) detecting and separating individual spots, (2) subtracting background, (3) creating a reference gel and (4) matching the spots to the reference gel, (5) modifying the reference gel, (6) normalizing the gel measurements for comparison, (7) calibrating for isoelectric point and molecular weight markers, and moreover, (8) constructing a database containing the measurement r...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
Decoding 2-D Maps by Autocovariance Function
This chapter describes a mathematical approach based on the study of the 2-D autocovariance function (2-D ACVF) useful for decoding the complex signals resulting from the separation of protein mixtures. The method allows to obtain fundamental analytical information hidden in 2-D PAGE maps by spot overlapping, such as the number of proteins present in the sample and the mean standard deviation of the spots, describing the separation performance. In addition, it is possible to identify ordered patterns potentially present in spot positions, which can be related to the chemical composition of the protein mixture, such as post...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
Sources of Experimental Variation in 2-D Maps: The Importance of Experimental Design in Gel-Based Proteomics
The success of proteomic studies employing 2-D maps largely depends on the way surveys and experiments have been organized and performed. Planning gel-based proteomic experiments involves the selection of equipment, methodology, treatments, types and number of samples, experimental layout, and methods for data analysis. A good experimental design will maximize the output of the experiment while taking into account the biological and technical resources available. In this chapter we provide guidelines to assist proteomics researchers in all these choices and help them to design quantitative 2-DE experiments. (Source: S...
Source: Springer protocols feed by Protein Science - November 27, 2015 Category: Biochemistry Source Type: news
Systems Analysis for Interpretation of Phosphoproteomics Data
Global phosphoproteomics investigations yield overwhelming datasets with up to tens of thousands of quantified phosphosites. The main challenge after acquiring such large-scale data is to extract the biological meaning and relate this to the experimental question at hand. Systems level analysis provides the best means for extracting functional insights from such types of datasets, and this has primed a rapid development of bioinformatics tools and resources over the last decade. Many of these tools are specialized databases that can be mined for annotation and pathway enrichment, whereas others provide a platform to genera...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Search Databases and Statistics: Pitfalls and Best Practices in Phosphoproteomics
Advances in mass spectrometric instrumentation in the past 15 years have resulted in an explosion in the raw data yield from typical phosphoproteomics workflows. This poses the challenge of confidently identifying peptide sequences, localizing phosphosites to proteins and quantifying these from the vast amounts of raw data. This task is tackled by computational tools implementing algorithms that match the experimental data to databases, providing the user with lists for downstream analysis. Several platforms for such automated interpretation of mass spectrometric data have been developed, each having strengths and weakness...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
From Phosphosites to Kinases
Kinases play a pivotal role in propagating the phosphorylation-mediated signaling networks in living cells. With the overwhelming quantities of phosphoproteomics data being generated, the number of identified phosphorylation sites (phosphosites) is ever increasing. Often, proteomics investigations aim to understand the global signaling modulation that takes place in different biological conditions investigated. For phosphoproteomics data, identifying the kinases central to mediating this response is key. This has prompted several efforts to catalogue the immense amounts of phosphorylation data and known or predicted kinase...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Resources for Assignment of Phosphorylation Sites on Peptides and Proteins
Reversible protein phosphorylation is a key regulatory posttranslational modification that plays a significant role in major cellular signaling processes. Phosphorylation events can be systematically identified, quantified, and localized on protein sequence using publicly available bioinformatic tools. Here we present the software tools commonly used by the phosphoproteomics community, discuss their underlying principles of operation, and provide a protocol for large-scale phosphoproteome data analysis using the MaxQuant software suite. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
Phosphoprotein Detection by High-Throughput Flow Cytometry
Phospho flow cytometry is a powerful technique for the detection of protein phosphorylation events that, like Western blotting, relies on phospho-epitope-specific antibodies. In contrast to the latter, however, multidimensional and directly quantifiable data is obtained at the single-cell level allowing separate analysis of small cell populations in complex cellular mixtures. Furthermore, up to 30 phospho-specific antibodies or antibodies identifying other posttranslational modifications in combination with cell surface markers can be analyzed in a single experiment. Utilizing a technique called fluorescent cell barcoding ...
Source: Springer protocols feed by Protein Science - November 19, 2015 Category: Biochemistry Source Type: news
