Abstract
Kinomics is an emerging field of science that involves the study of global kinase activity. As kinases are essential players in virtually all cellular activities, kinomic testing can directly examine protein function, distinguishing kinomics from more remote, upstream components of the central dogma, such as genomics and transcriptomics. While there exist several different approaches for kinomic research, peptide microarrays are the most widely used and involve kinase activity assessment through measurement of phosphorylation of peptide substrates on the array. Unfortunately, bioinformatic tools for analyzing kinomic data are quite limited necessitating the development of accessible open access software in order to facilitate standardization and dissemination of kinomic data for scientific use. Here, we examine and present tools for data analysis for the popular PamChip® (PamGene International) kinomic peptide microarray. As a result, we propose (1) a procedural optimization of kinetic curve data capture, (2) new methods for background normalization, (3) guidelines for the detection of outliers during parameterization, and (4) a standardized data model to store array data at various analytical points. In order to utilize the new data model, we developed a series of tools to implement the new methods and to visualize the various data models. In the interest of accessibility, we developed this new toolbox as a series of JavaScript procedures that can be utilized as either server side resources (easily packaged as web services) or as client side scripts (web applications running in the browser). The aggregation of these tools within a Kinomics Toolbox provides an extensible web based analytic platform that researchers can engage directly and web programmers can extend. As a proof of concept, we developed three analytical tools, a technical reproducibility visualizer, an ANOVA based detector of differentially phosphorylated peptides, and a heatmap display with hierarchical clustering.
Highlights
Kinases are fundamental to cellular life; they provide essential regulation and function in nearly every pathway
The kinome toolbox is a series of tools utilized for the processing and visualization of PamChip kinomic peptide arrays
The library is a series of packages that can be utilized to convert data between levels and to enrich the kinome objects with a rich software development kit (SDK) for easier use
Summary
Kinases are fundamental to cellular life; they provide essential regulation and function in nearly every pathway. A variety of isolation techniques are used in conjunction with tandem MS to quantify and accurately measure kinase activity[7]. While these MS techniques are continuing to develop and gaining popularity, peptide arrays remain the more commonly used kinomic approach. This procedure can be applied to each image separately. Since our kinome toolbox handles the signal and background intensities separately, we noted that the background itself is a function of the corresponding signal—in other words, adjacent higher intensity spots will influence the background intensity. Based on the technical reproducibility results, and the lack of an experiment-specific correction factor (ce), we recommend utilizing both background correction in conjunction with ls(Á)
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