RESUMO
Peptide microarrays consisting of defined phosphorylation target sites are an effective approach for high throughput analysis of cellular kinase (kinome) activity. Kinome peptide arrays are highly customizable and do not require species-specific reagents to measure kinase activity, making them amenable for kinome analysis in any species. Our group developed software, Platform for Integrated, Intelligent Kinome Analysis (PIIKA), to enable more effective extraction of meaningful biological information from kinome peptide array data. A subsequent version, PIIKA2, unveiled new statistical tools and data visualization options. Here we introduce PIIKA 2.5 to provide two essential quality control metrics and a new background correction technique to increase the accuracy and consistency of kinome results. The first metric alerts users to improper spot size and informs them of the need to perform manual resizing to enhance the quality of the raw intensity data. The second metric uses inter-array comparisons to identify outlier arrays that sometimes emerge as a consequence of technical issues. In addition, a new background correction method, background scaling, can sharply reduce spatial biases within a single array in comparison to background subtraction alone. Collectively, the modifications of PIIKA 2.5 enable identification and correction of technical issues inherent to the technology and better facilitate the extraction of meaningful biological information. We show that these metrics demonstrably enhance kinome analysis by identifying low quality data and reducing batch effects, and ultimately improve clustering of treatment groups and enhance reproducibility. The web-based and stand-alone versions of PIIKA 2.5 are freely accessible at via http://saphire.usask.ca.