Mammalian host response to pathogens is associated with fluctuations in high abundant proteins in body fluids as well as in regulation of proteins expressed in relatively low copy numbers like cytokines secreted from immune cells and endothelium. Hence, efficient monitoring of proteins associated with host response to pathogens remains a challenging task. In this paper we present a targeted proteome analysis of a panel of 20 proteins that are widely believed to be key players and indicators of bovine host response to mastitis pathogens. Stable isotope labeled variants of two concordant proteotypic peptides from each of these 20 proteins were obtained through the QconCAT method. We present the quantotypic properties of these 40 proteotypic peptides, and discuss their application to research in host pathogen interactions. Our results clearly demonstrate a robust monitoring of 17 targeted host-response proteins. Twelve of these were readily quantified in a simple extraction of mammary gland tissues, while the expression levels of the remaining proteins were too low for direct and stable quantification; hence their accurate quantification requires further fractionation of mammary gland tissues.

The Protein Information and Property Explorer 2 (PIPE2) is an enhanced software program and updated web application that aims at providing the proteomic researcher a simple, intuitive user interface through which to begin inquiry into the biological significance of a list of proteins typically produced by MS/MS proteomic processing software. PIPE2 includes an improved interface, new data visualization options, and new data analysis methods for combining disparate, but related, data sets. In particular, PIPE2 has been enhanced to handle multi-dimensional data like protein abundance, gene expression, and/or interaction data. The current architecture of PIPE2, modeled after that of the Gaggle (a programming infrastructure for interoperability between separately developed software tools), contains independent functional units that can be instantiated and pieced together at the user’s discretion to form a pipelined analysis workflow. Among these functional units is the Network Viewer component, which adds rich network analysis capabilities to the suite of existing proteomic web resources. Additionally, PIPE2 implements a framework within which new analysis procedures can be easily deployed and distributed over the World Wide Web. PIPE2 is available as a web service at http://pipe2.systemsbiology.net/.

Background

Since its inception, proteomics has essentially operated in a discovery mode with the goal of identifying and quantifying the maximal number of proteins in a sample. Increasingly, proteomic measurements are also supporting hypothesis-driven studies, in which a predetermined set of proteins is consistently detected and quantified in multiple samples. Selected reaction monitoring (SRM) is a targeted mass spectrometric technique that supports the detection and quantification of specific proteins in complex samples at high sensitivity and reproducibility. Here, we describe ATAQS, an integrated software platform that supports all stages of targeted, SRM-based proteomics experiments including target selection, transition optimization and post acquisition data analysis. This software will significantly facilitate the use of targeted proteomic techniques and contribute to the generation of highly sensitive, reproducible and complete datasets that are particularly critical for the discovery and validation of targets in hypothesis-driven studies in systems biology.

Result

We introduce a new open source software pipeline, ATAQS (Automated and Targeted Analysis with Quantitative SRM), which consists of a number of modules that collectively support the SRM assay development workflow for targeted proteomic experiments (project management and generation of protein, peptide and transitions and the validation of peptide detection by SRM). ATAQS provides a flexible pipeline for end-users by allowing the workflow to start or end at any point of the pipeline, and for computational biologists, by enabling the easy extension of java algorithm classes for their own algorithm plug-in or connection via an external web site.

This integrated system supports all steps in a SRM-based experiment and provides a user-friendly GUI that can be run by any operating system that allows the installation of the Mozilla Firefox web browser.

Conclusions

Targeted proteomics via SRM is a powerful new technique that enables the reproducible and accurate identification and quantification of sets of proteins of interest. ATAQS is the first open-source software that supports all steps of the targeted proteomics workflow. ATAQS also provides software API (Application Program Interface) documentation that enables the addition of new algorithms to each of the workflow steps. The software, installation guide and sample dataset can be found in http://tools.proteomecenter.org/ATAQS/ATAQS.html