Bioinformatics for LC-MS/MS-based proteomics.

Mass spectrometry instrumentation has continued to develop rapidly in the last two decades, enabled in part by advances in microelectronic hardware controllers and computerized control and data acquisition systems. The wealth and complexity of data produced by a modern instrument is such that the data can no longer be analyzed manually. Computerized data analysis has become de rigueur and the bioinformatics field has expanded to provide software applications for all aspects of the data analysis needed by LC-MS/MS. The bioinformatics field is evolving rapidly and software applications are continually being improved or replaced for existing applications as well as developed to support new types of experiments and analysis enabled by modern instrumentation. Entire books have been written on MS data analysis in proteomics but this review will be necessarily brief. In this chapter we will review the bioinformatics software applications available for different LC-MS/MS analysis tasks.

PIGOK: Linking protein identity to gene ontology and function.

Here we introduce a computer database that allows for the rapid retrieval of physicochemical properties, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes information about a protein or a list of proteins. We applied PIGOK analyzing Schizosaccharomyces pombe proteins displaying differential expression under oxidative stress and identified their biological functions and pathways. The database is available on the Internet at http://pc4-133.ludwig.ucl.ac.uk/pigok.html.

Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform.

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye and analysed by two-dimensional difference gel electrophoresis. Gel images analysed off-line, using the DeCyder image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.