In recent years mass spectrometry-based proteomics became very important and now it is the
leading approach employed in high-throughput analysis. Its relevance increased thanks to availability of
genome-sequence database and the development of high sensitivity instruments allows a rapid and automated
proteins profiling. The need to analyze complex biological samples at a large-scale level required the
development of computational tools to analyze and statistically evaluate data generated from mass
spectrometry (MS) experiments. These aspects have stimulated the young emerging field of bioinformatics in
proteomics to introduce new software and algorithms to handle large and heterogeneous data sets and to
improve the knowledge of discovery process. This review discusses of the most recent progresses in
bioinformatics tools useful in mass spectrometry-based proteomics. In particular we will be focusing on
software applications applied to proteomics profiling biomarker discovery and cluster analysis. Finally since
most known mechanisms leading to biological processes involve different molecules here are reported the
most recent methodologies to investigate biological systems through their underlying interactions with
particular attention to protein-protein interaction.