Using synthetic peptides to benchmark peptide identification software and search parameters for MS/MS data analysis

Abstract

Tandem mass spectrometry and sequence database searching are widely used in proteomics to identify peptides in complex mixtures. Here we present a benchmark study in which a pool of 20,103 synthetic peptides was measured and the resulting data set was analyzed using around 1800 different software and parameter set combinations. The results indicate a strong relationship between the performance of an analysis workflow and the applied parameter settings. We present and discuss strategies to optimize parameter settings in order to significantly increase the number of correctly assigned fragment ion spectra and to make the analysis method robust.

Keywords

• Mass spectrometry
• Data analysis
• Classical database search
• Synthetic peptides
• Search engine