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

•We developed a system to systematically analyze mass spectrometry data using different parameters and search engines.•Parameters and search engines were combined in over 1800 different ways.•High variability of the results is observed for individual search engines.•Combining several engines gives higher robustness toward sub-optimal parameters.

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.

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