The experiment with complex biological matrix and heavy peak overlap. A GC-MS experiment recorded on foetal calf serum spiked with a mix of 17 known compounds. The total ion chromatogram (TIC) is shown in dotted line, and individual ion chromatograms (ICs) in the m/z range 50–550 are overlaid in full line. The true multi-component GC-MS peaks, identified manually by an experienced operator, are shown as filled triangles at the bottom of the graph. The broad peak observed in the retention time range 464-469 s is an overloaded urea peak. The four packages have each picked different numbers of peaks, with AMDIS and XCMS picking many more peaks than either PyMS or AnalyzerPro. Both PyMS and AnalyzerPro performed similarly to an experienced operator, with some false positive peaks reported. In the area near 476 s both software reported several weak signals, not annotated by the human operator. For the list of detected signals see Additional file 4.