msPurity: Automated Evaluation of Precursor Ion Purity for Mass Spectrometry-Based Fragmentation in Metabolomics

Tandem mass spectrometry (MS/MS or MS²) is a widely used approach for structural annotation and identification of metabolites in complex biological samples. The importance of assessing the contribution of the precursor ion within an isolation window for MS² experiments has been previously detailed in proteomics, where precursor ion purity influences the quality and accuracy of matching to mass spectral libraries, but to date, there has been little attention to this data-processing technique in metabolomics. Here, we present msPurity, a vendor-independent R package for liquid chromatography (LC) and direct infusion (DI) MS² that calculates a simple metric to describe the contribution of the selected precursor. The precursor purity metric is calculated as "intensity of a selected precursor divided by the summed intensity of the isolation window". The metric is interpolated at the recorded point of MS² acquisition using bordering full-scan spectra. Isotopic peaks of the selected precursor can be removed, and low abundance peaks that are believed to have limited contribution to the resulting MS² spectra are removed. Additionally, the isolation efficiency of the mass spectrometer can be taken into account. The package was applied to Data Dependent Acquisition (DDA)-based MS² metabolomics data sets derived from three metabolomics data repositories. For the 10 LC-MS² DDA data sets with > ±1 Da isolation windows, the median precursor purity score ranged from 0.67 to 0.96 (scale = 0 to +1). The R package was also used to assess precursor purity of theoretical isolation windows from LC-MS data sets of differing sample types. The theoretical isolation windows being the same width used for an anticipated DDA experiment (±0.5 Da). The most complex sample had a median precursor purity score of 0.46 for the 64,498 XCMS determined features, in comparison to the less spectrally complex sample that had a purity score of 0.66 for 5071 XCMS features. It has been previously reported in proteomics that a purity score of <0.5 can produce unreliable spectra matching results. With this assumption, we show that for complex samples there will be a large number of metabolites where traditional DDA approaches will struggle to provide reliable annotations or accurate matches to mass spectral libraries.