Half decimal place rule

Many MS2 spectra in bottom-up proteomics experiments remain unassigned. To improve proteome coverage, we applied the half decimal place rule (HDPR) to remove non-peptidic molecules. The HDPR considers the ratio of the first digit after the decimal point to the full molecular mass and results in a relatively small permitted mass window for most peptides. Although the HDPR has been described previously, it has never been integrated into an acquisition strategy using high resolution mass spectrometers. The HDPR was applied to three technical replicates of an in-solution tryptic digest of HeLa cells which were analysed by LC-MS using a Q Exactive mass spectrometer.