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Application of the half decimal place rule to increase the peptide identification rate
Christian J Koehler1, Ravi Chand Bollineni1, Bernd Thiede1
1Department of Biosciences, University of Oslo, Oslo, Norway.
Rationale:
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 digits after the decimal point to the full molecular mass and results in a relatively small permitted mass window for most peptides.
Methods:
First, the HDPR mass filter was calculated for the human and other proteomes. Subsequently, the HDPR was applied to three technical replicates of an in-solution tryptic digest of HeLa cells which were analysed by liquid chromatography/mass spectrometry (LC/MS) using a quadrupole-orbitrap mass spectrometer (Q Exactive). In addition, the same sample was analysed three times with a fixed exclusion list. The exclusion list was based on only choosing doubly charged ions for fragmentation.
Results:
The peptide spectrum match (PSM) rate increased by 2-4% applying HDPR filters from 0.1-0.25 Da and 75-150 ppm, respectively. Excluding all MS2 events by applying an HDPR filter of doubly charged ions, we were able to improve PSMs by 0.9% and the PSM rate by 2.5%.
Conclusions:
An algorithm to filter precursors based on the HDPR was established to improve the targeting of the acquisition of MS2 spectra in data-dependent acquisition (DDA) experiments. According to our data, a total gain of PSMs of 1-5% might be achievable if the HPDR filter would already be applied during MS data acquisition. Copyright © 2016 John Wiley & Sons, Ltd.
