Effect of High-Accuracy Precursor Masses on Phosphopeptide Identification from MS³ Spectra

This contribution investigates the impact of precursor ion accuracy on the number of phosphopeptides identified from neutral loss-triggered MS3 spectra acquired on high-accuracy instruments. We show that replacing the MS3 precursor (parent) ion mass by a highly accurate MS2 precursor (grandparent) mass increases the number of identified phosphopeptides 3- to 5-fold. The number of unique phosphopeptides identified in addition to the MS2 data increases from 6% to 24%. Our results illustrate that the sensitivity of phosphopeptide identification depends critically on the mass tolerance setting applied in the database search. This study demonstrates that MS3 parent ion masses should always be substituted by high-accuracy grandparent ion masses and that error tolerance settings should be set as low as instrument accuracy permits to yield the best possible identification sensitivity.