Mass measurement and precursor mass assignment are independent processes in proteomic data acquisition. Due to misassignments to C-13 peak, or for ot
her reasons, extensive precursor mass s
hifts (i.e., deviations of t
he measured from calculated precursor neutral masses) in LC-MS/MS data obtained wit
h t
he
hig
h-accuracy LTQ-Orbitrap mass spectrometers
have been reported in previous studies. Alt
houg
h computational met
hods for post-acquisition reassignment to monoisotopic mass
have been developed to curate t
he MS/MS spectra prior to database searc
h, a simpler met
hod for estimating t
he fraction of spectra wit
h precursor mass s
hift so as to determine w
het
her t
he data require curation remains desirable. Here, we provide t
he evidence t
hat an easy approac
h, w
hic
h applies a large precursor tolerance (2.1 Da or
hig
her) in
SEQUEST searc
h against a forward and decoy protein sequence database and t
hen filters t
he data wit
h PeptideProphet peptide identification probability (
p 鈮?#xA0;0.9), could detect most of t
he MS/MS spectra containing inaccurate precursor masses. Furt
hermore, t
hroug
h t
he implementation of artificial mass s
hifts on 4000 randomly selected MS/MS spectra, w
hic
h originally
had accurate precursor mass assigned by t
he mass spectrometers, we demonstrated t
hat t
he accuracy of t
he precursor mass
has almost negligible influence on t
he efficacy and fidelity of peptide identification.<
h4 class="
h4">Biological significance
h4>
Integral precursor mass shift is a known problem and thus proteomic data should be handled and analyzed properly to avoid losing important protein identification and/or quantification information. A quick and easy approach for estimating the number of MS/MS spectra with inaccurate precursor mass assignments would be helpful for evaluating the performance of the instrument, determining whether the data requires curation prior to database search or should be searched with specific search parameter(s). Here we demonstrated most of the MS/MS spectra with inaccurate mass assignments (integral or non-integral changes) that could be easily identified by database search with large precursor tolerance windows.