Systematic Assessment of the Effect of Internal Library in Targeted Analysis of SWATH-MS

Targeted analysis of sequential window acquisition of
all theoretical
mass spectra (SWATH-MS) requires the spectral library, which can be
generated by shotgun mass spectrometry (MS) or by the pseudo-spectra
files directly obtained from SWATH-MS data. The external library generated
by shotgun MS is employed in most SWATH-MS research. However, performance
of the internal library, which is constructed by pseudo-spectra files,
in the targeted analysis of SWATH-MS has not been systemically evaluated.
Here, we show that up to 40% of the peptides detected by the internal
library were not overlapped with those detected by the external library
for most SWATH-MS data sets. However, the internal library did not
identify extra phosphopeptides compared with the external library
for phosphoproteomic SWATH-MS data. Therefore, the internal library
should be incorporated into the external library for targeted analysis
of nonphosphoproteomic SWATH-MS, given that it can significantly increase
the number of peptides of SWATH-MS without requiring additional instrument
measurement time.