Investigation of Effects of the Spectral Library on Analysis of diaPASEF Data

Targeted
analysis of data-independent acquisition (DIA) data needs
a spectral library, which is generated by data-dependent acquisition
(DDA) experiments or directly from DIA data. A comparison of the DDA
library and DIA library in analyzing DIA data has been reported. However,
the effects of different spectral libraries on the analysis of diaPASEF
data have not been investigated. Here, we generate different spectral
libraries with varying proteome coverage to analyze parallel accumulation–serial
fragmentation (diaPASEF) data. Besides, we also employ the library-free
strategy. The library, constructed by extensive fractionation DDA
experiments, produces the highest numbers of precursors and proteins
but with a high percentage of missing values. The library-free strategy
identifies 10–20% fewer proteins than the library-based method
but with a high degree of data completeness. A further study shows
that the library-free strategy, although it identifies fewer proteins
than the library-based method, leads to similar biological conclusions
as the library-based method.