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.