Impact of the Identification Strategy on the Reproducibility of the DDA and DIA Results

Data-independent acquisition (DIA)
is a promising technique for the proteomic analysis of complex protein
samples. A number of studies have claimed that DIA experiments are
more reproducible than data-dependent acquisition (DDA), but these
claims are unsubstantiated since different data analysis methods are
used in the two methods. Data analysis in most DIA workflows depends
on spectral library searches, whereas DDA typically employs sequence
database searches. In this study, we examined the reproducibility
of the DIA and DDA results using both sequence database and spectral
library search. The comparison was first performed using a cell lysate
and then extended to an interactome study. Protein overlap among the
technical replicates in both DDA and DIA experiments was 30% higher
with library-based identifications than with sequence database identifications.
The reproducibility of quantification was also improved with library
search compared to database search, with the mean of the coefficient
of variation decreasing more than 30% and a reduction in the number
of missing values of more than 35%. Our results show that regardless
of the acquisition method, higher identification and quantification
reproducibility is observed when library search was used.