With a multitude of DIA approaches developed recently, many of their features are not yet well explored. In this study we make use of a biologically relevant sample, the synaptosome, as a model to compare the spectral libraries generated by (1) DDA solely on Orbitrap (OT), (2) Orbitrap for MS1 scan and ion trap for MS/MS acquisition (IT), and (3) directDIA derived from DIA data. Next, we compared the number of spectral library features recovered, and quality of quantification, by DIA and WiSIM-DIA, and suggest future improvement.