Label-free quantitative proteomics technology utilizes the unique dual TIMS design of the timsTOF series mass spectrometer and the ddaPASEF® acquisition mode to achieve protein qualitative and quantitative analysis. The ddaPASEF acquisition mode combines data-dependent acquisition (DDA) with PASEF (Parallel Accumulation–Serial Fragmentation) technology. In the ddaPASEF acquisition mode, precursor ions with high ion intensity are selected from the MS1 spectra to set the mass spectrum scan range to specific narrow mass windows. Subsequently, the selected target precursor ions are sequentially fragmented in the MS2 spectra, recording their retention time, mass-to-charge ratio, ion intensity, and ion mobility. Finally, protein qualitative and quantitative analysis is conducted based on this information. Label-free quantitative proteomics technology offers simplicity in operation, independence from expensive labeling reagents, reduced data analysis complexity, and approximately 12-fold improved detection sensitivity compared to traditional label-free methods.