Updated project metadata. Data independent acquisition methods have become increasingly popular in mass spectrometry (MS)-based proteomics because they enable parallel and continuous acquisition of fragment spectra. However, these advantages come with the challenge of correctly reconstructing the precursor-fragment relationship in these highly multiplexed spectra for reliable identification and quantification. Here we introduce a scan mode for the combination of trapped ion mobility spectrometry (TIMS) with parallel accumulation – serial fragmentation (PASEF) that naturally and continuously follows the ion cloud in ion mobility and peptide mass dimensions as opposed to the step functions we employed before. Termed synchro-PASEF, it increases fragment yield several fold at sub-second cycle times. The quadrupole selection window moves synchronously through the mass and ion mobility range, deconvoluting the DIA spectra and defining precursor-quadrupole relationships. In this process, the quadrupole slices through the peptide precursors, which separates fragment ion signals of each precursor into adjacent synchro-PASEF scans. This precisely defines precursor – fragment relationship in ion mobility and mass dimensions. Importantly, the two parts of the fragment ion transitions in synchro-PASEF provide a further dimension of specificity via a lock and key mechanism. This is especially advantageous for quantification, where signals from interfering precursors in the dia selection window only affect the top or bottom part of the fragment ion, allowing them to be filtered out. In this paper, we establish the defining features of synchro-PASEF and explore its potential for proteomics analyses.