Synchro-PASEF Allows Precursor-Specific Fragment Ion Extraction and Interference Removal in Data-Independent Acquisition

Data-independent acquisition (DIA) methods have become increasingly popular in mass spectrometry–based proteomics because they enable continuous acquisition of fragment spectra for all precursors simultaneously. However, these advantages come with the challenge of correctly reconstructing the precursor–fragment relationships in these highly convoluted spectra for reliable identification and quantification. Here, we introduce a scan mode for the combination of trapped ion mobility spectrometry with parallel accumulation—serial fragmentation (PASEF) that seamlessly and continuously follows the natural shape of the ion cloud in ion mobility and peptide precursor mass dimensions. Termed synchro-PASEF, it increases the detected fragment ion current several-fold at sub-second cycle times. Consecutive quadrupole selection windows move synchronously through the mass and ion mobility range. 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 relationships in ion mobility and mass dimensions and effectively deconvolutes the DIA fragment space. Importantly, the partitioned parts of the fragment ion transitions provide a further dimension of specificity via a lock-and-key mechanism. This is also advantageous for quantification, where signals from interfering precursors in the DIA selection window do not affect all partitions of the fragment ion, allowing to retain only the specific parts for quantification. Overall, we establish the defining features of synchro-PASEF and explore its potential for proteomic analyses. [Display omitted] •Synchro-PASEF is a highly efficient scan mode for MS-based proteomics.•It allows very fast cycle times, amplifying the fragment signal three-fold.•Fragments are directly linked to precursors via precursor slicing.•Precursor slicing enables deconvolution to pure fragmentation spectra.•Synchro-PASEF unites the benefits of data-dependent and data-independent acquisition. The novel scan mode synchro-PASEF efficiently follows the natural shape of the precursor cloud in the m/z and the trapped ion mobility space. This manifests in short cycle times and high sampling frequency of the eluting peptides and fragment signal amplification. Additionally, the seamlessly movement of the quadrupole nearly universally slices the precursor in the ion mobility dimension. The slicing position adds tremendous