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 prote-omics because they enable continuous acquisition of fragment spectra for all precursors simultaneously. How-ever, these advantages come with the challenge of correctly reconstructing the precursor-fragment relation-ships 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 frag-mentation (PASEF) that seamlessly and continuously fol-lows 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 de-fines 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.

Automated summary

Data-independent acquisition (DIA) methods have benefits of continuous acquisition of fragment spectra for all precursors simultaneously, but reconstructing the precursor-fragment relationships in convoluted spectra poses challenges. In this study, synchro-PASEF scan mode is introduced, combining trapped ion mobility spectrometry and parallel accumulation-serial fragmentation (PASEF) to accurately define precursor-fragment relationships and deconvolute the DIA fragment space. The synchro-PASEF method increases fragment ion current and provides a lock-and-key mechanism for specificity in quantification. Overall, this research establishes the defining features of synchro-PASEF and explores its potential for proteomic analyses.