midiaPASEF maximizes information content in data-independent acquisition proteomics

Data-independent acquisition (DIA) approaches provide comprehensive records of all detectable precursor and fragment ions. Here we introduce midiaPASEF, a novel DIA scan mode using mobility-specific micro-encoding of overlapping quadrupole windows to optimally cover the ion population in the ion mobility-mass to charge plane. Using overlapping ion mobility-encoded quadrupole windows, midiaPASEF maximizes information content in DIA acquisitions which enables the determination of the precursor m/z of each fragment ion with a precision of less than 2 Th. The MIDIAlyzer pipeline integrates algorithms for multidimensional peak detection and for machine-learning-based classification of precursor-fragment relationships. MIDIAlyzer enables fully automated processing and multidimensional deconvolution of midia-PASEF files and exports highly specific DDA-like MSMS spectra which are suitable for de novo sequencing and can be searched directly with established tools including PEAKS, FragPipe and Mascot. midiaPASEF acquisition identifies over 40 unique peptides per second and provides powerful library-free DIA analyses including phosphopeptidome and immunopeptidome samples. ### Competing Interest Statement S.B., J.D, J.K. F.K., O.R. are employed by Bruker Daltonics. S.T. is the inventor of a patent of MIDIA technology (US11474072B2), which is held by Bruker Daltonics.