Here, we characterize the performance of midia-PASEF, a novel DIA scan mode using mobility-specific scanning of overlapping quadrupole windows to optimally cover the ion population in the ion mobility-mass to charge plane for various sample types, including phosphopeptidome and mixed proteome samples. Using diagonal scanning, midia-PASEF covers the entire precursor ion mass range without sacrificing cycle time, thereby maximizing information content in DIA acquisitions. Scan-specific encoding of quadrupole positions enables the determination of the precursor m/z of each fragment ion by dedicated precursor predoction algorithms. To process the resulting high-complexity datasets, we developed the Snakemake-based midiaID pipeline. midiaID integrates a set of specifically developed algorithms for multidimensional peak detection and for machine-learning-based classification of precursor-fragment relationships, which are stored in the format of a bipartite graph. Using machine learning approaches to refine the MIDIA graph based on initial database results, we significantly improved the specificity of precursor-fragment relationships, thereby surpassing the spectral purity of DDA in our deconvoluted MIDIA-MSMS spectra as indicated by PEAKS database search score distributions. The midiaID pipeline enables fully automated processing and multidimensional deconvolution of midia-PASEF files and exports highly specific DDA-like MSMS spectra with a fragment ion mass accuracy below 10 ppm. In contrast to DDA, midiaPASEF is non-stochastic and thus generates detailed detection profiles of each fragment ion in retention time, ion mobility and quadrupole isolation m/z dimensions, which facilitates the highly specific deconvolution and scoring of precursor-fragment relationships.

Resulting deconvoluted midia-PASEF-MSMS spectra are exported as .mgf files which are suitable for de novo sequencing and can be searched directly with established tools including SAGE, PEAKS, FragPipe and Mascot. In short gradients, midia-PASEF acquisition identifies over 100 unique peptides per second and provides powerful library-free DIA analyses including phosphopeptidome and immunopeptidome samples. midiaPASEF data are also suitable for peptide centric approaches and can be interrogated using spectral libraries. In summary, midia-PASEF provides all benefits of DIA acquisitions, including efficient ion sampling, high duty cycle and excellent reproducibility.