Background: The recently introduced DIA acquisition scheme, midia-PASEF, integrates the concepts and benefits of dia-PASEF, scanning quadrupole and overlapping quadrupole window acquisition. Using an overlapping ion mobility-encoded quadrupole window scheme facilitates fragment-precursor ion assignment and increases duty cycle as compared to fixed window schemes. midia-PASEF acquisition improves the precision of precursor-fragment relationships to <2 Th and resulting deconvoluted MIDIA-MS/MS fragment ion spectra display DDA-like quality with fragment ion mass accuracies below 10 ppm. In the present work, we conducted an in-depth evaluation of the midia-PASEF workflow for the analysis of phosphoproteomic samples.

Methods: Mouse brains were lysed and tryptically digested. Phosphopeptides were enriched using TiO₂ tips (GL Sciences, Japan) according to the manufacturer´s protocol. Phosphopeptides were analysed by LC-MS on mass spectrometers from the Bruker timsTOF series coupled to nanoElute LC systems (Bruker Corporation). Samples were separated on a reversed phase C18 column (IonOpticks, 25 cm x 75 μm 1.7 μm). Data were acquired using parallel accumulation serial fragmentation (PASEF) enhanced data-dependent (DDA) and data-independent (DIA) methods such as dia-PASEF, as well as midia-PASEF applying different isolation schemes for precursor ion selection. Data were analysed applying different software tools including PEAKS, Spectronaut and our in-house developed pipeline for midia-PASEF data processing.

Results: We evaluated and compared different midia-PASEF acquisition schemes applying different quadrupole isolation window widths (36 Th, 24 Th, 20 Th). In the original midia-PASEF method, quadrupole isolation window positions are shifted 12 Th as compared to the previous frame during acquisition resulting in an overlap of 24 Th between two neighboring frames. Here, we additionally tested the impact of changing the window overlap to 12 Th and 10 Th. The performance of the different midia-PASEF methods was compared to PASEF and dia-PASEF applying different tools for data processing. We could demonstrate that midia-PASEF provides, upon deconvolution, DDA-like MS/MS spectra that can be analyzed using either algorithms tailored for DDA data processing or our in-house developed pipeline, which has been optimized for the analysis of midia-PASEF data. Best results could be obtained applying a 36x24 Th window scheme achieving higher and similar phoshoproteome coverage as compared to dia-PASEF and dda-PASEF, respectively.

Conclusions: Evaluation of midia-PASEF for the analysis of phosphoproteomic samples revealed best results for a 36x24 Th-window scheme and subsequent processing using our in-house developed midia-PASEF processing pipeline, midiaID, combined with MS2 rescore.