Systematic optimization and benchmarking of synchro-PASEF for high-throughput phosphoproteome profiling
High-throughput data-independent acquisition (DIA) workflows paired with short chromatographic separations are increasingly adopted for systems biology and clinical proteomics. However, narrower peak widths from rapid separations demand faster mass spectrometer cycle times to maintain quantitative depth and reproducibility. The synchro-PASEF acquisition mode on timsTOF mass spectrometers diagonally scans across ion mobility and m/z space, enabling efficient sampling of the precursor ion cloud with shortened cycle times. While synchro-PASEF has demonstrated competitive identification depth for global protein abundance samples compared to conventional dia-PASEF, its performance for phosphoproteomics-where the precursor ion cloud is characteristically broader and bimodally distributed-has not been evaluated. Here, we systematically optimized synchro-PASEF methods for phosphoproteomics and benchmarked performance against two dia-PASEF methods across three sub-hour separations. We found that synchro-PASEF performance depends critically on balancing diagonal window number, total isolation width, and gradient length, with longer gradients favoring more windows for selectivity and shorter gradients favoring fewer windows to preserve sampling frequency. An optimized configuration quantified over 19,000 localized phosphosites using a 23-minute separation. Retention time summation (RTsum) with a factor of 2 increased phosphopeptide identifications by 5-20% and reduced phosphosite-level coefficients of variation by up to 30% across all dia-PASEF and synchro-PASEF methods tested. Using {beta}2-adrenergic receptor (B2AR) activation as a signaling model, we demonstrate that label-free DIA phosphoproteomics can be used to model phosphoproteomics dose-response relationships, showing that synchro-PASEF and dia-PASEF produce highly concordant phosphoproteomic responses, with comparable numbers of responding phosphosites, similar effect sizes, and nearly identical predicted protein kinase A (PKA) substrates downstream of the activated B2AR. While synchro-PASEF did not surpass optimized dia-PASEF in identification depth, its comparable biological performance and amenability to post-acquisition optimization through RTsum support its utility for high-throughput phosphoproteomics. This work provides a transferable framework for synchro-PASEF method optimization and demonstrates the broad utility of retention time summation for PASEF-based phosphoproteomics workflows. HighlightsO_LISystematic benchmarking of synchro-PASEF for typical phosphoproteomics workflows. C_LIO_LIRT summation improves IDs and quantitative precision for dia-PASEF and synchro-PASEF C_LIO_LIdia-PASEF and synchro-PASEF capture dose-response phosphosignaling with comparable performance C_LIO_LIProvides transferable framework for high-throughput DIA method design C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/734570v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@f83d3forg.highwire.dtl.DTLVardef@17d2e11org.highwire.dtl.DTLVardef@15b61eeorg.highwire.dtl.DTLVardef@7a667e_HPS_FORMAT_FIGEXP M_FIG C_FIG