{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T13:47:32Z","timestamp":1782827252773,"version":"3.54.5"},"posted":{"date-parts":[[2026,6,27]]},"group-title":"Biochemistry","reference-count":45,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,27]],"date-time":"2026-06-27T00:00:00Z","timestamp":1782518400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000026","name":"National Institute on Drug Abuse","doi-asserted-by":"crossref","award":["R01DA056354"],"award-info":[{"award-number":["R01DA056354"]}],"id":[{"id":"10.13039\/100000026","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100011098","name":"Stanford Bio-X","doi-asserted-by":"crossref","award":["IIP12-21"],"award-info":[{"award-number":["IIP12-21"]}],"id":[{"id":"10.13039\/100011098","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100005492","name":"Stanford University","doi-asserted-by":"crossref","award":["350788"],"award-info":[{"award-number":["350788"]}],"id":[{"id":"10.13039\/100005492","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"crossref","award":["T32GM154663"],"award-info":[{"award-number":["T32GM154663"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,27]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  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\n                  <jats:italic>m\/z<\/jats:italic>\n                  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\u2013where the precursor ion cloud is characteristically broader and bimodally distributed\u2013has 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.\n                <\/jats:p>\n                <jats:p>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\u201320% and reduced phosphosite-level coefficients of variation by up to 30% across all dia-PASEF and synchro-PASEF methods tested. Using \u03b22-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.<\/jats:p>\n                <jats:sec id=\"s1\">\n                  <jats:title>Highlights<\/jats:title>\n                  <jats:list list-type=\"bullet\">\n                    <jats:list-item>\n                      <jats:p>Systematic benchmarking of synchro-PASEF for typical phosphoproteomics workflows.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>RT summation improves IDs and quantitative precision for dia-PASEF and synchro-PASEF<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>dia-PASEF and synchro-PASEF capture dose-response phosphosignaling with comparable performance<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Provides transferable framework for high-throughput DIA method design<\/jats:p>\n                    <\/jats:list-item>\n                  <\/jats:list>\n                <\/jats:sec>\n                <jats:sec id=\"s2\">\n                  <jats:title>Graphical Abstract<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"734570v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.06.26.734570","type":"posted-content","created":{"date-parts":[[2026,6,28]],"date-time":"2026-06-28T10:30:43Z","timestamp":1782642643000},"source":"Crossref","is-referenced-by-count":0,"title":["Systematic optimization and benchmarking of synchro-PASEF for high-throughput phosphoproteome profiling"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0971-9711","authenticated-orcid":false,"given":"Dain R.","family":"Brademan","sequence":"first","affiliation":[{"name":"Department of Molecular & Cellular Physiology, Stanford School of Medicine, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5830-5347","authenticated-orcid":false,"given":"Angelina A.","family":"Mullarkey","sequence":"additional","affiliation":[{"name":"Department of Molecular & Cellular Physiology, Stanford School of Medicine, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9439-288X","authenticated-orcid":false,"given":"Mia L.","family":"Greeson","sequence":"additional","affiliation":[{"name":"Department of Molecular & Cellular Physiology, Stanford School of Medicine, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6773-2594","authenticated-orcid":false,"given":"Sarah","family":"Szvetecz","sequence":"additional","affiliation":[{"name":"Khoury College of Computer Sciences, Northeastern University, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1728-1104","authenticated-orcid":false,"given":"Olga","family":"Vitek","sequence":"additional","affiliation":[{"name":"Khoury College of Computer Sciences, Northeastern University, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6363-2644","authenticated-orcid":false,"given":"Emily E.","family":"Blythe","sequence":"additional","affiliation":[{"name":"Department of Genetics, Cell Biology, and Development, University of Minnesota, Twin Cities, Minneapolis, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0896-5910","authenticated-orcid":false,"given":"Ruth","family":"H\u00fcttenhain","sequence":"additional","affiliation":[{"name":"Department of Molecular & Cellular Physiology, Stanford School of Medicine, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026063006050661000_2026.06.26.734570v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-024-02558-2"},{"key":"2026063006050661000_2026.06.26.734570v1.2","doi-asserted-by":"crossref","unstructured":"Frommelt, F. , Federico, U. , Aebersold, R. , and Fossati, A . 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