biostudies-literatureUnknownShuken SRNIA NIH HHSNCI NIH HHSNIGMS NIH HHSThe GoDig platform enables sensitive, multiplexed targeted pathway proteomics without manual scheduling or synthetic standards. Here we present GoDig 2.0, which increases sample multiplexing to 35-fold, improves time efficiency and reduces scan delays for higher success rates, and allows flexible spectral and elution library generation from different mass spectrometry data types. GoDig 2.0 measures 2.4× more targets than GoDig 1.0, quantifying >99% of 800 peptides in a single run. We compiled a library of 23,989 human phosphorylation sites from a phosphoproteomic dataset and used it to profile kinase signaling differences across cell lines. In human brain tissue, we established a hyperphosphorylated tau assay including pTau127, revealing potential biomarkers for Alzheimer's disease. We alsbioRxiv : the preprint server for biology2025.09.10.675380https://www.ebi.ac.uk/biostudies/studies/S-EPMC12458933biostudies-literatureNext-Generation Multiplexed Targeted Proteomics Quantifies Post-Translational Modifications, Compound-Protein Interactions, and Disease Biomarkers with High Throughput.PMC12458933R01 GM132129K22 CA282268K99 AG088297R01 GM067945Shuken SRDawson SLGassaway BMBeard CRPaulo JAYu QFrere GAGygi SPCanterbury JDZuniga NRMcNerney MWOoi KHfalseNext-Generation Multiplexed Targeted Proteomics Quantifies Post-Translational Modifications, Compound-Protein Interactions, and Disease Biomarkers with High Throughput.The GoDig platform enables sensitive, multiplexed targeted pathway proteomics without manual scheduling or synthetic standards. Here we present GoDig 2.0, which increases sample multiplexing to 35-fold, improves time efficiency and reduces scan delays for higher success rates, and allows flexible spectral and elution library generation from different mass spectrometry data types. GoDig 2.0 measures 2.4× more targets than GoDig 1.0, quantifying >99% of 800 peptides in a single run. We compiled a library of 23,989 human phosphorylation sites from a phosphoproteomic dataset and used it to profile kinase signaling differences across cell lines. In human brain tissue, we established a hyperphosphorylated tau assay including pTau127, revealing potential biomarkers for Alzheimer's disease. We als2025-01-01T00:00:00Z2025 Sep2026-06-30T03:23:17.411Z2026-06-30T03:16:12.234ZS-EPMC124589334100062510.1101/2025.09.10.675380