IsoPS-DIA: Dual Functionality of Absolute Targeted Quantification and Global Proteome Profiling

Current gene testing reveals only the mutation status yet lacks protein expression of the actual drug target. A comprehensive evaluation requires methods that integrate the absolute quantification of mutant proteins with global profiling of downstream signaling and resistance pathways. Here, we present an isotope pair-separated data-independent acquisition (IsoPS-DIA) strategy with a dual functionality of multiplexed absolute quantification and global proteome profiling in a single run. IsoPS-DIA features a dual-window design: narrow consecutive windows separate light/heavy-isotope-labeled peptide pairs to reduce coisolation interference and maximize usable fragment ions, while wide variable windows capture proteome-wide information. Using EGFR mutations (L858R, G719A, Del19) in lung cancer cell lines as a model, IsoPS-DIA achieved subfemtomole sensitivity (LOQ 36-222 amol), excellent linearity across 4 orders of magnitude (R² = 0.998-0.999), and high reproducibility (median CV ∼ 3%). For the first time, the method quantified endogenous EGFR and KRAS driver mutations alongside their wild-type counterparts, revealing allele-specific expression heterogeneity not captured by genomic variant allele frequency (VAFs). Simultaneously, IsoPS-DIA achieved >6,000 protein coverage across six cell lines, uncovering variability in EGFR signaling cascades and actionable variants such as KRAS-G12S. Benchmarking against parallel reaction monitoring (PRM), fixed scanning window (Fix-DIA) and variable scanning windows DIA (Var-DIA) confirmed IsoPS-DIA's superior accuracy and reproducibility without compromising proteome coverage. IsoPS-DIA is compatible with both Orbitrap and quadrupole time-of-flight mass spectrometry (Q-TOF) platforms using standard software, and we provide an open-source window-design tool to facilitate adoption. These results demonstrate the unique capability of IsoPS-DIA to bridge genotype and proteotype through precise, scalable, and reproducible quantification, offering a broadly applicable platform for precision oncology and other applications.