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

High Resolution Image Download MS PowerPoint Slide 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 ( R 2 = 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.