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

## TL;DR

IsoPS-DIA is a new mass spectrometry method that simultaneously measures specific mutant proteins and profiles the entire proteome, improving precision in cancer research.

## Contribution

IsoPS-DIA introduces a dual-window DIA strategy for accurate absolute quantification and global proteome profiling in one experiment.

## Key findings

- IsoPS-DIA achieved subfemtomole sensitivity and high reproducibility in quantifying EGFR and KRAS mutations.
- The method revealed allele-specific expression heterogeneity not captured by genomic variant allele frequencies.
- IsoPS-DIA provided >6,000 protein coverage across six cell lines, uncovering variability in EGFR signaling and actionable variants.

## Abstract

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.