演題概要

2P-33（3A-O1-1015）

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IsoPS-DIA Mass Spectrometry: Simultaneous Absolute Quantification and Global Proteome Profiling

(¹IoC, Academia Sinica・ ²NTU)
^oChiu, Huan-Chi¹・ Chan, Hsin-Ju¹・ Chen, Li-Yu¹・ Hsu, Hsiang-En¹・ Chen, Yu-Ju^1,2

We present a novel DIA approach termed Isotope Pairs-Differentiated Data Independent Acquisition (IsoPS-DIA), which uses narrow windows (3-5 m/z) for precise absolute quantification of endogenous light and isotope-labeled heavy internal standard peptides, along with wider windows for global profiling. To establish IsoPS-DIA, we selected 25 target peptides representing FDA-approved drug targets in non-small cell lung cancer (NSCLC), such as EGFR, mutant EGFR, KRAS, PDL1, ERBB2, MET…, as a model study. IsoPS-DIA quantified 15 targeted peptides in NSCLC cell lines. Compared to conventional DIA (Fix-DIA) DIA, IsoPS-DIA showed a 6-fold increase in signal-to-noise ratio with similar protein identification coverage (4622 proteins vs. 4199 proteins). Additionally, compared to Fix-DIA and variable window DIA (Var-DIA), IsoPS-DIA demonstrated a lower average relative error (28.8%) that is comparable to PRM results (23.4%). This represents an 11.8-fold and 5.8-fold reduction in relative error compared to Fixed-DIA and Var-DIA. Finally, applying IsoPS-DIA to NSCLC tissue samples from mouse xenograft models revealed distinct expression levels of mutant and wild-type EGFR and other drug proteins, while the global profiling revealed differential activation in the NSCLC pathway and unexpected upregulation of immune-related pathways. In conclusion, IsoPS-DIA offers additional functionality for targeted protein absolute quantification beyond global proteome profiling.