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

Introduction
Genomic alterations drive disease initiation
and progression, and
many targeted therapies have been developed to target these actionable
mutations. The U.S. Food and Drug Administration (FDA) has approved
numerous small-molecule inhibitors and biologics for precision treatment.
For example, EGFR mutations (L858R and Del19), present
in 40–50% of Asian lung cancers, are widely used therapeutic
targets, while KRAS G12C
mutations, found in ∼30% of Caucasian cases, have recently
led to inhibitor approval. These advances
highlight how genomic testing has transformed oncology by guiding
targeted therapy.
,
Beyond cancer, SARS-CoV-2 protein
variants also influence immune escape and vaccine effectiveness, demonstrating the broader impact of protein mutation-specific
biology. However, DNA-based testing remains the standard for treatment
decisions, which cannot determine whether mutant proteins are expressed
at levels sufficient for therapeutic intervention. Because the drug response depends on both mutation status
and protein abundance, there is a pressing need for assays that directly
quantify druggable mutant proteins. Such protein-level measurements
would complement genomic testing and strengthen the molecular basis
for treatment decisions.
Data-independent acquisition (DIA)
has recently emerged as a powerful
proteomics strategy, offering higher reproducibility, fewer missing
values compared with data-dependent acquisition (DDA), and greater
multiplexing capacity than targeted MS. Since the early conceptualization
,
and development of Sequential Window Acquisition of All Theoretical
Mass Spectra (SWATH-MS), DIA has advanced
substantially through innovations in acquisition schemes and hardware,
leading to improved proteome coverage.
−

Recent efforts have
also integrated DIA with targeted MS modes for achieving both targeted
protein detection and global profiling. For example, Martínez-Val
et al. introduced Hybrid-DIA, which designed isotopically labeled
peptides triggering PRM and DIA scans in orbitrap enabled by application
programming interface (API),
,
while Sanner et al.
reported a mixed DIA–PRM workflow that alternates between DIA
and PRM. Both methods enhance target
sensitivity and quantitative performance, while retaining global profiling
capacity.
Despite these advances, absolute protein quantification
by DIA
remains limited. Stable isotope-labeled peptides with calibration
curves remain the gold standard, and
several studies have attempted to integrate them into DIA workflows.
Liu et al. quantified 41 plasma glycoproteins using SWATH-MS with
isotope dilution. Kim et al. applied
DIA for relative quantification of RAS mutations
in biopsy samples. Husson et al. recently
introduced Top3-ID-DIA for host cell protein quantification with performance
comparable to SRM. However, under conventional
DIA, coisolation of light and heavy peptide pairs generates overlapping
fragment ions, complicating spectral deconvolution and reducing quantification
performance. The restricted number of unique fragments further constrains
quantification precision, particularly for low-abundance proteins
and for distinguishing mutants from wild-type peptides.
Proteogenomics
integrates genomic data into reference databases
to enable detection of protein variants,
,
yet distinguishing mutant from wild-type peptides remains challenging
in shotgun proteomics due to high sequence similarity, low mutant
abundance, and the complexity of peptide mixtures. Targeted MS methods
such as selected/multiple reaction monitoring (SRM/MRM) and PRM provide
the current gold standard for absolute quantification of predefined
numbers of analytes with high specificity and sensitivity.
,
Only a few studies have applied these approaches to protein variants.
Tan et al. used SRM to validate single amino acid variant peptides
identified from multisearch engines. Chen
et al. quantified BRAF V600E mutants using an MRM
assay. Despite their sensitivity, targeted
MS methods are limited by a low multiplexing capacity and lack of
global proteome coverage.
In this work, we introduce an Isotope Pairs-Separated DIA (IsoPS-DIA)
strategy with dual functionality for simultaneous absolute targeted
protein quantification and global proteomic profiling. Unlike conventional
DIA, IsoPS-DIA employs dual scanning windows: (i) consecutive narrow
windows that resolve light/heavy isotopic peptide pairs for accurate
quantification and (ii) wide windows that enable global proteome profiling.
Using lung cancer as a model, IsoPS-DIA quantified endogenous levels
and activation pathways of clinically relevant driver mutations, including EGFR (L858R, Del19, G719A) and KRAS (G12S).
To maximize the detectability across mutation sites, we further incorporated
a multiple-protease digestion strategy. Benchmarking against conventional
DIA with fixed scanning window (Fix-DIA), variable Q1 isolation windows
DIA (Var-DIA) and PRM-MS demonstrated
the superior ability of IsoPS-DIA to quantify protein variants, their
wild-type counterparts, and signaling pathways linked to EGFR therapy.
Notably, this is a generic method compatible with both the Orbitrap
and Q-TOF platforms without the need for specialized software. To
the best of our knowledge, IsoPS-DIA represents the first DIA-MS approach
capable of absolute quantification of endogenous protein variants.
Finally, we provide an open-source window-design tool to facilitate
adoption of DIA design.

Experimental Section

Materials
and Reagents
Triethylammonium bicarbonate
buffer (TEABC), tris­(2-carboxyethyl) phosphine (TCEP), urea, and S-methylmethanethiosulfonate
(MMTS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lysyl
Endopeptidase (LysC, mass spectrometry grade) was purchased from Wako
Pure Chemical Industries, Ltd. (Osaka, Japan). Trypsin (modified,
sequence grade) was purchased from Promega (Madison, WI, USA). Endoproteinase
Glu-C (sequencing grade) was purchased from Roche (Basel, Switzerland).
The BCA protein assay kit was obtained from Pierce (Rockford, IL,
USA). C18 ZipTip Pipette Tips were purchased from Merck Millipore
(Burlington, USA). EGF Receptor internal standard peptides were chemically
synthesized with or without C-terminal lysine-(13C6
15N2) and glutamic acid-(13C5
15N1) to a 95% isotopic enrichment
by Mission Biotech (Taipei, Taiwan).

Cell Culture and Sample
Preparation
The human lung
adenocarcinoma cell lines A549 (EGFR wild-type),
PC9 (EGFR Del19), and H1975 (EGFR-L858R/T790M) were purchased from ATCC (Virginia, USA) and grown
in RPMI-1640 medium. The H3255 (EGFR-L858R), CL97
(EGFR-G719A/T790M), and CL68 (EGFR Del19/T790M) cell lines were kindly provided by Prof. Sung Liang
Yu (The Department of Clinical Laboratory Sciences and Medical Biotechnology,
National Taiwan University, Taiwan) and grown in RPMI-1640 medium.
RPMI-1640 medium was supplemented with 0.375% (w/v) HEPES, 0.22% (w/v)
sodium bicarbonate, 0.01% (w/v) sodium pyruvate, 10% (v/v) FBS, and
1% (v/v) penicillin–streptomycin–amphotericin solution
at 37 °C with 5% CO2. The NSCLC cells were subjected
to membrane protein extraction followed by gel-assisted digestion
based on our previously reported protocol. To construct the calibration curve for absolute quantification of
mutant and wild-type peptides, light synthetic peptides were pooled
with serial dilutions (0.5, 1, 5, 25, 100, 500 fmol/μL), while
heavy synthetic peptides were pooled with fixed concentration (25
fmol/μL). Furthermore, digested peptides from mouse lung tissues
were also added to each calibrator to mimic the complex background
in the samples and used in DIA-methods comparison.

LC-MS/MS Analysis
LC-MS/MS analyses were performed
on an Orbitrap Fusion Lumos Tribrid or an Orbitrap Eclipse Tribrid
mass spectrometer coupled to an Ultimate 3000 RSLCnano system (Thermo
Fisher Scientific, Bremen, Germany). To ensure consistent retention
times, all digests were spiked with iRT peptides (Biognosys AG, Schlieren,
Switzerland) and separated on a C18 column (Waters, CSH, 130 Å,
1.7 μm, 75 μm × 250 mm) at 300 nL/min. The mobile
phases were (A) 0.1% formic acid in water and (B) 0.1% formic acid
in acetonitrile (ACN). The 60 min gradient was as follows: 1–10%
B in 3 min, 10–15% in 37 min, 15–25% in 8 min, 25–45%
in 5 min, ramp to 90% in 2 min, and wash at 90% for 4 min. Common
MS1 settings were 390–1250 m/z, 120,000 resolution, AGC target 4e5, and maximum injection time
(IT) 50 ms. HCD collision energy was set at 25%. The MS1 windows in
the range of 400–1000 m/z for the three DIA methods are as follows: IsoPS-DIA: 40 variable isolation windows (4–60 Th); Fix-DIA: 40 fixed windows of 15 Th; and Var-DIA: variable window
sizes (9–63 Th) determined by MS1 ion intensity using swathTUNER. Full acquisition window details are listed in Supporting Table S1. MS2 scans were acquired
at 15,000 resolutions with AGC 4e5 and IT 22 ms. PRM:
Targeted isolation windows of 1.6 Th are set for precursor ions. Scheduled
MS2 scans were acquired at 50,000 resolution, AGC 5e4, and IT 86 ms.
An additional MS1 scan was collected at 30,000 resolution, AGC 4e5,
and IT 54 ms.
The LC-MS settings of IsoPS-DIA for the Q-TOF
analyzer are listed in Supporting Method 1.

Spectral Library Construction and DIA Data Processing
Protein
identification was achieved against the human reference proteome
from UniProtKB/Swiss-Prot (Homo sapiens, release 2020.05; 20,295 entries). Full-length sequences of EGFR
mutants (G719A, Del19, and L858R) and KRAS-G12S were appended to construct
a custom mutation database. Mutant peptide spectral libraries were
generated from DDA data acquired on 38 pooled samples of light- and
heavy-labeled peptides spiked into PC9 membrane digests, searched
in Proteome Discoverer version 2.5 (Thermo Fisher Scientific, Bremen,
Germany). DIA raw files were processed in Spectronaut v19.0 (Biognosys
AG, Schlieren, Switzerland) using the direct DIA+ workflow with default
settings unless specified. The mouse tissue samples were searched
against UniProtKB/Swiss-Prot (Mus musculus, release 2021.04; 17,074 entries) with the following parameters:
Trypsin/P as the protease, peptide length of 7–52 residues,
≤2 missed cleavages, methylthio (C) as a static modification,
and variable modifications of protein N-terminal acetylation, methionine
oxidation, and asparagine/glutamine deamidation (≤3 per peptide).
False discovery rates were controlled at 1% for precursors and proteins
(experiment-wise) and 1% for proteins (run-wise). The NSCLC cell line
samples were searched against human reference proteomes and used Lys-C/P
as the protease; other parameters are the same as above.

Absolute Protein
Quantification
The PRM and DIA raw
files were imported into Skyline and
searched against mutation-specific spectral libraries. Absolute peptide
abundances were determined from light-to-heavy (L/H) peak area ratios
of the extracted ion chromatograms. Calibration curves were constructed
for each peptide using linear regression (y = mx + b), with performance assessed by coefficient
of determination (R
2) and residual standard
deviation (Sy|x). Limits of detection (LOD) and quantification (LOQ)
were calculated as LOD = 3Sa/b and LOQ = 10Sa/b, where Sa is the standard
deviation of blanks and b is the regression slope. For calibration
curves, the regression model was assessed for linearity and reliability
using R
2 and Sy|x values. Calculation
of the relative error follows the equation Relative Error = (Actual
Value – Measured Value)/Actual Value.

Statistical Analysis
Peptide spectral similarity between
endogenous, library, and isotopic spectra was assessed in Skyline
using cosine similarity scores based on the dot product of the precursor
and fragment relative intensities. Signal-to-noise ratios and coefficients
of variation across Fix-DIA, Var-DIA, IsoPS-DIA, and PRM were compared
using Wilcoxon signed-rank tests in R. Reproducibility of relative
quantification among the DIA methods was evaluated by Spearman correlation.
For global proteomic comparison across six lung cancer cell lines,
protein intensities were log2-transformed, median-centered,
and missing values were imputed per cell line with MissForest. Differential expression across cell lines was
tested using Kruskal–Wallis with Benjamini–Hochberg
FDR correction (adjusted p < 0.05). Quantitative
comparison between cell lines was performed by using Wilcoxon rank-sum
with FDR correction; proteins with ≥2-fold change and adjusted p ≤ 0.05 were considered significantly upregulated.

Bioinformatic Analysis
Pathway enrichment analysis
was conducted on significantly differentially expressed proteins using
the STRING database by UniProt IDs, and
enrichment was performed based on the Reactome Pathway Database using the Homo sapiens gene set as the background universe. Reactome pathways with adjusted p-values <0.05 were considered enriched. The top enriched
pathways per cell line were visualized to facilitate functional interpretation.

Determination of EGFR and KRAS Variant Allele Frequencies in
Isogenic Cell Lines
EGFR and KRAS variant allele frequencies
(VAFs) were obtained either from the Cell Model Passports database (for CL68 cells) or determined experimentally
using the MassARRAY system (for H3255,
H1975, CL97, PC9, and A549 cells). For sample preparation, genomic
DNA was extracted using QIAamp kits and subjected to single nucleotide
extension. Analysis was performed on a Bruker Autoflex MALDI-TOF MS
platform. VAF was calculated as the mutant peak height relative to
the total peak height. The method was validated for precision, accuracy,
and sensitivity following TFDA-LDTS guidelines using FFPE, PBMC, and
plasmid samples. The laboratory maintains external accreditation through
CAP and EMQN proficiency testing.

Results
and Discussion

Design of IsoPS-DIA Strategy
In
conventional DIA, fixed
wide acquisition windows (10–20 Th) pose two major challenges
for absolute protein quantification. First, coisolation of light and
heavy peptides within the same window produces nearly identical fragment-ion
patterns, leaving few unique fragment ions for quantification. For
example, when using 13C6
15N2-lysine labeling at the C-terminus, all b-ions of
the light/heavy pair are identical (Figure
A), restricting quantifiable ions for mutant
peptides. Second, wide windows allow coelution of abundant nontarget
peptides, causing ion suppression, lower signal-to-noise ratios, and
reduced accuracy. To address these issues, IsoPS-DIA employs a dual-function
acquisition design. (i) For absolute targeted quantitation, narrow
4 Th windows separate isotopic pairs of targeted peptides into two
adjacent windows (Figure
B), enabling independent full fragment-ion series and reducing
ion suppression. The calibration curve was constructed by using different
amounts of isotopic pairs of light and heavy peptides. (ii) For global
proteomic profiling, wide variable windows (up to 60 Th) maximize
proteome coverage and provide flexibility to cover target-specific
pathways. This hybrid design achieves both enhanced accuracy of absolute
protein quantification and comprehensive proteome profiling in single
LC-MS/MS run (Figure
C). To obtain the absolute quantification of targeted peptides, heavy-isotope
internal standards are spiked into each sample. The light-to-heavy
ratios were obtained by extracted ion chromatograms of fragment ions
and fitted to external calibration curves to derive peptide concentrations
by linear interpolation. For global proteome profiling, relative protein
abundances are obtained from Spectronaut, which quantifies peptides
using fragment-ion chromatograms and selects the top three abundant
peptides to derive protein-level abundance.
For demonstration, our model study focused on clinically
relevant EGFR mutations responsive to targeted therapies:
point mutation
L858R (exon 21, 39–47%), E746–A750 (accounting for ∼69%
of exon 19 deletions, abbreviated Del19), and another common mutation
G719X (X = A, C, S, D, exon 18, 2–3%).
−

Six NSCLC cell lines representing
these genotypes (H3255-L858R, CL97-G719A/T790M, H1975-L858R/T790M,
PC9-Del19, CL68-Del19/T790M, and A549-EGFR WT/KRAS-G12S)
were selected, collectively covering 81–99% of clinical EGFR mutations. In-silico digestion identified LysC and
GluC as optimal proteases to generate mutant- and wild-type-containing
peptides with favorable MS detectability (Table S2). For absolute quantification,
heavy-isotope-labeled peptides (13C/15N-labeled
C-terminal lysine or glutamic acid) were spiked as internal standards
to optimize DIA settings, evaluate quantitation performance, and construct
spectral libraries.
To streamline window configuration, we developed
an interactive
web-based IsoPS-DIA design tool on GitHub (https://github.com/Isaac-Chiu/IsoPS-DIA-Window-designer). The Python tool allows users to customize acquisition windows
based on precursor m/z and retention
time with an R script provided to prepare input files from Skyline
exports. Using this tool, we designed 36 windows across 400–1000 m/z, including 16 narrow targeted windows
(4 Th) for eight isotopic pairs of EGFR wild-type,
L858R, G719A, and Del19 peptides, and 20 broader windows for untargeted
global profiling. For instance, L858R light (454.25 m/z) and heavy (458.26 m/z) peptides were placed in adjacent 453–457 and 457–461 m/z windows, enabling independent fragmentation
to collect full sets of fragment ions for quantification (Figure
B). Full window details
are provided in Supporting Table S1.

Enhanced Detection and Quantitation Performance by IsoPS-DIA
We first evaluated the performance of fragment-ion detection of
mutant peptides using IsoPS-DIA versus conventional DIA with a fixed
scanning window (Fix-DIA, 15 Th). Light and heavy synthetic peptides
(25 fmol) spiked into 100 ng of mouse tissue digests (with iRT peptides)
were analyzed. The feature to detect full series of fragments in IsoPS-DIA
was demonstrated in the example of EGFR Del19 peptides
(746ELREATSPK754) (Figure
A). Fix-DIA coisolated the light (515.78 m/z) and heavy (519.79 m/z) precursors within the 505–520 m/z window, producing shared y- and b-ions and leaving only a few unique low-abundance y-ions (y3- y6, y8) suitable for quantification (Figure
B); only y3 and y8
showed >5% relative intensity, which limited their utility for
robust
quantification. In contrast, IsoPS-DIA separated the light and heavy
peptides into adjacent windows, enabling independent fragmentation
to detect a full series of fragments. This resulted in 10 unambiguous
and quantifiable fragment ions (b3–b7, y3–y6, and y8)
(Figure
C,D). Compared
to Fix-DIA, IsoPS-DIA yielded a 5-fold increase in quantifiable fragment
ions, with all y-ions exhibiting 3 to 47% higher intensities.
We next benchmarked IsoPS-DIA
against PRM, Fix-DIA, and Var-DIA
across three concentration levels (0.75, 2.5, and 7.5 fmol) of synthetic
peptides spiked into mouse lung digests. As expected, PRM showed the
highest signal-to-noise ratios (SNR) for extracted ion chromatograms
(XICs), and IsoPS-DIA achieved comparable profiles (Figure
A). Among the DIA methods,
IsoPS-DIA exhibited superior fragment peak profiles with reduced background
interference. For the EGFR-G719A mutant and wild-type peptides, SNRs
reached 21.4 and 12.5 with IsoPS-DIA, compared to 1.7 and 2.7 with
Fix-DIA, and 6.3 and 6.7 with Var-DIA, respectively (Figure
A). Across all targeted peptides,
IsoPS-DIA improved median SNRs by 1.4–2.9-fold over Fix-DIA
and 1.9–2.5-fold over Var-DIA (Figure
B). Notably, at the lowest concentration
(0.75 fmol), all seven target peptides were detected with higher SNRs
by IsoPS-DIA, whereas 6–7 peptides fell below LOQ (SNR <
10) with Fix-DIA or Var-DIA (Figure
C and Table S3). For four
peptides, IsoPS-DIA SNRs exceeded other methods by >2-fold.
Absolute quantification performance was then assessed
by using
calibration curves constructed from light peptides (0–25 fmol)
spiked with heavy internal standards (25 fmol). Two quality control
(QC) samples, 2.5 and 7.5 fmol, were prepared to evaluate the absolute
quantification performance. All methods achieved good linearity (average R
2 > 0.99), though Fix-DIA showed slightly
lower
linearity (Table S4). Notable deviation
was observed for peptide 455EISDGDVIISGNK467 due to background interference in low calibrators, observed in the
0, 1, and 5 fmol calibrators (Figure S1). At the 7.5 fmol QC level, conventional Fix-DIA showed a pronounced
deviation for the 455EISDGDVIISGNK467 peptide
(127% RE; Figure S2A), whereas the other
approaches had lower error ranges (Table S5). At the 2.5 fmol QC level, the four methods exhibited comparable
accuracy, with average relative errors (REs) of 10.6, 17.8, 21.3,
and 15% for Fix-DIA, IsoPS-DIA, Var-DIA, and PRM, respectively. Among
the analytical merits, varied degrees of reproducibility were observed
among the four methods. IsoPS-DIA demonstrated markedly superior reproducibility,
particularly at low concentrations, as reflected by the low CVs at
the 2.5 fmol QC level (2.2%), which is comparable to PRM (2.9%) and
outperformed the other DIA approaches (Var-DIA: 15.3%, Fix-DIA: 30.3%)
(Figure
C).
In summary, IsoPS-DIA achieved
PRM-like sensitivity while maintaining
DIA’s broad proteomic coverage. Its narrow isolation windows
enhance fragment-ion yield, SNR, and reproducibility, especially at
low targeted protein concentrations.

IsoPS-DIA Quantified Endogenous EGFR Mutant
and Downstream Signaling Proteome
After performance validation,
IsoPS-DIA was applied to six NSCLC cell lines: five harboring EGFR mutations (H3255–L858R, CL97–G719A/T790M,
H1975–L858R/T790M, PC9–Del19, CL68–Del19/T790M)
and A549 cells with wild-type EGFR and KRAS-G12S. A spectral library was built from synthetic EGFR mutant peptides (L858R, G719A, Del19), which were not previously
reported in public repositories. Cosine similarity scores were used
to distinguish wild-type and mutant peptides, which showed <20%
spectral similarity in DIA data (Figure S3). All targeted peptides were confidently identified in their respective
cell lines by using the library. For example, the L858R peptide (853ITDFGRAK860) was confidently detected in H3255
with high spectral similarity to its synthetic standard (Ldotp = 0.902, Figure S4A, Table S6), and Del19 peptide (737KVKIPVAIKTSPKANKE753) in CL68 cells (Ldotp = 0.823 and consistent coeluting profiles, Figure S4B). Wild-type EGFR peptides (L858, E746-A750)
and other variants (G719A, G719-WT) also showed strong spectral matches
(Ldotp >0.81) and consistent coelution profiles (Figure S4C–F). Importantly, the use of narrow scanning
windows in IsoPS-DIA provided richer fragment patterns. Low spectral
similarity (<20%) between wild-type and mutant peptides supported
high specificity in their identification and quantification.
Absolute quantification was then performed to determine mutant-to-wild-type
EGFR and KRAS ratios by calibration curves across 0.5–500 fmol
(R
2 = 0.998–0.999) (Figure S5). IsoPS-DIA achieved subfemtomole sensitivity
with LODs ranging from 36 to 222 amol and LOQs from 121 to 741 amol
(Table S7) and high reproducibility with
median CV = 2.4% (Table S8). Quantification
revealed distinct expression patterns across the cell lines (Figure
A). In H3255, L858R
was quantified at 84.97 ± 2.84 fmol/μg versus 27.16 ±
0.76 fmol/μg WT (mutant-to-WT ratio = 3.1), while H1975 showed
much lower L858R concentration (1.22 ± 0.04 vs 4.82 ± 0.10,
ratio = 0.3), reflecting >10-fold variation. For the Del19 deletion,
CL68 showed higher mutant expression (28.70 ± 1.01 vs 12.41 ±
0.27 fmol/μg), whereas PC9 expressed mutant and WT at comparable
levels (9.49 ± 0.25 vs 8.55 ± 2.03 fmol/μg). G719A
was dominantly expressed in CL97 (34.09 ± 0.75 vs 15.74 ±
0.13 fmol/μg WT). IsoPS-DIA’s multiplexing capability
also enabled quantification of KRAS and its G12S
variant, a hotspot in ∼4.4% of lung cancers.
,
In A549 cells, both WT and G12S forms were detected at comparable
levels (7.58 ± 0.08 vs 6.67 ± 0.06 fmol/μg) (Figure
B). To the best of
our knowledge, this is the first report of simultaneous absolute quantification
of multiple endogenous EGFR driver mutations with
their wild-type counterparts.
To evaluate how allele-specific
protein expression corresponds
to genomic mutation burden, we quantified the ratios of the absolute
concentration of mutant proteins across six cell lines and compared
them with their respective genomic variant allele frequencies (VAFs)
obtained by MassARRAY (Table S8). Overall,
all mutant proteins were confidently quantified by IsoPS-DIA, demonstrating
the method’s ability to directly measure functional, expressed
oncoproteins. Genomic VAFs were uniformly high (>70–95%)
across
EGFR mutant lines (H3255, H1975, PC9, CL68, CL97) and KRAS mutant
A549, reflecting clonal or near-clonal mutations. In contrast, protein-level
mutant ratios were systematically lower than genomic VAFs showed substantial
variability (20–76% for EGFR mutants; 53% for KRAS-G12S), indicating
that the protein expression of mutant vs wild-type alleles is not
strictly proportional to DNA VAF. The 46% discrepancy observed in
KRAS-G12S (A549) likely results from sequence homology between Ras
isoforms (HRAS and NRAS) at residues 6–16, which inflates the
wild-type peptide signal. Notably, high EGFR expressors (H3255, CL68,
and CL97) have similar VAF between gene and protein levels (<20%
discrepancies), whereas low-expressors (H1975 and PC9) exhibited discrepancies
between 43 and 54%. Powered by multiprotease digestion and IsoPS-DIA,
this study provides the first absolute quantification of mutant protein
ratios in these isogenic lines. These findings suggest differential
transcription, translation, or protein stability between alleles,
highlighting that genomic mutation burden does not fully predict the
abundance of the mutant protein actually present in cells. Protein-level
VAF reveals functional mutant oncoprotein expression not predicted
by DNA VAF, improving assessment of therapeutic target abundance.
Beyond target quantification, IsoPS-DIA enabled global proteome
profiling to map the downstream signaling proteome. Using direct DIA
analysis with Spectronaut, 6,183 proteins were quantified across all
cell lines, including 48 components of EGFR-related signaling pathways
(Ras-Raf-MEK-ERK, PI3K-AKT-mTOR, JNK, Rho-ROCK, SRC-JAK-STAT) (Figure
C). Distinct expression
patterns reflected pathway activation status: L858R mutant lines showed
stronger downstream signaling. For example, H3255 exhibited high ErbB
family protein levels (EGFR, ERBB2, and ERBB3), consistent with elevated
L858R expression and aggressive proliferative signaling. These findings
agree with the reports that L858R tumors are more aggressive than
Del19 deletions. Pathway Z-scores confirmed
higher relative enrichment scores for EGFR-related pathways in H3255
and CL97 cells compared to those in H1975 cells (Figure
D), correlating with EGFR mutant
abundance. In contrast, A549 displayed lower Z-scores and reduced
expression of proteins in the EGFR pathway. These findings support
a direct association between EGFR mutation abundance and protein expression
of enriched downstream pathways.
In summary, the abundance and
type of EGFR or KRAS mutation strongly
influence the downstream protein
expression levels of NSCLC EGFR-related signaling pathways. IsoPS-DIA
uniquely provides absolute quantification of drug targets together
with global pathway profiling, enabling functional stratification
of oncogenic signaling and revealing actionable vulnerabilities.

Quantitative Global Profiling of EGFR Genotype-Dependent Proteomic
Landscape in NSCLC Cell Lines
Finally, we benchmarked the
global profiling performance of IsoPS-DIA, Fix-DIA, and Var-DIA using
six PC9 cell lines. All methods were configured with 40 MS2 events
covering the 400–1000 m/z range. Despite IsoPS-DIA allocating more windows to separate isotopic
paired light and heavy peptides, it provided comparable identifications
using direct DIA search: 3936 protein groups for Fix-DIA, 4,130 for
Var-DIA, and 4,091 for IsoPS-DIA and precursor ions (Fix-DIA: 40,577;
Var-DIA: 40,215; IsoPS-DIA: 34,673) in 100 ng mouse tissue peptide
samples (Figure S6A,B). Importantly, triplicate
analyses of IsoPS-DIA achieved superior quantification reproducibility,
with 78% of protein groups showing CVs < 10%, compared to 60% for
Fix-DIA and 63.7% for Var-DIA (Figure S6A). Triplicates also showed high correlations (Spearman ρ > 0.995; Figure S9C), indicating that
narrower windows in IsoPS-DIA do not compromise proteome coverage
or sensitivity.
We next applied IsoPS-DIA to six NSCLC cell
lines, identifying 6183 protein groups in total, with 5357–5735
proteins detected per single-shot run using direct DIA search (Figure
A). Differentially
expressed proteins (DEPs) were determined after normalization, imputation,
and statistical filtering (Kruskal–Wallis H test, adjusted p < 0.05), followed by hierarchical clustering. As shown
in Figure
B, PC9 and
CL68, both harboring Del19 deletions, clustered together and exhibited
similar proteomic profiles compared to A549 (KRAS-G12S mutation),
while H3255 (L858R) and CL97 (G719A) form another cluster. H1975,
with the lowest EGFR expression and mutant-to-wild-type protein expression
ratio, displayed a distinct proteomic signature (Figure
B).
These clustering patterns were concordant with pathway-level
comparisons
(Figure
D), demonstrating
the agreement between global proteomic shifts and enriched EGFR-related
signaling pathways. In total, 3166 DEPs were identified across the
six cell lines (Wilcoxon signed-rank, adjusted p <
0.05). Reactome pathway enrichment analysis of upregulated proteins
revealed the top ten enriched pathways for each cell line (Figure
C). H3255 cells exhibited
enrichment in PI3K-AKT-related pathways, while CL97 cells showed enrichment
in endocytosis-related processes. The analysis further highlighted
common functional categories, such as membrane trafficking, vesicle-mediated
transport, and ER/Golgi-associated pathways, in these cell lines,
reinforcing the complementary insights gained from IsoPS-DIA. In addition,
distinct signatures were also observed: H1975 displayed strong enrichment
in cell cycle and RNA metabolism pathways (FDR – log10 >
60),
while A549 showed mitochondrial pathway activationreflecting
EGFR-independent signaling alterations in NSCLC.
To demonstrate
the general applicability of IsoPS-DIA across different
instrument platforms, we applied the method for the three of the same
cell lines using a Q-TOF instrument (TripleTOF 5600). The calibration
curves of seven EGFR peptides showed excellent linearity with R
2 values exceeding 0.993 (Figure S7A). We further quantified the L858R mutation peptide
and its corresponding wild-type peptide in A549, H3255, and PC9 cell
lines, obtaining 80 fmol per μg peptide from cell sample from
L858R-bearing H3255 cell. The overall expression levels are consistent
with those measured on the Orbitrap platform (Figure S7B). In addition, DIA analysis enabled the identification
of 1872, 1316, and 2330 proteins in A549, H3255, and PC9 cells, respectively
(Figure S7C). Together, these results highlight
the compatibility of the IsoPS-DIA strategy with the Q-TOF instrument.
In summary, IsoPS-DIA uniquely integrates the absolute quantification
of EGFR and KRAS mutant proteins with unbiased global proteomic profiling.
While genotypes are defined by DNA sequencing, our results revealed
marked variability in mutant versus wild-type protein abundance, suggesting
allele-specific expression that may affect the therapeutic response.
In parallel, downstream proteomic profiling uncovered functional pathway
alterations, providing a systems-level framework to evaluate tumor
draggability beyond the genotype alone.

Conclusions
In
this study, IsoPS-DIA was developed to offer dual functionality
in a single LC-MS/MS run: (i) multiplexed absolute quantification
with strength to distinguish mutations and (ii) simultaneous global
proteome profiling to read out downstream signaling and resistance
pathways. Methodologically, the key advance is the dual-window scheme:
narrow 4 Th windows resolve light/heavy pairs into adjacent scans,
eliminating coisolation and overlapping fragment spectra that confound
isotope-dilution DIA, while wide variable windows preserve proteome
coverage. This design yields richer unique fragments and higher S/N,
enabling subfmol sensitivity of LODs (36–222 amol), low LOQs
(121–741 amol), high linearity (R
2 ≈ 0.998–0.999), and low CVs (∼2–3%),
approaching PRM-like precision without sacrificing DIA-level breadth
of >5000 proteins (dependent on the instrument).
Compared
with SRM/PRM, which remains the gold standard for absolute
quantification but scales poorly and lacks global proteome information,
IsoPS-DIA retains high multiplexing and system-wide readouts. Relative
to Hybrid-DIA and mixed DIA–PRM modes that alternate or trigger
PRM scans, our approach does not rely on instrument-specific APIs
or duty-cycle trade-offs for targeted events, and it directly supports
absolute (not just relative) quantification. IsoPS-DIA’s isotope
separation removes spectral deconvolution ambiguity for light and
heavy peptides and increases the number and intensity of quantifiable
ions. Thus, IsoPS demonstrated improved accuracy at low abundancecritical
for endogenous variant peptides and combined with a multiprotease
strategy to maximize mutant site detectability for distinguishing
mutant vs wild-type forms. Practically, IsoPS-DIA is applicable to
different platforms (Orbitrap/Q-TOF) and uses standard software. To
facilitate adoption, we developed an open-source, web-based IsoPS-DIA
window-design tool (https://github.com/Isaac-Chiu/IsoPS-DIA-Window-designer), written in Python and freely available to the community. The tool
customizes DIA scanning windows based on precursor m/z and retention time with an R script for preparing inputs from Skyline exports. By releasing this
resource, we aim to facilitate broad adoption of IsoPS-DIA and its
application to mutant proteins, as well as other targeted or discovery
proteomics studies.
Application of IsoPS to NSCLC cell lines
enabled the first direct
quantification of endogenous EGFR mutant and wild-type proteins, revealing
heterogeneous expression patterns and mutation-to-wild-type ratios
that cannot be inferred from gene testing data alone. These findings
highlight the value of protein-level measurements to uncover allele-specific
expression and post-transcriptional regulation, which are critical
for predicting therapeutic responses to EGFR-targeted therapies. This
IsoPS strategy established a reproducible and scalable assay as a
powerful complement to conventional gene testing in clinical and translational
research.
While IsoPS-DIA performed robustly for 16 peptides
tested, limitations
include a finite number of narrow windows per run (target-set sizing
and duty-cycle schedule). Advancement in high-speed mass spectrometers
will help scale up to larger target panels or make strategic adjustments
to maintain analytical quality. The latter may include optimizing
window schemes through multiplexing, adjusting m/z coverage, or increasing the LC gradient time. Given its
narrow-window design, IsoPS-DIA is optimally suited for panels of
25–35 target peptides in light–heavy pairs. Future work
will focus on extending this platform to broader target sets and clinical
sample types, enabling comprehensive quantitative readouts of oncogenic
mutations and their functional proteomic consequences.

Supplementary Material