Phosphopeptidome Profiling of Human Plasma for Hepatocellular Carcinoma Biomarker Discovery.

Introduction
Hepatocellular carcinoma (HCC) is a common
primary liver cancer
and an alarming global public health concern. More than 900,000 new cases are detected each year, according to
GLOBOCAN 2020. About 830,000 deaths happen
yearly, making HCC the sixth utmost commonly diagnosed cancer and
the third top origin of cancer-related deceases globally. The occurrence of this malignancy has been growing
gradually in both developing and developed countries, as a result
of various risk issues. Chronic viral infections caused by HBV and
HCV remain to account for an extensive part of cases, particularly
in areas with high endemicity. Moreover,
metabolic and lifestyle factors as well as obesity, consumption of
alcohol, insulin resistance, nonalcoholic fatty liver disease, and
type 2 diabetes contribute pointedly to the worldwide disease burden.
−

Current HCC monitoring guidelines primarily recommend abdominal
ultrasound imaging, every six months in combination with serum alpha-fetoprotein
(AFP) testing for individuals at high risk, typically those with cirrhosis
or chronic hepatitis B or C. However,
these methods have important drawbacks. In noncancer liver conditions
like chronic hepatitis or cirrhosis, AFP levels can be high making
it hard to rely on this test alone and
ultrasounds can miss early signs of HCC about 47% of the time. Markers such as des-γ-carboxy prothrombin
(DCP), and AFP-L3, combined with other
factors including sex, age, and AFP perform better than alone AFP,
−

but across diverse patient populations none of them provide the
reliability required for early detection. Therefore, there is a pressing need to find new, noninvasive markers
that accurately detect HCC in patients with liver cirrhosis (CIRR),
because liver cirrhosis often occurs alongside it and makes diagnosis
tougher.
Plasma, which serves as an accessible “liquid
biopsy,”
offers insight into both tissue-specific alterations and overall physiological
status, that makes it valuable resource for discovery of biomarker. Nonetheless, studying the plasma proteome remains
technically challenging for its wide dynamic range and the dominance
of a few highly abundant proteins that can obscure the detection of
low-abundance biomarkers. Recent technological
advancements, such as high-resolution mass spectrometry, enhanced
chromatographic separation techniques, and robust quantitative workflows,
have helped a more detailed investigation of the plasma proteome. Furthermore, multimarker panels identified from
proteomic analyses have demonstrated better performance compared to
AFP alone in distinguishing HCC from chronic or cirrhosis hepatitis. In addition to conventional proteomic workflows
based on tryptic digests, body fluids inherently contain low molecular
weight peptides collectively referred to as the endogenous peptidome. These endogenous peptides arise from protein
secretion, proteolytic processing, or protein degradation, and considered
to deliver “snapshots” of pathological or physiological
states. Endogenous peptides offer several
advantages as biomarkers: they are normally stable, can be measured
directly without enzymatic digestion, and frequently reflect disease
associated protease activity.

Recent
research on profiling of endogenous peptidome in serum has
detected several significantly altered peptides in HCC vs CIRR. Particularly,
a peptide derived from immunoglobulin heavy constant gamma 4 exhibited
perfect discrimination between HCC from CIRR in a discovery cohort. Phosphoproteomic studies in HCC have also reported
dysregulated phosphorylation pathways in tumor tissues and experimental
models, highlighting alterations in cell-cycle control, metabolic
regulation, and kinase.
,
While these studies
provide insight into intracellular signaling changes, they are primarily
tissue-based or model-based and do not capture circulating phosphorylated
peptides. Profiling the endogenous phosphopeptidome may therefore
offer a minimally invasive means of detecting systemic phosphorylation
and proteolytic changes associated with HCC. The endogenous phosphopeptidome,
phosphorylated peptides naturally existing in circulation remains
largely unexplored. Profiling the endogenous phosphopeptidome could
offer the combined advantages of both approaches: the accessibility
of endogenous peptides and the biological specificity conferred by
phosphorylation. This provides a powerful and novel window for exploratory
systemic signaling alterations associated with HCC. However, the endogenous
phosphopeptidome poses significant technical challenges. The abundance
of phosphorylated endogenous peptides is predictable to be even lower
than that of nonphosphorylated peptides, demanding highly sensitive
enrichment strategies and high-resolution detection methods.
Herein, we report profiling of human plasma endogenous phosphopeptidome
in a cohort of 60 patients with HCC (n = 20), CIRR
(n = 20), and healthy controls (CTL) (n = 20) to identify HCC candidate biomarkers. Endogenous peptides
were released from yellow plasma, and enriched for phosphopeptides
prior to analysis on a nanoLC-Orbitrap LUMOS mass spectrometer. Label-free
quantification followed by statistical evaluation using one-way ANOVA
identified the significant alterations across the three groups. Multivariate
analysis revealed distinct clustering of HCC from CIRR and CTL, while
functional annotation highlighted pathways relevant to liver disease
progression and tumor biology. By integrating phosphopeptidome profiling
with quantitative and bioinformatic analyses, this work provides the
first systematic characterization of circulating endogenous phosphopeptides
in HCC and establishes their potential as a novel reservoir of minimally
invasive candidate biomarkers for HCC.

Materials and Methods

Experimental
Design
The overall experimental workflow
for this study is summarized in Figure
. Plasma samples obtained from HCC, CIRR, and CTL were
processed under acidic condition to release endogenous peptides followed
by desalting and enrichment of released phosphopeptides. The samples
were then analyzed on a nano-LC coupled with Orbitrap Fusion Lumos
Tribrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA,
USA). Data were processed in Proteome Discoverer (PD) for annotation
and label-free quantification. Rigorous quality control was performed
using HeLa digest standards injected after each batch of HCC, CIRR,
and CTL plasma samples. For each QC run, 200 ng of HeLa digest was
analyzed under identical conditions on the mass spectrometer. Statistical
and bioinformatics analyses of the processed LC-MS/MS data included
ANOVA with Tukey’s post hoc tests, chi-squared testing for
presence/absence patterns, and Gene Ontology (GO) enrichment and pathway
mapping, as well as protein–protein interaction (PPI) network
analysis using STRING and Ingenuity Pathway Analysis (IPA).

Study Cohort
Plasma
samples from 60 subjects, comprising
20 patients with HCC, 20 patients with liver cirrhosis, and 20 healthy
volunteers were selected for this study from our repository in such
a way that the three groups do not differ significantly in gender,
age, or other clinical characteristics. Also, none of the subjects
had alcohol-related liver disease or nonalcoholic fatty liver disease,
thereby reducing potential confounding factors and enabling a clearer
assessment of disease-specific molecular alterations. Detailed demographic
and clinical characteristics of all subjects are summarized in Table
. All participants
were recruited from MedStar Georgetown University Hospital (MGUH)
and Howard University Hospital, and the study protocol was approved
by the Georgetown University Institutional Review Board (IRB). Written
informed consent and Health Insurance Portability and Accountability
Act (HIPAA) authorization were obtained from all participants prior
to enrollment. For HCC cases, diagnoses were confirmed by diagnostic
imaging and histopathological examination, with clinical staging determined
according to the tumor node metastasis (TNM) classification system.
Peripheral venous blood was collected using sterile vacuum tubes without
anticoagulants. Samples were centrifuged at room temperature, first
at 1000 × g for 10 min, followed by a second centrifugation at
2500 × g for 10 min. The resulting serum was carefully collected,
aliquoted in the presence of protease inhibitors, and stored at –
80 °C until further analysis.

Materials
All
reagents were analytical or LC–MS
grade unless otherwise noted. Trifluoroacetic acid (TFA; Thermo Fisher
Scientific, Cat. No. 85183), formic acid (FA; Thermo Fisher Scientific,
Cat. No. A117–50), and acetonitrile (ACN; Thermo Fisher Scientific,
Cat. No. A955–4) were used as LC–MS grade solvents,
and ultrapure water was produced using a Milli-Q purification system
(MilliporeSigma). Endogenous peptides were isolated using Amicon Ultra
0.5 mL centrifugal filter units (10 kDa MWCO; MilliporeSigma, Cat.
No. UFC501096), desalted using Oasis HLB 1 cc Vac Cartridges (10 mg;
Waters, Cat. No. WAT094226), and enriched for phosphopeptides with
the Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific,
Cat. No. A32992). All microcentrifuge tubes and pipet tips were MS-compatible
(Eppendorf or Thermo Fisher Scientific). HeLa Protein Digest Standard
(Thermo Fisher Scientific, Cat. No. 88328) was used for routine quality
control injections. NanoLC–MS/MS analyses were performed using
a Dionex UltiMate 3000 RSLCnano system coupled to an Orbitrap Fusion
Lumos Tribrid mass spectrometer (Thermo Fisher Scientific). Chromatographic
separation was achieved using an Acclaim PepMap RSLC C18 analytical
column (75 μm × 250 mm, 2 μm, 100 Å; Thermo
Scientific, Cat. No. 164942) and a PepMap C18 trap column (75 μm
× 20 mm, 3 μm, 100 Å; Thermo Scientific, Cat. No.
164535). Endogenous phosphopeptide identification and label-free quantification
were performed using Proteome Discoverer 3.0 (Thermo Fisher Scientific).
Functional enrichment analysis was conducted using Ingenuity Pathway
Analysis (IPA; Qiagen), and statistical analyses, including ANOVA
and PCA, were performed in R version 4.3.0.

Sample Preparation
Endogenous peptides were extracted
from 100 μL of human serum using our previously reported method
with minor modifications. Samples were
acidified with 500 μL of 1% trifluoroacetic acid (TFA), vortexed,
and heated at 95 °C for 15 min to disrupt peptide–protein
interactions. After cooling to room temperature, mixtures were filtered
through preconditioned with 1% TFA Amicon Ultra-0.5 centrifugal units
(10 kDa MWCO) and centrifuged at 14,000 × g for 20 min at 10
°C, followed by two washes with 100 μL of 1% TFA. Filtered
peptides were desalted using Oasis HLB 1 cc Vac Cartridges (10 mg),
conditioned with acetonitrile and equilibrated with 0.1% TFA in water.
Peptides were loaded, washed twice with 0.1% TFA in water, and eluted
with 70% acetonitrile containing 0.1% TFA, then dried under vacuum.
For endogenous phosphopeptide enrichment, dried peptides were resuspended
in 100 μL of loading buffer from the Fe-NTA Phosphopeptide Enrichment
Kit (Thermo Fisher Scientific). Fe-NTA spin columns were equilibrated
with 200 μL of loading buffer and centrifuged at 1,000 ×
g for 1 min. Peptide samples (100 μL) were applied to the columns
and incubated for 30 min at room temperature with gentle agitation.
Columns were washed twice with 200 μL of wash buffer and once
with 200 μL of water to remove unbound peptides, and captured
endogenous phosphopeptides were eluted twice with 70 μL of elution
buffer. Eluates endogenous phosphopeptides were dried and reconstituted
with 3% ACN in 0.1% FA and peptide concentration was determined with
nanodrop before nano-LC-MS/MS.

Nano-LC-MS/MS Data Acquisition
Data were acquired using
a Dionex 3000 UltiMate Nano LC system interfaced to a LUMOS terebrid
mass spectrometer (Thermo Scientific, San Jose, CA, USA) equipped
with Easy-Spray ESI source (Thermo Fisher Scientific). One μg
of peptides from each sample was injected into the LC system for analysis.
To achieve preconcentration and cleanup, peptides were first passed
through a C18 Acclaim PepMap trap column (75 μm × 20 mm,
3 μm, 100 Å, Thermo Scientific) before being transferred
to a C18 Acclaim PepMap RSLC column (75 μm × 250 mm, 3
μm, 100 Å, Thermo Scientific). A multistage gradient was
used with a total run time of 120 min. The mobile phase A was water
and 0.1% formic acid (FA), while mobile phase B was 100% acetonitrile
(ACN) and 0.1% FA. The column oven temperature was maintained at 35
°C throughout the run. During the initial 5 min, the mobile phase
B was held constant at 4% with a 300 nL/min flow rate. It gradually
increased to 35% over the next 120 min with a flow rate of 220 nL/min.
Between 125 and 133 min, mobile phase B was further increased to 95%
with a flow rate of 250 nL/min, and this composition was maintained
for 5 min. Finally, the mobile phase B was reduced to 4% with a flow
rate of 300 nL/min and held constant until the end of the run. The
separated peptides were directed to the mass spectrometer at a voltage
of 2.2 kV. The full MS scan was performed with a scan range of 370–1850 m/z and the analytes were detected in the
Orbitrap at a resolution of 12K. The top 3 most intense ions were
selected for MS2 fragmentation in a high-energy collision dissociation
(HCD) cell, with a normalized collision energy (NCE) of 28 at a resolution
of 6K, with a dynamic exclusion of 30 ms.

Endogenous Phosphopeptide
Annotation
Endogenous phosphopeptide
identification and label-free quantification (LFQ) was performed in
Proteome Discoverer 3.0 (Thermo Scientific, Waltham, MA, USA) using
Sequest HT against the human UniProt database (July 2024). The workflow
included mass recalibration, Minora Feature Detector, Standard Spectrum
Selector, and Percolator nodes. Precursor and fragment mass tolerances
were set to 10 ppm and 0.02 Da, respectively, with no enzyme specificity
and phosphorylation on serine, threonine, and tyrosine (STY) as a
dynamic modification. High-confidence identifications were filtered
at a 1% false discovery rate using Percolator.

Data Analysis
Clustering within each sample group was
assessed by principal component analysis (PCA) using LFQ intensities
of all endogenous phosphopeptides to identify potential outliers.
Peptides detected in less than 70% of at least one patient group were
left aside for subsequent analysis based on absent and present calls.
Abundance values for the remaining endogenous phosphopeptides were
log2-transformed, and missing values inherent to data-dependent
acquisition (DDA) shotgun proteomics were imputed using the K-nearest
neighbor (KNN) feature-wise method. Data were normalized by the median
to reduce inter-run variation. Following significance analysis using
ANOVA, differential expression of endogenous phosphopeptides in HCC
vs CIRR as well as HCC vs CTL was assessed. Endogenous phosphopeptides
with a fold change >1.5 and false discovery rate (FDR) < 0.05
were
considered significant. Precursor proteins of differentially expressed
endogenous phosphopeptides were further analyzed for pathway and functional
correlations using Ingenuity Pathway Analysis (IPA) software (Qiagen
Inc., Germantown, MD, USA). Differential expression of endogenous
phosphopeptides detected in fewer than 70% of the samples in each
group was evaluated by assessing change in peptide occurrence in HCC
vs CIRR and HCC vs CTL via Pearson’s chi-squared test.

Results
The QC injections demonstrated high reproducibility
across the
entire acquisition period. The distribution of log-transformed protein
abundances showed tight clustering across all runs (Figure S6), while the number of proteins identified per injection
remained stable with minimal variation (Figure
c). These findings confirm the robustness
of the nano-LC-MS workflow and validate that the observed differences
between groups reflect biological rather than technical variation.
Comprehensive profiling revealed a rich plasma
phosphopeptidome
across the 60 samples. In total, 1,365 unique endogenous phosphopeptides
corresponding to 549 precursor phosphoproteins were identified (Supplementary Table S2). Correlation analysis
across the 60 plasma samples demonstrated high reproducibility and
internal consistency of the data set. Pairwise Pearson correlation
coefficients for normalized peptide intensities were consistently
high (r > 0.9 for most comparisons), confirming
that
the endogenous phosphopeptidome was measured with excellent technical
stability across all runs (Figure S1).
Group-wise abundance analysis further illustrated the global behavior
of the endogenous phosphopeptidome. At the protein level, phosphoprotein
intensities were broadly distributed across all three groups, but
distinct abundance shifts were observed in disease cohorts compared
with CTL (Figure S5). At the peptide level,
overall phosphopeptide abundance profiles showed similar trends (Figure S4). Box plots revealed that HCC samples
contained a greater number of high-intensity peptides compared with
CIRR and CTL groups, as shown in Figure S3 by the most individual points (outliers) above the upper whisker.
Partial least-squares–discriminant analysis (PLS-DA) confirmed
distinct clustering of the diseased group from heathy controls (Figure
b).
Consistency
of phosphopeptide and precursor protein identification
was demonstrated by bar plots showing little variation in counts per
sample (Figure
d–e).
At the peptide level, 803 phosphopeptides were shared across all groups,
while subsets were uniquely enriched: 40 in HCC, 119 in CIRR, and
134 in CTL subjects, with 188 shared between HCC and CTL and 53 shared
between HCC and CIRR (Figure
g). At the protein level, 284 phosphoproteins were common
to all groups, while 84 were unique to CTL, 64 to CIRR, and 14 to
HCC (Figure
f). The
identified phosphoproteins exhibited a broad distribution of physicochemical
properties (Figure S2). Sequence coverage
values ranged from low to moderate, reflecting partial but consistent
coverage across the plasma proteome. The molecular weight (MW) distribution
spanned a wide range, with most proteins detected between ∼
10–100 kDa. The isoelectric point (pI) profile was evenly distributed
across acidic, neutral, and basic proteins. At the peptide level,
the endogenous phosphopeptides demonstrated wide variation in molecular
features (Figure S3). The charge state
distribution was dominated by doubly and triply charged peptides.
The retention time (RT) distribution showed uniform peptide elution
across the chromatographic gradient. The monoisotopic mass (MH+) values were broadly distributed, and the peptide length
distribution ranged from short motifs (∼7–10 residues)
to longer sequences (>20 residues), capturing the heterogeneity
of
circulating phosphopeptides. These features confirm that the data
set captures a representative and chemically diverse view of the circulating
phosphopeptidome.
To focus on biologically robust signals, peptides
were filtered
for 70% presence within each group 421 peptides were retained for
differential expression analysis. One-way ANOVA identified 69 significantly
altered peptides across HCC, CIRR, and CTL groups (FDR < 0.05, Supplementary Table S1 and Figure S7). Representative dot plots of significantly upward
and downward trend when comparing all the three groups are shown in Figure
.
Pairwise comparisons provided
further insight.
In the HCC vs CIRR,
statherin (P02808, DSSEEKFLR & DSSEEKFL) displayed the strongest
upregulation in HCC (fold change 5.26 and 3.16), while ApoA1 peptides
such as GHKEVTKEVVTS showed fold changes greater than 5. VSLGSPSGEVSHPRKT
(P02765) was also significantly higher in HCC (fold change 2.0). In
the HCC vs CTL comparison, several phosphopeptides were significantly
elevated. These included SLPGESEEMMEEVD from Interalpha-trypsin
inhibitor heavy chain (FC = 2.9) and YEYDELPAKDD from Complement C4-A (FC = 3.0), both involved in inflammatory
and acute-phase pathways. Conversely, multiple peptides showed decreased
abundance in HCC, such as NSPLDEENLTQENQDRGTHVDLG from Alpha-1-antichymotrypsin (FC = – 2.8), TFEEVSGNVSPGTRREYHTE from Fibrinogen
alpha chain (FC = – 1.8), and GSKGPLDQLEKGGETAQSAD
from Heparin cofactor 2 (FC = – 2.3).
Representative dot plots of significantly upregulated and downregulated
peptides are shown in Figures
(HCC, CIRR, and CTL) and (HCC vs
CIRR), with complete results presented in Table
.
Presence/absence testing by chi-squared
analysis further
highlighted
discriminatory peptides (Table
). PKLSPHKVQG (Q9GZP8) was uniquely detected in HCC but absent
the samples from cirrhotic patients and healthy control volunteers.
PRPGSTGTWNPGSSERGSAGHWTSESS and STFESKSYKMADEAGSEADHEGTH (P02671)
was enriched in patients with cirrhosis but not detected in HCC cases.
PLGEEDLPSEED (Q16790) and PDAKVEEEPEEEPEETA (P14625) were found over
50% HCC but absent in healthy controls. Such presence/absence patterns
add another dimension to the discriminatory potential of the plasma
phosphopeptidome.
Functional enrichment analysis of the significantly
altered endogenous
phosphoproteins revealed strong clustering into biologically relevant
categories (Figure S8). At the cellular
component (GO) level, enriched terms included blood microparticles,
platelet alpha granule lumen, extracellular matrix, and exosome compartments,
reflecting the secretory and circulating nature of the identified
phosphoproteins. Enrichment in endoplasmic reticulum and vesicle lumen
further underscored the contribution of secretory pathway proteins
to the circulating phosphopeptidome. At the molecular function (GO)
level, the dominant terms included peptidase regulator activity, enzyme
inhibitor activity, and serine-type endopeptidase inhibitor activity,
consistent with the overrepresentation of serpins and protease regulatory
proteins among the altered candidates. Additional enrichments in glycosaminoglycan
binding, collagen binding, and extracellular matrix structural constituents
highlight the functional involvement of phosphoproteins in extracellular
interactions and tissue remodeling. Pathway analysis using Ingenuity
Pathway Analysis (IPA) confirmed activation of several disease-relevant
signaling cascades. The top canonical pathways included coagulation
system, integrin signaling, LXR/RXR activation, acute-phase response
signaling, and post-translational protein phosphorylation (Figure
). Additional pathways
such as syndecan interactions, integrin–cell surface interactions,
and calcium-mediated platelet activation were also significantly represented,
suggesting links to vascular remodeling and immune modulation. The
protein–protein interaction (PPI) network analysis highlighted
tightly connected modules formed by apolipoproteins (APOA1, APOC3),
complement factors (C4A), serpins (SERPINA3, ITIH2), and structural
proteins (FN1, SPARC, collagens). These clusters converge on key regulators
such as integrins, TGF-β, and lipid metabolism proteins, with
LXR/RXR emerging as a central hub. The network topology underscores
the coordinated regulation of lipid transport, coagulation, and immune
signaling, all of which are processes strongly implicated in hepatocellular
carcinoma pathogenesis.

Diagnostic
Performance
To evaluate the diagnostic potential
of the identified endogenous phosphopeptides, receiver operating characteristic
(ROC) curve analysis was performed. In the HCC vs CTL comparison,
several peptides demonstrated strong discriminatory power shown in Figure
b. The peptide PPGAPHTEEEGAE
exhibited the highest diagnostic accuracy with an AUC of 0.913, indicating
excellent sensitivity and specificity. Other peptides, including YEYDELPAKDD
(AUC = 0.853), MGVVSLGSPSGEVSHPRKT (AUC = 0.830), and SLPGESEEMMEEVD
(AUC = 0.805), also showed robust classification performance. Additional
candidates such as NSPLDEENLTQENQDRGTHVDLG (AUC = 0.785) and GSKGPLDQLEKGGETAQSAD
(AUC = 0.773) demonstrated moderate but consistent diagnostic potential.
In the HCC vs CIRR comparison shown in Figure
a, the peptide DSSEEKFLR achieved the highest
diagnostic performance with an AUC of 0.968, reflecting excellent
discrimination between the two groups. Its truncated variant DSSEEKFL
also performed well with an AUC of 0.820. Other peptides, including
VSLGSPSGEVSHPRKT (AUC = 0.778) and GHKEVTKEVVTS (AUC = 0.763), showed
moderate diagnostic accuracy. By contrast, alpha-fetoprotein (AFP),
based on clinical serum data rather than MS analysis, yielded only
modest discrimination with an AUC of 0.648, performing considerably
worse than the MS-derived endogenous phosphopeptides.

Discussion
This study represents, to our knowledge,
the first comprehensive
characterization of the plasma endogenous phosphopeptidome in HCC,
benchmarked against CIRR and CTL. By applying a digestion-free workflow
with enrichment of phosphorylated peptides, we captured 1,365 unique
endogenous phosphopeptides corresponding to 549 precursor proteins.
The high reproducibility of our data set, reflected in strong correlations
across 60 plasma samples (r > 0.9) and consistent
HeLa QC performance, validates the robustness of this approach for
studying phosphorylation-dependent plasma signatures. The observation
that PLS-DA separated the disease groups from healthy controls with
minimal overlap demonstrates that the circulating phosphopeptidome
contains strong disease-specific information. These findings extend
earlier serum and plasma proteomics studies that focused on the digested
proteome rather than the endogenous peptidome. Villanueva et al. reported
large-scale proteomic alterations in HCC serum, identifying apolipoproteins
and complement proteins as candidate biomarkers, while Kimhofer et al. highlighted proteomic and metabolomic
markers linked to coagulation and immune signaling. Our study builds on this knowledge by demonstrating that
phosphorylation-specific endogenous peptides, which represent direct
in vivo proteolytic and signaling states, can differentiate HCC from
both CIRR and CTL individuals. Complement C4-A (YEYDELPAKDD) and Interalpha-trypsin
inhibitor heavy chain H4 (SLPGESEEMMEEVD) were markedly elevated, consistent with activation of complement and
acute-phase response pathways in HCC plasma. In contrast, peptides from Alpha-1-antichymotrypsin (NSPLDEENLTQENQDRGTHVDLG),
Heparin cofactor II (GSKGPLDQLEKGGETAQSAD), and Fibrinogen alpha chain
(TFEEVSGNVSPGTRREYHTE) were reduced, indicating impaired
protease inhibition and anticoagulant regulation. Together, these
shifts point to coordinated dysregulation of complement coagulation
and acute-phase signaling, highlighting the plasma endogenous phosphopeptidome
as a sensitive indicator of early hepatocellular carcinoma–associated
biochemical changes. The HCC vs CIRR comparison highlighted statherin-derived
peptides (DSSEEKFLR and DSSEEKFL) are highly discriminatory. Statherin, a phosphorylated protein,
is classically associated with saliva, but its presence in plasma
at a very low level
,
and strong upregulation in HCC
is novel, suggesting an unexplored role in systemic signaling or tumor
biology. The parallel increase of Fibrinogen alpha chain (GHKEVTKEVVTS)
and Alpha-2-HS-glycoprotein (VSLGSPSGEVSHPRKT) further supports activation
of the coagulation and acute-phase response systems, both of which
are tightly linked to hepatic inflammation, matrix remodeling, and
tumor progression. These findings align with prior observations that
fibrinogen and fetuin-A (AHSG) undergo extensive phosphorylation during
chronic liver injury, modulating cell adhesion and signaling cascades
associated with oncogenic transformation.
The distinct presence–absence
patterns of endogenous phosphopeptides
across disease states likely reflect underlying alterations in protease
activity, extracellular matrix turnover, and phosphorylation-driven
signaling during liver disease progression. The exclusive appearance
of PKLSPHKVQG in HCC plasma suggests tumor-specific proteolytic processing
or kinase-mediated modification events that generate unique circulating
fragments absent in cirrhotic or healthy control states. Conversely,
the disappearance of fibrinogen-derived peptides such as PRPGSTGTWNPGSSERGSAGHWTSESS
and STFESKSYKMADEAGSEADHEGTH in HCC compared with cirrhosis may indicate
progressive degradation or altered secretion of coagulation-related
proteins as fibrosis transitions to malignancy. Together, these qualitative
shifts in phosphopeptide occurrence highlight that the circulating
phosphopeptidome not only encodes quantitative abundance changes but
also captures dynamic molecular turnover events, offering mechanistic
insight into tumor-associated protease dysregulation and matrix remodeling
in HCC. Such binary patterns have been used in other body-fluid proteomics
studies as robust diagnostic indicators, particularly when quantitative
fold changes may be subtle.

The
ROC analyses provide strong translational support for these
observations. Individual peptides demonstrated diagnostic accuracies
that substantially outperformed AFP, the current clinical standard.
In particular, PPGAPHTEEEGAE discriminated HCC from healthy controls
with an AUC of 0.913, while statherin-derived DSSEEKFLR distinguished
HCC from cirrhosis with an AUC of 0.968. These values exceed the typical
performance of AFP (AUC 0.6–0.7 in most clinical studies),
reinforcing the potential of endogenous phosphopeptides as superior
biomarkers. Importantly, the top-performing peptides mapped back to
biological pathways identified in enrichment analyses, further validating
their mechanistic plausibility. For example, fibrinogen- and alpha-1-antichymotrypsin–derived
peptides link directly to coagulation and acute-phase pathways, while
ApoA1- and statherin-derived peptides align with lipid metabolism
and extracellular remodeling.
The functional enrichment and
pathway analysis of differentially
expressed endogenous phosphoproteins revealed that HCC progression
is strongly reflected in the circulating phosphopeptidome. GO enrichment
at the cellular component level highlighted extracellular compartments
including blood microparticles, platelet alpha granule lumen, and
exosomes. This finding underscores the role of secretory and vesicle-associated
proteins in shaping the plasma phosphopeptidome, consistent with recent
reports that extracellular vesicles serve as important carriers of
post-translationally modified peptides in cancer and liver disease.
,
At the molecular function level, the enrichment of peptidase regulator
activity and serine-type endopeptidase inhibitor activity points to
a central role of protease–antiprotease balance in the systemic
circulation of HCC patients. The predominance of serpins and related
protease inhibitors in our data set aligns with previous proteomics
studies linking dysregulated protease activity to liver injury, tissue
remodeling, and tumor microenvironment dynamics. The additional enrichment of extracellular matrix and glycosaminoglycan
binding functions further indicates that circulating phosphoproteins
are closely tied to processes of matrix turnover and fibrosis, both
of which are fundamental to cirrhosis and HCC development. Ingenuity
Pathway Analysis (IPA) provided deeper insight into disease-relevant
signaling networks. The coagulation system and acute-phase response
signaling emerged among the most significant pathways, highlighting
systemic inflammation and hemostatic imbalance as key features of
HCC biology. This is consistent with earlier plasma proteomics and
clinical data showing that hypercoagulability and inflammatory mediators
contribute to both cirrhosis progression and hepatocarcinogenesis. Importantly, the enrichment of the LXR/RXR axis
suggests altered lipid metabolism and nuclear receptor signaling in
the plasma phosphopeptidome of HCC patients. LXR/RXR signaling has
been previously implicated in hepatic lipid regulation, immune modulation,
and tumor-promoting processes, and our
findings provide phosphopeptide-level evidence of its systemic dysregulation
in HCC. The protein–protein interaction (PPI) network analysis
supports these observations, revealing highly connected clusters composed
of apolipoproteins (APOA1, APOC3), complement components (C4A), serpins
(SERPINA3, ITIH2), and extracellular matrix proteins (FN1, SPARC,
collagens). The network topology emphasizes the interplay between
lipid transport, complement activation, and extracellular remodeling,
with integrins and TGF-β signaling nodes acting as central regulators.
These hubs are known to drive cell adhesion, migration, and immune
interactions in the tumor microenvironment. Together, the enrichment and network analyses demonstrate that the
altered plasma phosphopeptidome in HCC captures a convergent signature
of inflammation, coagulation, matrix remodeling, and lipid signaling.
Overall, this systems-level view extends beyond individual peptide
markers, revealing that endogenous phosphopeptides map onto coordinated
biological processes relevant to HCC pathogenesis. These results suggest
that the plasma phosphopeptidome may serve not only as a diagnostic
biomarker reservoir but also as a window into the systemic signaling
changes that accompany liver cancer progression. This study has several
strengths, including the digestion-free workflow that preserved in
vivo phosphorylation, rigorous QC to ensure reproducibility, and the
inclusion of cirrhosis controls, which addresses the critical diagnostic
challenge of distinguishing HCC from cirrhosis. Limitations include
the modest cohort size and the absence of validation in an independent
population. Future studies should validate these candidate peptides
using targeted assays such as PRM or MRM in larger cohorts, ideally
including early stage HCC patients.

Conclusions
This
study provides the first in-depth
characterization of the
plasma endogenous phosphopeptidome in hepatocellular carcinoma (HCC),
revealing its strong potential as a minimally invasive source of circulating
biomarkers. Using high-resolution LC–MS/MS and integrative
statistical analysis, we identified several phosphopeptides with outstanding
diagnostic performance. The statherin-derived peptide DSSEEKFLR achieved
an AUC = 0.968 for distinguishing HCC from cirrhosis far exceeding
AFP (AUC = 0.648) while its truncated variant DSSEEKFL (AUC = 0.820)
reinforces the findings. Additional candidates, including PPGAPHTEEEGAE
(NST1), YEYDELPAKDD (C4A), SLPGESEEMMEEVD (ITIH4), and VSLGSPSGEVSHPRKT
(AHSG), also displayed high accuracy (AUC > 0.80). Together, these
findings highlight a novel phosphopeptide signature that captures
disease-specific proteolytic and phosphorylation events, offering
a promising foundation for blood-based diagnostics and clinical monitoring
of HCC. While these findings are promising, validation of the candidate
biomarkers via absolute quantification in a larger sample size is
needed. Future work will focus on validating these candidates using
targeted assays and assessing their performance across broader clinical
populations.

Supplementary Material