Biology subjects

Ueda, K.

Publications and source records attributed to Ueda, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Molecular communication enables cooperative genomic RNA replication in all-aqueous droplet colonies

Multicellular organization enables biological functions to be distributed among specialized cells and coordinated through intercellular communication. Integrating this organizational principle with genome replication would link functional division of labor to the propagation of genetic information. Here, we show that genomic RNAs with complementary functions can cooperatively replicate across communicating artificial compartments. We constructed multicell-like colonies from all-aqueous droplets formed by phase separation of two incompatible polymers and stabilized at their interfaces by liposomes and amyloid-like proteins. The droplets assembled spontaneously while remaining permeable to protein-sized macromolecules. Two genomic RNAs encoding a replication enzyme and a metabolic enzyme were distributed in distinct colony-forming droplets and cooperatively replicated through cell-free translation and reciprocal molecular communication. These findings establish that genome replication can be collectively supported by communicating artificial compartments and provide a route toward multicell-like systems that coordinate spatially distributed genetic functions.

synthetic biology↗

bioRxiv2026

Modulating supplementary motor area excitability enhances groove-related pleasure during music listening

Pleasurable urge to move to music is often referred to as groove. Although previous studies have shown an association between the supplementary motor area (SMA) and the groove experience, its causal role remains unclear. Here, we investigated whether the SMA is causally involved in groove experience during music listening using repetitive transcranial magnetic stimulation. Fifteen healthy participants completed three sessions on separate days: excitatory stimulation (intermittent theta burst stimulation; iTBS) over the SMA, inhibitory stimulation (continuous theta burst stimulation; cTBS) over the SMA, and sham stimulation (iTBS or cTBS) over the vertex. After each stimulation session, participants listened to five high-groove and five low-groove musical excerpts and rated urge-to-move and pleasure on a 0-100 scale. Heart rate was additionally recorded as an exploratory physiological measure during music listening. Linear mixed-effects models (LMM) showed that pleasure ratings, but not urge-to-move ratings, were higher following both iTBS and cTBS compared with sham stimulation. In exploratory LMMs, reduced log-transformed heart rate variability (HRV) significantly predicted higher pleasure ratings. These findings suggest that SMA stimulation modulates the pleasurable component of the groove experience, likely via network-level mechanisms rather than a simple linear relationship between SMA excitability and pleasure. They also raise the possibility that reduced parasympathetic activity, reflected by lower HRV, mediates the stimulation-related increase in musical pleasure. Future studies should investigate the causal roles of other brain regions as well as clarify the directionality between autonomic changes and the groove experience.

neuroscience↗

bioRxiv2026

Macrophage extracellular traps promote maladaptive cardiac remodelling and heart failure via PAD4-dependent mechanisms

AimsThe activation of inflammatory cells, particularly macrophages, plays a pivotal role in the pathogenesis of cardiac remodelling and heart failure. Emerging evidence indicates that extracellular traps released from inflammatory immune cells contribute to the progression of various pathologies. However, the clinical relevance and mechanistic role of macrophage extracellular traps (METs) in heart failure remain to be elucidated. Methods and ResultsEndomyocardial biopsy specimens from 69 patients with heart failure were analysed by fluorescent immunostaining to identify and quantify METs. The numbers of METs per myocardial tissue area in patients with heart failure showed a negative correlation with left ventricular (LV) ejection fraction and a positive correlation with LV end-diastolic diameter. Patients with higher MET counts had significantly lower event-free survival from the composite cardiac events. In a murine model of pressure overload by transverse aortic constriction (TAC), METs were most abundantly observed at 3 days post-TAC and remained detectable throughout the 4-week observation period. In vitro, time-dependent MET formation was induced by an intrinsic trigger of mitochondrial DNA in bone marrow-derived macrophages from wild-type (WT) mice, but not in peptidyl arginine deiminase 4 (PAD4)-deficient macrophages, indicating that PAD4 activity is indispensable for MET formation. The recipient mice transplanted with bone marrow cells from PAD4 knockout mice showed more preserved cardiac function, reduced myocardial fibrosis, and improved survival in response to TAC, compared to those transplanted with WT mice. Ex vivo analyses demonstrated that conditioned medium containing METs from WT macrophages induced fibroblast-to-myofibroblast transition via Toll-like receptor 4 signalling. ConclusionsPAD4-dependent MET formation from bone marrow-derived macrophages represents a novel driver of cardiac remodelling. Targeting MET formation may offer a potential therapeutic strategy for heart failure. Translational PerspectiveMacrophage extracellular traps (METs) are abundant in myocardial tissue from patients with heart failure with reduced ejection fraction and are associated with adverse left ventricular remodelling and worse clinical outcomes. These findings support myocardial MET burden as a potential tissue biomarker to improve risk stratification in heart failure patients. In mice, pressure overload induces MET formation, and hematopoietic PAD4 deficiency suppresses myocardial METs, attenuates fibrosis, preserves cardiac function, and improves survival. Mechanistically, mitochondrial DNA-enriched cardiomyocyte-derived exophers trigger PAD4-dependent METs, which activate cardiac fibroblasts through TLR4 signalling. Suppressing METs represents a potential therapeutic strategy to attenuate the progression of heart failure. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/711858v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@11b9b57org.highwire.dtl.DTLVardef@17386f7org.highwire.dtl.DTLVardef@1a1f002org.highwire.dtl.DTLVardef@942bbc_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2025

Predictive Networks Generate Motion-Induced Color Illusions

Subjective color (SC) refers to chromatic perception induced by achromatic stimuli, as classically demonstrated using Benhams top. Despite nearly two centuries of investigation, the underlying neural mechanisms remain unclear. We developed artificial neural network (ANN) models trained on natural videos using predictive learning to investigate SC generation. These models successfully reproduced artificial subjective color (ASC) despite the absence of explicit color information in the training stimuli. Critically, ASC characteristics were systematically influenced by the colors of moving objects in the training videos. Using progressively simplified stimuli, ranging from natural scenes to 3D computer graphics to 2D animations, we demonstrated that the colors of moving objects are the primary determinant of ASC properties. We propose that predictive learning establishes color associations with motion patterns, causing the arcs on Benhams top to be perceived as moving objects with their corresponding colors. These findings suggest that cortical predictive mechanisms may complement retinal processes in generating subjective color phenomena.

neuroscience↗

bioRxiv2025

Mycophenolate mofetil reduces the branching of microglial processes

Microglia, the resident immune cells in the central nervous system, play important roles not only in immune response but also in neurogenesis, synaptogenesis, and neural circuit formation. Microglia also surveil the brain environment via elongation and retraction of their processes. Previously, we found that the purine salvage pathway is involved in the regulation of morphology and dynamics of the microglial cell line BV2. Here, we show that intraperitoneal administration of mycophenolate mofetil (MMF), an inosine monophosphate dehydrogenase (IMPDH) inhibitor, reduces microglial branching during postnatal development. Imaging mass spectrometry analysis revealed that MMF administration decreases guanosine nucleotides in the brain. Interestingly, despite the essential role of guanosine nucleotides in cellular proliferation, MMF administration did not significantly affect microglial proliferation. On the other hand, MMF administration attenuated the level of GTP-bound forms of RhoA and Rac1 small GTPases. Notably, MMF administration decreased the number of branches, while process length remained unaffected. Since microglial branching affects microglial complexity and diversity, our findings suggest that guanosine nucleotide production is essential for generating proper microglial diversity.

neuroscience↗

bioRxiv2025

Development of a liquid biopsy for bladder cancer using a mutant protein panel in urinary extracellular vesicles

Non-muscle invasive bladder cancer often relapses after cystoscopic surgery, necessitating rigorous monitoring for recurrence through invasive and painful cystoscopy. To develop a novel non-invasive and cancer-specific diagnostic method, somatic mutant proteins in urinary extracellular vesicles (EVs) were for the first time investigated using a proteogenomics pipeline consisting of whole exome sequencing and LC/MS. The analysis of bladder cancer tissues, cultured tissue-derived EVs, and urinary EVs from five patients identified 11,207, 9,809, and 5,828 unique proteins, respectively. Notably, 39, 32, and 4 mutant proteins were found in each of the sample sets. Furthermore, mass spectrometric absolute quantification measurements were conducted using prospectively collected urine samples, revealing that the levels of all monitored mutant proteins (LCP1_D321H, TKT_K102N, and PLCD1_R639H) exhibited a clear correlation with the cystoscopic tumor burden. Therefore, the presence of mutant proteins in Evs presents an ideal approach for liquid biopsy, serving as a non-invasive urine test for bladder cancer. TeaserMutant proteins in urine were identified using a proteogenomic pipeline, which may serve as a novel method for monitoring bladder cancer.

cancer biology↗

bioRxiv2025

Functional and structural profiling of circulation via genetically encoded modular fluorescent probes.

Sustained labeling of fluids is crucial for their investigation in animal models. Here, we introduce a mouse line (Alb-mSc-ST), where blood and interstitial fluid are labeled with the red fluorescent protein mScarlet and SpyTag. The SpyTag-SpyCatcher technology is exploited to monitor circulating fluid properties by biosensors or detect blood-brain barrier disruption. This approach represents a valuable tool for studying vascular structure, permeability and microenvironment in body organs in vivo.

neuroscience↗

bioRxiv2024

MitoNEET reduces the mitochondrial oxidative phosphorylation during epithelial-mesenchymal transition

Mitochondrial functions range from catabolic to anabolic, which are tightly coordinated to meet cellular demands for proliferation and motility. MitoNEET is a mitochondrial outer membrane protein with a CDGSH domain and is involved in mitochondrial function. Epithelial-to-mesenchymal transition (EMT) is the process in which cells lose their epithelial characteristics and acquire mesenchymal traits, such as motility, which is a vital step for organism development and wound-healing. Cellular motility is associated with high ATP consumption owing to lamellipodia formation, which is supported by upregulated oxidative phosphorylation (OXPHOS) capacity. However, how mitoNEET is involved in the regulation of OXPHOS capacity and subsequent cellular motility remains unclear. Here we show that loss of mitoNEET regulation during EMT impairs both OXPHOS enhancement and cell motility in non-transformed NMuMG mouse mammary gland epithelial cells. We found that mitoNEET is downregulated during EMT, and that the aberrant expression of mitoNEET abolishes the upregulation of OXPHOS, leading to the inhibition of cell motility. Furthermore, we found that mitoNEET topology may be crucial for the regulation of the mitochondrial electron transfer chain, suggesting an additional regulatory pathway for OXPHOS capacity. Our results demonstrate that mitochondrial OXPHOS capacity during EMT is partly regulated by the dynamics of the outer membrane protein. We believe that our findings are the first step towards understanding the mechanisms by which mitochondrial outer membrane protein topology affects organelle functions.

cell biology↗

bioRxiv2023

Prenatal vitamin D deficiency alters immune cell proportions of young adult offspring through alteration of long-term stem cell fates

Vitamin D deficiency is a common deficiency worldwide, particularly among women of reproductive age. During pregnancy, it increases the risk of immune-related diseases in offspring later in life. However, exactly how the body remembers exposure to an adverse environment during development is poorly understood. Herein, we explore the effects of prenatal vitamin D deficiency on immune cell proportions in offspring using vitamin D deficient mice established by dietary manipulation. We found that prenatal vitamin D deficiency alters immune cell proportions in offspring by changing the transcriptional properties of genes downstream of vitamin D receptor signaling in hematopoietic stem and progenitor cells of both the fetus and adults. Our results suggest the role of cellular differentiation properties of the hematopoiesis as the long-term memories of prenatal exposure at the adult stage. Moreover, further investigations of the associations between maternal vitamin D levels and cord blood immune cell profiles from 75 healthy pregnant women and their term babies also confirm that maternal vitamin D levels in the second trimester significantly affect immune cell proportions in the babies. This highlights the importance of providing vitamin D supplementation at specific stages of pregnancy.

developmental biology↗

bioRxiv2023

p53 controls the nuclear entry and epigenetic modification of H3.1 by downregulating nuclear phosphatidic acid

Histones are key molecules of epigenetic regulation and inheritance, and are thought to be chaperoned and transported into the nucleus appropriately prior to being integrated into nucleosomes. H3.1 histone is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, as a new component of duplicating nucleosomes. Here we found that p53 is necessary to secure the normal behavior and modification of H3.1 in the nucleus during the G1/S phase, in which p53 increases C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1) levels and decreases enhancer of zeste homolog 2 (EZH2) levels in the H3.1 interactome. In the absence of p53, H3.1 molecules tended to be tethered at or near the nuclear envelope (NE), where they were predominantly trimethylated at lysine 27 (H3K27me3) by EZH2, without forming nucleosomes. This accumulation was likely caused by the high affinity of H3.1 towards phosphatidic acid (PA). p53 reduced nuclear PA levels by increasing levels of CTDNEP1, which activates lipin to convert PA into diacylglycerol. Induction of the TMEM255A gene by p53 linked p53 with CTDNEP1, in which TMEM255A stabilized CTDNEP1. We moreover found that the cytosolic H3 chaperone HSC70 attenuates the H3.1-PA interaction, and our molecular imaging analyses suggested that H3.1 molecules may be anchored around the NE after their nuclear entry. Our results expand our knowledge of p53 function in regulation of the nuclear behavior of H3.1 during the G1/S phase, in which p53 may primarily target nuclear PA and EZH2.

cell biology↗

bioRxiv2023

Differentiation of Large Extracellular Vesicles in Oral Fluid: Combined Protocol of Small Force Centrifugation and Pattern Analysis

Extracellular vesicles (EVs) in biofluids are highly heterogeneous entities in terms of their origins and physicochemical properties. Considering the application of EVs in diagnostic and therapeutic fields, it is of extreme importance to establish differentiating methods by which focused EV subclasses are operationally defined. Several differentiation protocols have been proposed; however, they have mainly focused on smaller types of EVs, and the heterogeneous nature of large EVs has not yet been fully explored. In this report, to classify large EVs into subgroups based on their physicochemical properties, we have developed a protocol, named EV differentiation by sedimentation patterns (ESP), in which entities in the crude large EV fraction are first moved through a density gradient of iodixanol with small centrifugation forces, and then the migration patterns of molecules through the gradients are analyzed using a non-hierarchical data clustering algorithm. Based on this method, proteins in the large EV fractions of oral fluids clustered into three groups: proteins shared with small EV cargos and enriched in immuno-related proteins (Group 1), proteins involved in energy metabolism and protein synthesis (Group 2), and proteins required for vesicle trafficking (Group 3). These observations indicate that the physiochemical properties of EVs, which are defined through low-speed gradient centrifugation, are well associated with their functions within cells. This protocol enables the detailed subclassification of EV populations that are difficult to differentiate using conventional separation methods.

molecular biology↗

bioRxiv2022

Emergence of linkage between cooperative RNA replicators encoding replication and metabolic enzymes thorough experimental evolution

The integration of individually replicating genes into a primitive chromosome is a key evolutionary transition in the development of life, allowing the simultaneous inheritance of genes. However, how this transition occurred is unclear because of the extended size of primitive chromosomes, which replicate slower than unlinked genes. Theoretical studies have suggested that a primitive chromosome can evolve in the presence of cell-like compartments, as the physical linkage prevents the stochastic loss of essential genes upon division, but experimental support for this is lacking. Here, we demonstrate the evolution of a chromosome-like RNA from two cooperative RNA replicators encoding replication and metabolic enzymes. Through their long-term replication in cell-like compartments, linked RNAs emerged with the two cooperative RNAs connected end-to-end. The linked RNAs had different mutation patterns than the two unlinked RNAs, suggesting that they were maintained as partially distinct lineages in the population. Our results provide experimental evidence supporting the plausibility of the evolution of a primitive chromosome from unlinked gene fragments, an important step in the emergence of complex biological systems. Author SummaryThe integration of genes into a chromosome is a fundamental genetic organization in all extant life. The assembly of unlinked genes during prebiotic evolution was likely a major evolutionary transition toward the development of a complex cell. Decades of theoretical studies have suggested a plausible evolutionary pathway to a primitive chromosome from replicating RNA molecules that harbor cooperative genes within a protocell structure. However, demonstrating the evolution of a primitive chromosome in an experimental setup is challenging. We previously developed a cooperative RNA replication system in which two types of RNAs co-replicate using their self-encoded replication and metabolic enzymes. Using this system, in the present study, we demonstrate the evolution of a linkage between the two cooperative RNA replicators in compartments. An evolved "linked" RNA harbored the entire region of both genes, accumulated distinct mutations, and retained the ability to replicate using the two proteins translated from itself. These experimental findings support a prebiotic evolutionary scenario, in which unlinked genes assembled into a single genomic structure.

evolutionary biology↗

bioRxiv2021

ABCA1 causes an asymmetric cholesterol distribution to regulate intracellular cholesterol homeostasis

Cholesterol is a major and essential component of the mammalian cell plasma membrane (PM) and the loss of cholesterol homeostasis leads to various pathologies. Cellular cholesterol uptake and synthesis are regulated by a cholesterol sensor in the endoplasmic reticulum (ER). However, it remains unclear how the PM cholesterol level is sensed. Here we show that the sensing depends on ATP-binding cassette A1 (ABCA1) and Aster-A, which cooperatively maintain the asymmetric transbilayer cholesterol distribution in the PM. ABCA1 translocates (flops) cholesterol from the inner to the outer leaflet of the PM to maintain a low inner cholesterol level. When the inner cholesterol level exceeds a threshold, Aster-A is recruited to the PM-ER contact site to transfer cholesterol to the ER. These results show unknown synergy between ABCA1 and Aster-A in intracellular cholesterol homeostasis.

cell biology↗

bioRxiv2021

Rational control of structural off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, a key parameter that largely dictates the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

biophysics↗

bioRxiv2021

Fast and ultra-sensitive glycoform analysis by supercritical fluid chromatography-tandem mass spectrometry

Therapeutic monoclonal antibodies (mAbs) are currently the largest and fastest growing category of biopharmaceuticals. Glycosylation of mAbs has a significant impact on their effector functions, as well as on their safety and pharmacokinetics. Heterogeneity of glycoforms is affected by various factors such as the producing cells and cell culture process. Therefore, accurate glycoform characterization is essential for drug design, process optimization, manufacturing, and quality control of therapeutic mAbs. In this study, we developed a fast, quantitative, and highly sensitive analytical platform for mAb glycan profiling by supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS). An 8-minute analysis of bevacizumab, nivolumab, ramucirumab, rituximab, and trastuzumab by SFC-MS detected a total of 102 glycoforms, with a detection limit of 5 attomole. The dynamic range of glycan abundance was over 6 orders of magnitude for bevacizumab analysis by SFC-MS, compared to 3 orders of magnitude for conventional fluorescence HPLC analysis. This method revealed the glycan profile characteristics and lot-to-lot heterogeneity of various therapeutic mAbs. We were also able to detect a series of structural variations in pharmacologically important glycan structures. SFC-MS-based glycoform profiling method will provide an ideal platform for in-depth analysis of precise glycan structure and abundance.

biochemistry↗

bioRxiv2021

Elevated METTL9 is associated with peritoneal dissemination in human scirrhous gastric cancers

Methylation, the most common chemical modification of cellular components such as DNA, RNA, and proteins, impacts biological processes including transcription, RNA processing, and protein dynamics. Although abnormal expression of methyltransferase can lead to various diseases including cancers, little is known about the relationship between methyltransferase and cancers. Here we aimed to understand the role of methyltransferase in cancer metastasis. We found that elevated methyltransferase-like 9 (METTL9) is closely associated with the acquisition of metastatic activity in human scirrhous gastric cancers. The stable knockdown of METTL9 via an shRNA vector technique in our original metastatic cells from scirrhous gastric cancer patients significantly inhibited migration and invasion. In metastatic cells, METTL9 protein is predominantly localized in mitochondria, and the METTL9 knockdown significantly reduced mitochondrial Complex I activity. METTL9 can be a promising molecular target to inhibit peritoneal dissemination of scirrhous gastric cancers. This report is the first to describe the relationship between METTL9 and cancer metastasis. HighlightsO_LIElevated METTL9 correlates with metastasis in human scirrhous gastric cancer. C_LIO_LIThis is the first report on the biological relationship between METTL9 and metastasis. C_LIO_LIMETTL9 protein localizes mainly in mitochondria in metastatic scirrhous gastric cancer. C_LIO_LIMETTL9 knockdown reduces mitochondrial Complex I activity to decrease cell migration and invasion in metastatic scirrhous gastric cancer. C_LIO_LIMETTL9 holds promise against peritoneal dissemination of scirrhous gastric cancer. C_LI

cancer biology↗

bioRxiv2021

Recruitment of Peroxin14 to lipid droplets affects triglyceride storage in Drosophila

The activity of multiple organelles must be coordinated to ensure cellular lipid homeostasis. This includes the peroxisomes which metabolise certain lipids and lipid droplets which act as neutral lipid storage centres. Direct organellar contact between peroxisomes and lipid droplets has been observed, and interaction between proteins associated with the membranes of these organelles has been shown, but the functional role of these interactions is not clear. In Drosophila cells, we identified a novel localization of a subset of three transmembrane Peroxin proteins (Peroxin3, Peroxin13, and Peroxin14), normally required for peroxisome biogenesis, to newly formed lipid droplets. This event was not linked to significant changes in peroxisome size or number, nor was recruitment of other Peroxin proteins or mature peroxisomes observed. The presence of these Peroxin proteins at lipid droplets influences their function as changes in the relative levels of Peroxin14 associated with the lipid droplet surface directly affected the presence of regulatory perilipin and lipases with corresponding effects on triglyceride storage. Summary statementInteractions between peroxisomes and lipid droplets is thought to help coordinate management of cellular lipids. Peroxin proteins are required for peroxisome biogenesis. A spectrum of effects on triacylglyceride storage was seen when each of the 12 conserved Peroxins are knocked down in the Drosophila fat body with Peroxin14 knockdown having the largest effect. When Drosophila S2 cells were cultured in excess oleic acid, Peroxin3, Peroxin13, and Peroxin14, but not other Peroxins were localized to lipid droplets independently of other peroxisome markers. The presence of Peroxin14 at the lipid droplet surface altered recruitment of perilipin and lipase proteins.

cell biology↗

bioRxiv2021

Differential immunopeptidome analysis revealed cancer specific amino acid usage of HLA class-I antigens and novel neoantigens of colorectal cancer

Understanding the properties of human leukocyte antigen (HLA) peptides (HLAp) is essential for cancer precision medicine, while the direct identification of HLAp from a tiny piece of clinical tissues by mass spectrometry (MS) is still confronted with technical challenges. Here, to overcome that hindrances, high-field asymmetric waveform ion mobility spectrometry (FAIMS) is introduced to conduct differential ion mobility (DIM)-MS by seamless gas-phase fractionation optimal for scarce samples. The global-immunopeptidomics analysis enables in-depth identification of 44,785 HLAp, including 2 neoantigens with KRAS-G12V and CPPED1-R228Q from stage IV colorectal cancer (CRC) tissues obtained from 17 subjects. Comparison of tissue-based personal immunopeptidome disclosed cancer-specific processing of HLAp. Since the direct identification of neoantigens from CRC tissues suggests more potential neoantigens have yet to been identified, the targeted-immunopeptidomics by screening-oriented parallel reaction monitoring (PRM) has been established and identified two more neoantigens with oncogenic KRAS. The targeted-immunopeptidomics analysis clarifies the presentation of aimed neoantigens, including neoantigens that are unpredictable by computational algorithms. The comprehensive-immunopeptidomics approach combining the FAIMS-assisted DIM-MS and the screening-oriented PRM contributes the direct and effective identification of HLAp and emerging neoantigens from clinical tissue samples, as an advanced strategy to identify the authentic and recurrent neoantigens for cancer immunotherapy.

cancer biology↗