Biology subjects

Luo, L.

Publications and source records attributed to Luo, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Pediatric brain charts reveal ADHD biotypes with distinct developmental, molecular, and therapeutic profiles

Attention-deficit/hyperactivity disorder (ADHD) exhibits profound clinical heterogeneity; however, its underlying neurobiological substrates remain unclear. Here, leveraging large - scale structural magnetic resonance imaging data from 12,663 typically developing children and 1,699 children with ADHD across Chinese and United States cohorts, we construct population specific pediatric brain charts via hierarchical Bayesian normative modeling to quantify individual deviations in cortical morphology. Data-driven clustering identifies two robust, cross - ethnically replicable ADHD biotypes: an infra - normal biotype characterized by widespread cortical thinning and a supra - normal biotype marked by cortical thickening. Multivariate analyses reveal distinct brain-behavior architectures: cortical deviations correlate preferentially with symptom severity in the infra-normal biotype but with both symptom severity and cognitive performance in the supranormal biotype. Clinically, the infra-normal biotype demonstrates a superior response to methylphenidate over atomoxetine, whereas the supra-normal biotype responds comparably to both. Transcriptomic analyses identify biotype-specific molecular signatures converging with ADHD genome-wide association study (GWAS) risk loci but exhibiting distinct expression patterns in the brain. These findings classify ADHD heterogeneity into neurobiologically distinct, clinically informed biotypes, providing a mechanistic framework for precision psychiatry in pediatric ADHD.

neuroscience↗

bioRxiv2026

PatientPheX: A Full-Text Biomedical Corpus for Patient-Specific Phenotype Information Extraction

Patient-specific phenotype information extraction identifiers and normalizes phenotype entities and links them with individual patients, supporting rare disease research and genotype-phenotype analysis. However, existing datasets rarely combine full-text biomedical content with detailed annotations of patients, phenotypes, related entities, and their associations. In this work, we present PatientPheX, a corpus for patient-specific phenotype information extraction comprising 200 full-text articles, primarily case reports and case series. The corpus provides mention annotations and normalization for seven biomedical entity types, together with explicit associations between patients and entities across sentences, paragraphs, and sections. Negated findings and composite phenotype mentions are also annotated. Corpus construction combines automated pre-annotation, large language model (LLM)-assisted verification, and manual review and adjudication. Benchmarks cover entity recognition and normalization and patient-phenotype association extraction using gold-standard and predicted entities. The overall document-level entity F1 was 86.95%, while phenotype F1 was 79.33%. The best relation extraction macro-F1 decreased from 84.14% with gold-standard entities to 54.98% with predicted entities, highlighting the combined challenges of phenotype extraction and patient attribution. PatientPheX provides a resource for developing and evaluating patient-centered information extraction methods and supports the construction of structured patient-level knowledge resources for phenotype-driven biomedical research.

bioinformatics↗

bioRxiv2026

Structural streamlining of axonemal doublet microtubules in Plasmodium male gametes

Male gametogenesis in malaria parasites produces up to eight flagellated gametes within approximately 15 min, requiring de novo construction of motile axonemes on an unusually short timescale. Here we combine cellular cryo-electron tomography, high-resolution cryo-EM, proteomics, and parasite genetics to define the molecular architecture and assembly states of the Plasmodium male gamete axoneme. The doublet microtubules retain the principal surface-associated motility modules but contain a markedly reduced luminal microtubule inner protein network. Their inner junction lacks PACRG and is instead organized by CFAP20 and CFAP52, whose combined loss disrupts doublet integrity and exflagellation. We further identify a calmodulin protein at the outer dynein arm docking region and show that outer dynein arms accumulate progressively during gametogenesis. These findings establish distinct form of axonemal remodeling in Plasmodium and reveal the ordered assembly of structural and motor modules during rapid male gamete formation.

cell biology↗

bioRxiv2026

Hypothalamic Cell Type Evolution across Vertebrates

The hypothalamus regulates vital functions and is highly conserved among vertebrates occupying diverse niches, but little is known about the evolution of its cell types. Using comparative single-cell transcriptomics across major vertebrate classes, we identified deeply conserved and clade-specific cell types. While homologous cell types express conserved transcription factors and neuropeptides, their regulatory linkages are often rewired across evolution. The paraventricular nucleus largely retains its neuropeptide expression and responds to dehydration with fine cellular resolution from amphibians to mammals, whereas lamina terminalis neurons are specific to terrestrial vertebrates and exhibit gene expression divergence in cell types activated by dehydration and heat. We propose that a conserved set of keystone cell types sustain essential functions across vertebrates, while flexible gene regulatory programs alongside numerous clade-specific cell types offer evolutionary fluidity.

evolutionary biology↗

bioRxiv2026

Endolysosomal acidification regulates intestinal injury and repair in Drosophila by augmenting JNK activation and innate immune signaling pathways

Vacuolar ATPases (V-ATPases) are highly conserved multi-subunit proton pumps that drive the acidification of intracellular vesicles, especially endosomes and lysosomes. Beyond enabling cargo degradation, endolysosomal acidification shapes signal transduction, acting both positively through receptor processing in endosomes and negatively through lysosomal turnover of pathway mediators. Whether endolysosomal acidification shapes the intestinal epithelial response to oxidative injury remains unclear. Here we use Drosophila to define the role of V-ATPases in intestinal injury driven by excessive oxidative stress. Consistent with lysosomal biogenesis programs induced by oxidant in other systems, we find that oxidant exposure increases V-ATPase subunit expression and abundance of acidified vesicles in the midgut. Yet this response is not protective. RNAi depletion of multiple V-ATPase subunits suppresses lethality induced by either hydrogen peroxide or paraquat exposure. These effects map to mature absorptive enterocytes rather than to intestinal progenitors. Enterocyte-specific depletion of Vha44 (subunit C of the V1 domain) preserves intestinal barrier function and suppresses the cell death, JNK pathway activation, and IMD/NF-{kappa}B reporter activity induced by oxidant exposure. Overexpression of the MAP3K Tak1 sensitizes animals to oxidative stress and drives JNK and IMD signaling, both of which require Vha44. Together, these findings identify endolysosomal acidification as an amplifier of pro-apoptotic JNK and IMD/NF-{kappa}B signaling, such that attenuating V-ATPase levels in mature enterocytes protects the intestinal epithelium from excessive oxidant burden.

cell biology↗

bioRxiv2026

Projectomic Organization of the Serotonin System of the Mouse Brain

The serotonin system innervates nearly the entire brain to support diverse functions, and its dysfunction is implicated in multiple psychiatric disorders. Although recent studies suggested that this anatomically diffuse system supports differentiated rather than uniform modulation, its overall organization is unclear. Here, using systematic whole-brain axon tracing and integrated analyses, we show that the serotonin projectome is organized by functional relatedness rather than physical proximity of its targets. Dorsal and median raphe serotonin neurons partition into five projectomic groups, each preferentially innervating the hippocampus, basal ganglia, cortex, medial interbrain, or brainstem/lateral thalamus, with within-group structure ranging from discrete subgroups to continuous variation. Spatial transcriptomic analyses reveal that each projectomic group corresponds to a distinct combination of transcriptomic clusters, with transcriptome and somatic position jointly predicting projectomic identity. Projection-specific serotonin depletion produces dissociable behavioral phenotypes. Together, projectomic identity emerges as a central axis linking axon collateralization, molecular diversity, and behavioral function.

neuroscience↗

bioRxiv2026

Physics-aware measurement-supervised deep learning enables point spread function inversion in soft X-ray tomography

Soft X-ray tomography (SXT) is an emerging modality for whole-cell 3D imaging in near-native states. However, the effective spatial resolution is limited by optical artifacts characterized by the point spread function (PSF). Standard reconstruction methods force a compromise between structural sharpness and noise, failing to fully resolve these depth-dependent artifacts. By embedding experimentally measured, depth-variant PSFs into a differentiable forward model, we demonstrate a physics-aware computational optimization that bypasses these limitations to recover high-frequency cellular ultrastructure. The structural fidelity was validated using split-tilt Fourier ring correlation (FRC), alongside an experimental bead phantom tomogram, providing supporting evidence that the recovered high-frequency features reflect genuine specimen structure rather than fabricated artifacts. Our method effectively increases FRC spatial resolution and recovers cellular ultrastructure. Furthermore, under sparse-angular subsampling, the framework maintained spatial resolution using half the projection angles, a computational proxy pointing toward the potential for reduced radiation exposure in future acquisitions. This hardware-free, computational approach offers a route toward mitigating the optical and dosimetric constraints that currently limit nanoscale soft X-ray tomography.

Cell Biology↗

bioRxiv2026

Development and Analytical Validation of a Smartphone-Based Quantitative Lateral Flow Immunoassay for Serum Cystatin-C

Cystatin-C is an important renal function biomarker, and conventional quantification requires centralized laboratory analyzers, which limits timely testing in primary care and resource-limited settings. To address this need, we developed and validated a simple, rapid, and quantitative smartphone-based (SP) lateral flow immunoassay (LFIA) for measuring serum Cystatin-C. The SP-LFIA platform consists of a colorimetric LFIA strip and a custom SP reader with uniform LED illumination and macro lens for image capture. Quantitative image analysis of the colorimetric signal is performed by a dedicated application using a pre-defined third order polynomial calibration model. Following systematic optimization, the assay demonstrated a wide quantitative range of 0.32-8.00 mg/L, with a limit of detection of 0.15 mg/L. Analytical validation conducted according to CLSI guidelines showed excellent precision, with intra- and inter-assay coefficients of variation below 10%, and no significant interference from bilirubin, triglycerides, hemoglobin, or rheumatoid factor. Accelerated stability testing confirmed robust strip performance after storage at 50 {degrees}C for 28 days. Method comparison using 100 clinical serum samples showed high agreement with a commercial PETIA reference method (R{superscript 2} = 0.993) and minimal bias. These results indicate that the developed smartphone-based LFIA provides a reliable, cost-effective, and practical tool for point-of-care Cystatin-C monitoring.

biochemistry↗

bioRxiv2026

Chemoinformatics-guided discovery of food-grade anionic stabilizers for phycocyanin under acidic conditions

Phycocyanin (PC) is the principal natural blue pigment used in functional beverages, but it rapidly loses color and aggregates under acidic conditions (pH {approx} 3). Experimental screening of stabilizers is costly and combinatorially intractable. Here we develop a chemoinformatics framework -- descriptor-based QSPR, a chemistry-prior heuristic, and virtual screening -- that learns from three rounds of commissioned screening (48 compounds, 6% hit rate) to predict stabilizer efficacy directly from molecular structure. In this genuinely small-data regime (3 positives), a LightGBM classifier built from 10 RDKit descriptors and 11 domain-expert charge/polymer features attained a leave-one-out AUC of 0.73, only marginally above a single-feature charge-density baseline (AUC 0.67); LOO sensitivity was 1/3 at threshold 0.5. A complementary chemistry-prior heuristic encoding anion-type priors substantially outperformed both, reaching AUC 0.95, indicating that explicit chemical knowledge captures information that descriptor-based ML cannot readily recover at this dataset size. SHAP analysis of the QSPR identified effective negative-charge density per unit, log molecular weight, polyphosphate identity, and functional-group density as the dominant features (jointly {approx}97% of mean |SHAP|), recovering the electrostatic-complexation mechanism without it being supplied as a prior. Virtual screening of 30 generally recognized as safe (GRAS) food additives nominated the pyrophosphate family -- led by sodium pyrophosphate decahydrate and sodium hexametaphosphate (SHMP), both at P {approx} 0.99 -- and the heuristic additionally flagged sodium phytate (IP{square}), which the descriptor model under-ranked at P = 0.045. Experimental validation at pH 3 and 46 {degrees}C for 7 days confirmed SHMP 2:1 (78.1 {+/-} 11.3% color retention), TSPP 2:1 (54.1 {+/-} 10.6%) and IP{square} 1:1 (52.7 {+/-} 9.0%), while a ternary IP{square} + STPP combination reached 83.8 {+/-} 11.9%, surpassing all single-component formulations. {zeta}-Potential measurements indicated a predominantly electrostatic origin for the protection (Pearson r = -0.82 between {zeta} and CR{square} {square} {square}; n = 24; p = 1 x 10{square} {square}). The framework, dataset and code are released to accelerate stabilizer discovery for other acid-sensitive food colorants and to provide a candid small-data benchmark.

biochemistry↗

bioRxiv2026

Pathogen-specific host responses define distinct pneumonia endotypes in the human lung

Pneumonia is the leading cause of death from infectious disease worldwide. The diagnosis and treatment of patients with pneumonia lag behind other major conditions, relying on syndromic definitions that lack molecular resolution and ignore underlying endotypes. We sought to test the hypothesis that dynamic pathogen-specific host responses in the alveolar space represent distinct pneumonia endotypes linked to different clinical features and outcomes. We prospectively enrolled a cohort of 690 patients (including immunocompromised patients) with known or suspected pneumonia receiving mechanical ventilation in whom the etiology of pneumonia was determined by gold-standard analysis of distal lung fluid obtained by bronchoalveolar lavage (BAL) combined with clinical adjudication. From these patients, we analyzed 792 BAL fluid samples, including 310 serial samples, using flow cytometry (482 patients) and single-cell RNA-sequencing (170 patients; 263 samples, complemented by 9 healthy controls and 25 post-COVID-19 patients, yielding [~]2.4 million single cells across 28 cell types), and extracted daily clinical data from the electronic health record (>15,000 patient-days). We used machine learning models to identify pathogen-specific host responses in the transcriptome of alveolar immune cells that were associated with changes in alveolar cell abundance and clinical features. Our results suggest that therapeutic strategies for pneumonia should be individualized to specific host-pathogen interactions.

immunology↗

bioRxiv2026

Tripeptidyl peptidase II is essential for maintaining cerebrovascular homeostasis of female mice and represents a novel therapeutic target for vascular dementia

Although cerebrovascular impairment is a known common driver of both Vascular dementia (VaD) and Alzheimers disease (AD), the underlying mechanisms remain poorly defined. Substantial evidence has demonstrated that brain-derived estrogen is essential for cerebrovascular health and neuroprotection. Consequently, therapeutic strategies that can replicate the beneficial effects of estrogen replacement therapy (ERT) in the brain while avoiding its peripheral risks are being actively pursued. Here, we demonstrate that tripeptidyl peptidase II (TPP2) is essential for cerebrovascular homeostasis in adult female mice by orchestrating intracellular Ca{superscript 2} distribution and local estrogen biosynthesis. Specifically, TPP2 deficiency triggers a vicious cycle of Ca{superscript 2} imbalance and estrogen deficiency, thereby disrupting the anticipatory unfolded protein response (UPR). This disruption consequently drives aberrant pexophagic flux and ultimately depletes ether-linked phosphatidylcholine (PC-O)--the essential building block of endothelial cell (EC) membranes. In addition, we identified PC-O as a key facilitator of choline uptake through FLVCR2 and therefore its deficiency causes subsequent choline depletion as well as significantly decreased acetylcholine (ACh). Consistent with the fact that choline is a building block of PC and that ACh is a primary driver of vascular dilation, TPP2 depletion leads to significant cerebrovascular degeneration characterized by narrowed lumens, decreased EC number, and abnormal aggregation of ECs within the blood vessel. Additionally, AAV-mediated specific expression of Far1 in ECs to promote ether-PC biosynthesis significantly increases cerebrovascular volume and diameter in hippocampi of adult female TPP2 knockout (T2KO) mice. Importantly, ectopic expression of Far1 in ECs not only significantly ameliorates memory impairment in global and conditional TPP2-depleted female mice, but also significantly improves memory performance of naturally aged female mice. In total, our findings establish TPP2 as a key determinant of cerebrovascular homeostasis in adult female mice, positioning it as a novel therapeutic candidate for the treatment of VaD and AD.

neuroscience↗

bioRxiv2026

Reciprocal repulsions enforce heterotypic dendrite segregation in an olfactory circuit

How dendrites of different neurons segregate into discrete spatial domains during neural circuit assembly is poorly understood. Here, using the Drosophila olfactory system, we found that heterophilic interactions between two cell-surface proteins Teneurin-m (Ten-m) and Capricious (Caps) drive dendrite segregation. Ten-m and Caps are expressed in largely inverse patterns across projection neuron (PN) types when PNs are establishing their dendritic territories. Loss of Ten-m in Ten-m+ PNs causes their dendrites to invade Caps+ territories, whereas loss of Caps in Caps+ PNs causes dendrite invasion into Ten-m+ territories. Structure-guided mutations that abolish Ten- m-Caps binding disrupt dendrite segregation, whereas the same mutation on Ten-m preserves its homophilic attraction in a synaptic partner matching assay. These results support a model in which mutual repulsions between two inversely expressed cell-surface proteins drive dendrite segregation into discrete glomerular territories.

neuroscience↗

bioRxiv2026

FOXA1 preserves cell polarity and restrains lysosome biogenesis in non-small cell lung adenocarcinoma

BackgroundThis study investigates the role of the pioneer transcription factor FOXA1 as a master gene in sustaining epithelial cell polarization in early-stage lung adenocarcinoma. The partial loss of FOXA1 is explored to determine if it will affect plasticity and progression of lung adenocarcinoma. The study also addresses the transcriptional circuitry that links polarity defects to lysosome homeostasis. MethodsA multiomics approach was used to define the status of the chromatin in epithelial and mesenchymal states of A549 adenocarcinoma cells obtained with a newly synthetized TGF-{beta} receptor inhibitor or TGF-{beta} respectively. The study leveraged ATAC-seq, RNA sequencing, Cut&Tag sequencing of FOXA1 and histone marks profiling. The functional impact of FOXA1 was examined by partial silencing in vitro and by heterozygous FOXA1 deletion in a KrasG12D mouse model. Three-dimensional organoid culture, high-resolution electron microscopy, spatial transcriptomics and multiplex immunohistochemistry assessed carcinoma cell polarity, proliferation, the tumor microenvironment and organelle content. Group differences were evaluated with two-tailed t tests or one-way analysis of variance. ResultsFOXA1 binding and expression were highest in cells harboring an epithelial phenotype. In mouse KrasG12D LUAD tumors FOXA1 marked polarized, CDH1-positive cells; heterozygous loss diminished CDH1, disrupted apical-basal architecture, lowered organoid-forming efficiency and remodeled the immune microenvironment. Spatial transcriptomics and ultrastructural analyses showed that FOXA1-deficient carcinoma cells accumulated lysosomes, down-regulated vesicle fusion genes of the SNARE family and activated the lysosomal CLEAR gene network. FOXA1 occupied enhancers of lysosome-associated genes and competed with the transcription factor TFE3, thereby suppressing transcription of cathepsin B and cathepsin C and restricting lysosome biogenesis. ConclusionsFOXA1 is a central regulator that preserves epithelial cell polarity and limits lysosome formation in lung adenocarcinoma. Targeting the FOXA1-TFE3-lysosome axis may affect tumor plasticity and provide new therapeutic opportunities.

cancer biology↗

bioRxiv2026

Topographic CA1 input shapes subicular spatial coding

Precise anatomical topographic mapping in the brain supports high-fidelity information transfer for computations such as sensory alignment and motor coordination1,2. As a brain structure central to learning, memory, and navigation, the hippocampal formation is likewise organized with striking topographic precision3-8. Within this circuit, the subiculum plays a critical role in forming the cognitive map by integrating spatial and non-spatial information from both CA1 and the entorhinal cortex (EC)9-17. Inputs from these regions follow a continuous topography such that distal subiculum receives projections from proximal CA1 (pCA1) and medial EC (MEC), whereas proximal subiculum receives projections from distal CA1 (dCA1) and lateral EC (LEC)3-5,7,8. This anatomical architecture suggests that subicular computations may depend critically on the fidelity of these circuit topographies. However, because CA1 and EC provide partially overlapping spatial information, it remains unclear to what extent CA1-to-subiculum topography, independent of ECs contribution, shapes the physiological properties of subicular neurons. Here, by selectively disrupting CA1-to-subiculum topographic projections in latrophilin-2 conditional knockout mice, we show that precise topography shapes the anatomical distribution of subicular spatial coding while preserving single-cell tuning. Disrupted CA1 input topography also selectively impairs subicular boundary vector coding and long-term network stability. Thus, CA1 input topography is critical for organizing subicular spatial coding and network dynamics. HighlightsO_LICA1[->]subiculum topography organizes spatial coding distribution in the subiculum. C_LIO_LIDisrupted CA1 input topography spares individual subicular place cell tuning. C_LIO_LIDisrupted CA1 input topography selectively impairs subicular boundary vector coding. C_LIO_LILong-term subicular ensemble reactivation requires precise CA1 input topography. C_LI

neuroscience↗

bioRxiv2026

Preclinical efficacy of a systemically-administered, second-generation STING agonist that promotes antitumour immunity in combination with radiotherapy

As potent triggers of innate immunity, STING agonists hold promise as active immunotherapeutic agents for cancer treatment. Second-generation STING agonists, suitable for systemic delivery, are being investigated in preclinical research and have entered clinical trials. Here, the novel synthetic STING agonist, BI-1703880 (STINGa), which was designed for intravenous delivery, was investigated for anti-tumour and immunological effects. We show that STINGa activates the STING pathway and results in a transient and dose-dependent upregulation and secretion of interferons and proinflammatory cytokines in vitro and in vivo. We show that intravenous administration of repeated dosing with low-dose STINGa is well tolerated. We report that radiotherapy (RT) and STING agonism synergizes to generate innate immune cell and CD8+ T cell responses that control tumour growth. Anti-tumour activity induced by combined RT / STINGa was reduced in mice lacking a functional immune system. RT / STINGa combination treatment also initiated development of protective immune memory. RT / STINGa upregulated PD-L1, PD-1 and CTLA-4 in the tumour microenvironment. Our findings show that combining RT / STINGa with immune checkpoint inhibitors further increases therapeutic benefit. Our data confirm STING as a therapeutic target in cancer and support the clinical development of BI-1703880 STING agonist, thereby suggesting radiotherapy as a potential combination for enhancing anti-tumour efficacy.

cancer biology↗

bioRxiv2026

A tunnel microtract organ for T cell progenitor homing is formed by neural crest morphogenesis via Sox10-Cdc42 axis

Thymic colonization by T cell progenitors (TCPs) is essential for adaptive immunity, yet the guiding tissues remain elusive. Here, we unveiled a tunnel microtract (TMT) as an organ indispensable in TCPs homing in both zebrafish and mouse. Disruption of TMT leads to compromised T cell development. Specifically, the zebrafish TMT were positioned bilaterally beneath the fifth branchial levator muscle, connecting the thymus and kidney. They are semi-coiled, non-vascular, non-lymphatic tubes of epithelial signatures. Impressively, Sox10 activates Cdc42 to promote F-actin remodeling in neural crest cells (NCCs), leading to precise elongation and tight packaging of a low-permeability TMT. Remarkably, a homologous CD34 TMT was observed in mice, which bilaterally enveloped the embryonic thymus and extended into the thyroid cartilage. Sox10-Cdc42 signalling functioned recapitulatively in NCC morphogenesis during its construction. These findings establish TMT as an unappreciated NCC-derived organ in TCPs homing, with implications in T cell development and immune disorders.

developmental biology↗

bioRxiv2026

Noncanonical loops regulated by EMF1 and cohesin-associated factors shape the distinct 3D genome architecture in plants

In mammals, cohesin-mediated chromatin loop extrusion is blocked at boundaries, where CTCF, WAPL, and PDS5s co-bind and regulate cohesin, establishing an impermeable barrier with no exceptions. In contrast, our study revealed that in Arabidopsis, some noncanonical contacts have emerged between the boundary flanking regions, which suggest that cohesin is not absolutely blocked but enables sliding beyond boundaries. We identified these interactions as noncanonical loops and clarified their regulatory proteins. Firstly, the plant-specific boundary binding protein EMF1 is essential for noncanonical loops formation. Surprisingly, although both Arabidopsis WAPLs and PDS5s inhibit loop extension as mammals, PDS5s do not co-bind and cooperate with WAPLs at boundaries. Instead, PDS5s bind to boundary-flanking regions enriched with H3K4me1 through their Tudor domains, restricting further extension of noncanonical loops. Consequently, pds5a/b/c mutants lose noncanonical loops, resulting in emergence of larger canonical loops but anchored on boundaries and co-upregulation of anchored gene pairs. Collectively, our findings reveal that the plant-specific noncanonical loop, regulated by plant-specific EMF1 and PDS5 proteins, is the fundamental structure of the distinctive chromatin organization of plants, which provides novel mechanistic insights into eukaryotic chromatin architecture regulation.

plant biology↗

bioRxiv2026

AbNovoBench: a resource and benchmarking platform for monoclonal antibody de novo sequencing

Monoclonal antibodies (mAbs) are critical in disease diagnostics and therapeutics, yet the performance of mass spectrometry (MS)-based de novo sequencing remains incompletely characterized due to limited antibody-specific datasets and the absence of a standardized benchmark framework. Here we present AbNovoBench, a comprehensive framework for evaluating data analysis strategies for mAb de novo sequencing. It features the largest high-quality dataset to date, generated in-house, comprising 1,638,248 peptide-spectrum matches from 131 mAbs across six species and 11 proteases, supplemented by eight mAbs with known full-length sequence for end-to-end reconstruction assessment. Employing a unified training dataset, we systematically benchmarked 13 deep learning-based de novo peptide sequencing algorithms and three assembly strategies across peptide sequencing metrics (accuracy, robustness, efficiency, error types) and assembly metrics (coverage depth, assembly score). AbNovoBench (https://abnovobench.com) provides an online platform enriched with curated antibody MS resources and pre-trained models, enabling customizable antibody sequencing workflows, accelerating antibody-specific algorithms development, and improving reproducibility in proteomics.

systems biology↗