Multiple Sclerosis

Longitudinal characterization of multiple sclerosis (MS) lesions remains constrained by the lack of frameworks capable of establishing consistent instance-level correspondences across time. Conventional segmentation approaches produce semantic lesion masks at each visit and therefore fail to capture the complex instance temporal patterns associated with lesion appearance, disappearance, splitting, or merging. This study presents a comparative evaluation of five strategies for automated tracking of spinal cord MS lesions in longitudinal MRI data from a multi-site cohort. The investigated strategies rely either on deformable registration or on a spinal anatomical reference system, and encompass overlap-based matching, coordinate-based Hungarian algorithm, gradient-boosted classification, and Siamese model classification. Tracking accuracy is quantified using instance-level true positives, false positives, and false negatives, allowing to assess the presence of one-to-many and many-to-one associations. Results show best performance for the registration-based overlap method. This study provides the first systematic analysis of lesion-instance correspondence in the spinal cord and outlines the strengths and limitations of registration-based and registration-free paradigms for longitudinal MS assessment. The code is available at http://github.com/ivadomed/longitudinal-sc-ms-lesion-tracking .

White matter hyperintensities (WMH), bright regions on Fluid-attenuated Inversion Recovery (FLAIR) scans are associated with cerebrovascular pathology and neurodegeneration. FLAIR is usually acquired with thick slices in clinical settings, giving it poor through-plane resolution. Super-resolution (SR) is a widely used method for recovering an isotropic volume from an anisotropic scan. Yet whether applying it prior to WMH segmentation preserves lesion content remains unknown: a model may erase small real lesions or hallucinate absent ones. We used 1-mm isotropic high-resolution (HR) FLAIR scans from 29 individuals in the ADNI cohort, each manually segmented for WMH by an expert. Then, we degraded each to simulated 3 and 5 mm through-plane acquisitions. Multi-contrast implicit neural representation (INR), a single-contrast self-supervised model (ECLARE), and cubic interpolation were used to upsample them onto the HR grid. WMH segmentation from a simulated thick slice and the original HR FLAIR set the floor and ceiling, respectively, for the per-lesion analysis. Of four WMH segmentation methods (WMH-SynthSeg, segcsvd, MARS-WMH, TrUE-Net), we ran the analysis under the most sensitive one to small lesions on HR (MARS-WMH) with the evaluation metrics of detection sensitivity, erasure rate (HR-detected lesions lost after reconstruction), and hallucination rate (predicted components absent from both the manual and HR segmentation). The dominant effect of SR was erasure of small real lesions, not hallucination, and it increased with slice thickness, though every reconstruction still improved lesion detection over the raw thick slice. ECLARE recovered small lesion signal best at both thicknesses, while the INR was no better than cubic interpolation.

Multiple sclerosis (MS) is diagnosed through clinical assessment, magnetic resonance imaging, laboratory evidence when appropriate, and exclusion of better explanations. Blood RNA expression data may contain disease associated immune signal, but a blood RNA classifier cannot be treated as a replacement for clinical diagnosis. This paper presents MS-MLB (Multiple Sclerosis Machine Learning Benchmark), a reproducible open benchmark for machine learning based MS research classification from whole blood RNA expression data. MS-MLB uses the public GSE17048 cohort, converts it into an MS versus healthy control task, and evaluates multiple algorithms under a shared, leakage controlled pipeline that a researcher can rerun without reconfiguring the evaluation. The evaluation includes nested cross-validation, an untouched stratified holdout set, bootstrap confidence intervals, ROC and precision recall analysis, calibration measurement, and an exploratory MS Research Score. In the final benchmark summary, Gradient Boosting ranked first by MS Research Score on the holdout set, with an MS Research Score of 93.83, AUC-ROC of 0.989, sensitivity of 0.950, specificity of 0.778, $F_{1}$ score of 0.927, and Brier score of 0.050. Prior studies have applied machine learning to MS blood transcriptomic data, including PBMC stage classification and whole blood diagnostic signature modeling. The contribution here is different and narrower. To our knowledge, MS-MLB is the first open benchmark focused on MS versus healthy control classification from GSE17048 whole blood RNA expression data with a documented external model submission pathway built into the framework. The score is intended for research comparison only and has not been clinically validated. The benchmark is accessible here: https://github.com/duckyquang/MS-MLB.

Molecular structure elucidation from tandem mass spectra (MS/MS) is a central inverse problem in analytical chemistry. Most existing approaches to MS/MS identification remain tied to reference libraries or predefined candidate sets, whereas de novo methods aim to generate structures directly from spectra. A common de novo route predicts a molecular fingerprint from the spectrum and then decodes structures from it, enabling decoder pretraining on large molecule-only corpora. However, this paradigm creates a training-inference mismatch: the decoder is trained on oracle fingerprints computed from molecules, but at inference it is queried with a noisy spectrum-induced fingerprint posterior that is typically collapsed to a single thresholded fingerprint. We introduce MS-GPT, which recasts fingerprint-mediated de novo elucidation as spectrum-induced posterior querying of a conditional molecule-language model. MS-GPT conditions a molecule-language model on fingerprints and formulas, then converts the spectrum-induced posterior into a band of fingerprint queries near the oracle-fingerprint manifold through active-bit density calibration. Candidates sampled across this band are pooled and ranked by generation-frequency consensus. A lightweight LoRA adapter further mitigates domain-specific posterior bias while preserving the pretrained molecular prior. On NPLIB1 and MassSpecGym, MS-GPT sets a new state of the art, reaching Top-1/Top-10 exact-match accuracy of 29.8%/41.1% and 23.9%/28.7%, respectively. Candidate-pool scaling shows that efficient autoregressive molecular generation continues to improve recall with a little additional inference cost. The source code and model checkpoints are available at https://github.com/VIKI623/MS-GPT.

Existing BraTS-GLI datasets provide a widely used benchmark for adult glioma MRI segmentation, but their task definition focuses on tumor subregions and does not systematically represent coexisting white matter hyperintensities (WMH). In joint segmentation settings, such unlabeled abnormalities introduce task-specific label noise by treating pathological regions as normal tissue. To address this limitation, we introduce BraTS-GLI Anatomy-Lesion, a controlled-access, labels-only derived resource built from the BraTS 2023-GLI training cohort. The resource provides 1,251 unified eight-class anatomy-lesion label sets aligned with the original four-modal MRI cases, including image-repair labels for 116 cases requiring repaired imaging inputs. The cohort is organized into a 394-case purified subset and an 857-case extended subset, with case-level metadata covering label source, image-repair requirements, quality-control status, access conditions, checksums, and release boundaries. Compared with the original BraTS-GLI annotations, the resource substantially expands foreground supervision by incorporating healthy brain tissues and previously unlabeled coexisting abnormalities within a unified label space. A validation study using MedNeXt and T1/FLAIR inputs suggests that WMH-aware supervision preserves healthy-tissue segmentation performance across both in-domain GLI and external WMH datasets, while improving sensitivity to coexisting lesions relative to noisy-control training. The resource is intended for scientific research and supports joint anatomy-lesion supervision, label-noise analysis, and reproducible evaluation. Data are available at https://www.synapse.org/Synapse:syn75210889/wiki/, and code is available at https://github.com/xyx200/brats-gli-anatomy-lesion-code. The data resource DOI is https://doi.org/10.7303/SYN75210889.

Safe navigation within the spinal subarachnoid space is constrained by its narrow, compliant, and delicate anatomy. Conventional catheters and continuum robots rely on proximal pushing, generating friction and shear along the tissue device interface that limit distal controllability and increase the risk of neural injury. Here, we present a 2 mm diameter eversion-growing robotic platform that enables friction minimised extension and steering within the human spinal subarachnoid space, validated through computational modelling, phantom experiments, and intact human cadaver studies. The robot integrates a miniature endoscope for real time intrathecal visualisation and advances by pressure driven tip eversion, localising motion to the distal tip while minimising translational sliding of the deployed body. Phantom experiments demonstrated reductions of 65.2% in mean interaction force and 48.0% in peak interaction force compared with matched push-based insertion. Physics based modelling showed that eversion based growth redistributed tissue loading, reducing local stress concentrations and interfacial shear relative to conventional insertion. In an intact human cadaver, the system achieved 150 mm of controlled intrathecal extension with concurrent fluoroscopic and endoscopic visualisation, providing access across multiple vertebral levels from a standard lumbar entry point. Postprocedural laminectomy and durotomy revealed no observable macroscopic disruption of the dura mater or surrounding neural structures. These results provide the first mechanically characterised and multimodally validated demonstration of eversion-based robotic navigation in intact human spinal anatomy, establishing a quantitative and procedural foundation for future intrathecal interventions. Further validation in larger anatomical cohorts and under physiological conditions will be required before clinical translation.

White Matter Hyperintensities (WMHs) are commonly observed in brain Magnetic Resonance Imaging (MRI) scans. They are associated with various neurological conditions, including vascular and inflammatory demyelinating diseases. Despite differing in etiology, WMHs from these conditions often appear similar on Fluid Attenuated Inversion Recovery (FLAIR) images. This similarity makes differential diagnosis challenging. In this work, we highlight the potential of combining attention-based segmentation with feature-driven classification. This approach supports more accurate and efficient classification between vascular and demyelinating white matter pathologies. For segmentation, we evaluate the effectiveness of attention mechanisms, specifically the Bottleneck Attention Module (BAM) and the Convolutional Block Attention Module (CBAM). We also test different architectures, particularly Attention U-Net. In addition, we explore advanced training strategies, such as patch-based learning and a 2.5D approach, to enhance lesion detection. After segmentation, we extract morphological features from the lesion masks. We then use them to classify WMHs based on their underlying cause. Our experiments utilize five publicly available datasets with diverse imaging protocols to promote model generalizability, despite limited sample sizes. The results suggest that attention-based segmentation and feature-driven classification offer a promising direction for discriminating vascular and demyelinating white matter lesions. Further validation in larger clinical cohorts is still needed.

Background: Digital health technologies allow for frequent, remote gait monitoring in people with multiple sclerosis (MS). However, to differentiate daily variability from actual disease progression in longitudinal data, established minimal clinically important differences (MCID) are required. Currently, there is limited literature defining these thresholds for digital gait metrics. Objective: To establish MCIDs for digital gait measures reflecting progression in MS. Methods: Digital gait measures were captured via daily, remote, smartphone-based Two-Minute Walk Tests in CONSONANCE (NCT03523858), a phase 3b study of ocrelizumab in progressive MS. Using an anchor-based approach, median changes from baseline at Week 96 on digital gait measures were computed for patients showing clinically meaningful worsening on either Timed 25-Foot Walk, Ambulation Score, Expanded Disability Status Scale, or 12-item Multiple Sclerosis Walking Scale. These changes were subsequently triangulated to derive the MCID estimates. Results: 243 patients with progressive MS (female: n=125 (51%); mean [SD] age: 49.3 [9.3]; mean [SD] EDSS: 4.8 [1.4]) had digital gait data available at baseline and Week 96. Median changes were generally consistent across anchors. Triangulated MCIDs are: Step Velocity = -0.16 m/s, Step Velocity Scaled to Walking Time = -0.18 m/s, Step Duration = 0.06 s, Step Length = -0.07 m, Total Number of Steps = -28, and Total Distance Walked = -24 m. Conclusion: These MCIDs provide a framework for interpreting meaningful gait changes and integrating digital measures into MS outcome evaluation. Beyond facilitating novel clinical trial endpoints to evaluate treatment efficacy, they enable objective, real-world monitoring to advance personalized patient care.

Reliable benchmarking is critical for developing machine learning models for tandem mass spectrometry (MS/MS) based molecule discovery. Subtle issues in experimental design and model evaluation procedures can degrade the trustworthiness of such benchmarks and lead to erroneous conclusions. We conduct a thorough review of model evaluation issues in the recent MS/MS machine learning literature, using the standard MassSpecGym benchmark suite as a case study to illustrate the impact of these issues. We find evaluation issues in at least 17 of 26 papers reporting MassSpecGym benchmark results in the first year of its adoption. We isolate three classes of failures: (i) data leakage, (ii) shortcut learning, and (iii) implementation bugs and metric divergence. Through extensive experimentation and code replication, we quantify the impact of these issues and show how they corrupt the evaluation standards MassSpecGym was designed to enforce. We distill our findings into recommendations generalizable to MS/MS challenges, benchmarks, and custom evaluation setups. We also release MassSpecGym v1.5, an implementation of our recommendations in the MassSpecGym benchmarking suite which addresses the failure modes identified in this audit. MassSpecGym v1.5 is publicly available at https://github.com/pluskal-lab/MassSpecGym.

Paramagnetic rim lesions (Rim$^+$) identified on susceptibility-sensitive MRI have recently emerged as a specific biomarker of chronic active inflammation in Multiple Sclerosis (MS) and are associated with long-term disability progression. However, susceptibility imaging and expert interpretation remain limited to specialized centers, visual assessment is time-consuming and variable, and the low prevalence of Rim$^+$ lesions poses severe class imbalance challenges for automated analysis. We propose a 3D multimodal deep learning framework for lesion-level Rim$^+$/Rim$^-$ classification from Quantitative Susceptibility Mapping (QSM) and FLAIR MRI. The architecture explicitly models modality asymmetry by treating QSM as the primary susceptibility-driven signal and conditioning it with FLAIR-derived structural context. To improve robustness under limited data, we employ self-supervised multimodal pretraining followed by supervised fine-tuning with contrastive regularization. The method was evaluated on a clinically acquired cohort of 88 people with MS with expert lesion annotations as reference standard. Results highlight improved performance compared to prior architectures, supporting the effectiveness of asymmetric multimodal modeling for automated chronic active lesion identification.

3D FLAIR MRI is widely recommended as one of the standard MRI sequences for brain imaging in multiple sclerosis (MS), but publicly available MS datasets remain relatively small and vary across scanners, acquisition protocols, and lesion patterns. This scarcity and variability hinder the development of robust neuroimaging machine learning models and are particularly challenging for generative models that aim to synthesize images while preserving small, sparse lesions. We propose Lesion-DDPM, a 3D conditional diffusion framework for lesion-aware FLAIR synthesis that incorporates multi-level anatomical mask injection together with a lesion-weighted reconstruction loss to emphasize lesion voxels while maintaining global brain structure. Using a curated subset of the MSLesSeg dataset, we compare Lesion-DDPM with representative state-of-the-art GAN- and diffusion-based models, assessing both image-generation metrics and downstream 3D U-Net segmentation. In our experiments, Lesion-DDPM achieved the lowest lesion-region reconstruction error among all methods. In a downstream 3D U-Net lesion segmentation task, a model trained only on Lesion-DDPM-generated scans and evaluated on real MRIs reached a Dice score of 0.616 compared with 0.569 for the best competing synthetic dataset. When Lesion-DDPM images were added to the real training set, the Dice score further increased to 0.685.

Leptomeningeal collaterals (LMCs) are an important prognostic factor in acute ischemic stroke. Existing automated methods rely on CT angiography (CTA), but individual LMCs are often too small to be resolved on CTA, limiting these methods to coarse collateral scoring. Digital subtraction angiography (DSA) visualizes individual collaterals at superior resolution, yet current assessment remains subjective, relying on manual grading scales that suffer from poor inter-rater agreement. We present a framework that formulates collateral detection as the classification of individual vessel segments on a graph derived from DSA. A hybrid graph-pixel architecture combines a topology-aware graph branch with a dense pixel branch, fused in a shared node-probability space. In a five-fold cross-validation setting, the fused model achieves a PR-AUC of 0.434, outperforming the graph-only (0.403) and pixel-only (0.362) baselines. To our knowledge, this is the first method to enable the individualization of LMCs in DSA, allowing for precise per-vessel quantitative assessment. This integration shifts DSA assessment toward objective evaluation, supporting future biomarker and pattern discovery for individual LMCs.

Background: The lateral ventricle choroid plexus (LVCP) is gaining recognition as a key imaging biomarker for multiple sclerosis (MS) related to physical disability and neuroinflammation. Yet, manual segmentation of the LVCP is highly tedious, restricting its use in broad clinical trials and longitudinal assessments. This research aims to develop a SwinUNETR-driven pipeline that leverages targeted intra- and peri-ventricular small patch sampling to automatically segment the LVCP in MS from both standalone and multi-modal MRI inputs. Methods: We retrospectively assessed 3T MRI scans across three sets of data stemming from two separate MS-dominant cohorts (Dataset 1: n=177; Dataset 2: n=177; expanded test set: n=388). Our method employed a SwinUNETR architecture trained on 32x32x32 voxel patches, benchmarking it against the 3D UXNET model. The primary metric for evaluation was the Dice Similarity Coefficient (DSC), supplemented by computational demand (GFLOPs) and the 95th percentile Hausdorff Distance (HD95). Results: On the extended test set, the SwinUNETR model secured a mean DSC of 0.868 (95% CI: 0.863-0.872) with MPRAGE and FLAIR combined, showing a statistically significant gain over UXNET (DSC: 0.858 [95% CI: 0.853-0.862], p<0.0001). When restricted to standalone FLAIR inputs, the transformer-based approach sustained a high DSC of 0.863, while the spatial localization of UXNET worsened considerably (HD95: 1.86 vs. 3.00 mm). Importantly, the proposed framework lowered computational load by 99% (91.8 vs. 22,080 GFLOPs). By integrating localized patch sampling with a SwinUNETR architecture, this methodology offers an accurate, robust, and statistically superior alternative to current leading models for LVCP segmentation. Its vast reduction in computational cost makes it ideal for widespread implementation in clinical and research environments.

Untargeted liquid chromatography-high-resolution mass spectrometry (LC-HRMS) detects thousands of molecular features per sample, yet only 2-20% receive confident structural annotations. A root cause of this "dark metabolome" is that tandem MS/MS acquisition is reactive: instruments select precursors only after ions appear, blind to what elutes next. We reframe chromatographic elution as an autoregressive sequence prediction task. Because reversed-phase elution order is governed by hydrophobicity, successive features form a physically constrained sequence, like tokens in language. We discretize the mass-to-charge (m/z) axis into 110 bins and train long short-term memory (LSTM) and Transformer models to predict the next eluting m/z bin from five annotation-free per-token features: m/z bin, mass defect, retention-time gap, polarity, and intensity rank. Trained on 15,242 features from four clinical lipidomics cohorts (342 plasma samples; SCIEX TripleTOF 6600+, Waters CSH C18), the LSTM reaches 98.4% top-1 accuracy (99.99% top-5; mean absolute error 3.6 Da) and the Transformer 98.0%. Ablation shows autoregressive context accounts for 55.5 percentage points while no single feature contributes more than 0.2 pp: the sequential pattern, not molecular properties, drives prediction. Models transfer across instruments sharing the method (r=0.999 on an independent Agilent 6530 dataset) but fail under a different column chemistry (5.1% top-1) or polarity mode (2.6%), confirming method- and mode-specificity. Fine-tuning on as few as two to five quality-control injections recovers held-out accuracy from 2.6% to nearly 50%, so cross-condition deployment needs minimal calibration. These results establish that elution sequences are highly predictable and lay the groundwork for predictive MS/MS acquisition to improve annotation coverage in untargeted metabolomics.

Image restoration models are increasingly applied to degraded medical scans, but in safety-sensitive settings they must improve image quality without uncontrolled modification of clinically important regions. This is especially relevant for intracranial CT and CT angiography (CTA), where small vessels and aneurysm-relevant cues lie near high-contrast anatomical boundaries. We frame medical image restoration as a conservative AI problem and present a residual-bounded 2.5D restoration framework trained on synthetically degraded CT/CTA inputs. The model adds a learned residual to the original center slice through an edit-control map that limits the magnitude and spatial extent of modification. We evaluate the framework using an aneurysm-relevant image-recovery matrix, paired comparison against a Gaussian baseline, Monte Carlo stability testing, anatomical localization of meaningful edits, and external evaluation on low-dose CT. On 50 out-of-distribution CT-CTA cases, the bounded model achieved a mean target gain of 0.0635, a mean PSNR of 37.51 dB, and an iatrogenic-edit rate of 4.0%. Across 1,000 Monte Carlo runs, it remained net positive in 85.4% of runs with no stably negative cases. On external low-dose CT, the model was directionally beneficial and produced a substantially smaller modification footprint than the baseline. Meaningful edits concentrated in brain and skull regions while unrelated anatomy showed negligible change. These findings provide preliminary computational evidence that residual-bounded restoration is feasible in boundary-sensitive vascular imaging, but they do not establish clinical diagnostic performance and require expert review and prospective validation before clinical use.

Multiple Sclerosis (MS) is a chronic autoimmune disease that can significantly reduce the quality of life of a patient. Existing treatment options can only help slow down the progression of the disease. Therefore, early detection and precise monitoring of disease progression are important. Deep learning offers state-of-the-art models for detecting and segmenting MS lesions in brain MRI scans. However, most of these models are evaluated using the Dice score, without accounting for lesion-wise detection and segmentation performance or other metrics that quantify model performance in cases that are complex or confusing for human annotators, or in cases that are essential for disease detection and progression monitoring. In this paper, we highlight the need to rethink the evaluation of MS lesion segmentation models. In this context, we first present problem fingerprinting in detail to highlight what neurologists look for in brain MRI scans for MS detection and progression monitoring, and which metrics are required to properly quantify model performance in these contexts. Additionally, we present an analysis of state-of-the-art models on two open-source datasets using these metrics to highlight their usability for real-world deployment in hospitals.

Multiple technologies that measure expression levels of protein mixtures in the human body offer a potential for detection and understanding the disease. The recent increase of these technologies prompts researchers to evaluate the individual and combined utility of data generated by the technologies. In this work, we study two data sources to measure the expression of protein mixtures in the human body: whole-sample MS profiling and multiplexed protein arrays. We investigate the individual and combined utility of these technologies by learning and testing a variety of classification models on the data from a pancreatic cancer study. We show that for the combination of these two (heterogeneous) datasets, classification models that work well on one of them individually fail on the combination of the two datasets. We study and propose a class of model fusion methods that acknowledge the differences and try to reap most of the benefits from their combination.

Multiple sclerosis (MS) expresses substantial clinical and radiological heterogeneity, which poses significant challenges for automatic lesion segmentation. The current deep learning-based SOTA is highly susceptible to changes in both distribution, e.g., changes in scanner; as well as the structure of inputs, evident in the current divide between cross-sectional and longitudinal approaches. We introduce TimeLesSeg, a unified contrast-agnostic framework designed to segment MS lesions regardless of the presence of a temporal dimension in its inputs, with a single convolutional neural network. Our approach models pathological priors through lesion masks, which are processed together with the current scan. Cross-sectional processing is enabled by exposing the model to training cases where no prior information is available, which are modeled with an empty mask, allowing it to operate seamlessly in both scenarios. To overcome the scarcity and inconsistency of longitudinal datasets, we propose a novel generative pipeline in which patterns of lesion evolution are simulated by stochastically deforming each individual lesion with morphological operations, producing realistic prior timepoints. In parallel, we achieve contrast agnosticism through Gaussian mixture model-based domain randomization, enabling the network to experience a wide spectrum of intensity profiles. Results on three publicly available and two in-house datasets show that TimeLesSeg outperforms the contrast-agnostic state of the art on single-modality inputs across overlap- and distance-based metrics. In longitudinal processing, our method outperforms SAMSEG, and captures lesion load dynamics more accurately than both the former and LST-AI. All source code related to the development of TimeLesSeg is available at https://github.com/NeuroADaS-Lab/TimeLesSeg.

Federated techniques such as federated learning and federated analysis have emerged as a powerful paradigm for enabling multi-center research on sensitive clinical data while preserving patient privacy. In this study, we introduce a simulation framework that emulates a real-world federated research project focused on the analysis of multiple sclerosis (MS) patient data. The project comprises two components: an image segmentation task and a clinical data analysis task, where federated variants of survival analysis and Principal Component Analysis (PCA) are employed. To capture the complexity and heterogeneity of real clinical datasets, we construct a federation of high-fidelity synthetic cohorts designed to mirror MS-related clinical and demographic characteristics, while the imaging component leverages publicly available real-world datasets. Our simulation replicates key elements of authentic federated workflows, including distributed data governance, site-specific preprocessing, model training across isolated nodes, and the secure aggregation of analytical outputs. This framework provides a realistic testbed for developing, evaluating, and benchmarking federated learning methods in the context of MS research.

Tandem mass spectrometry provides a high-throughput framework for identifying and quantifying proteins in complex biological samples. In computational proteomics, predicting peptide MS/MS spectra is a critical task, enabling downstream applications such as large-scale peptide identification and quantification. While deep learning architectures have substantially improved prediction accuracy, three evaluation challenges obscure the true progress of the field. First, inconsistent data preprocessing and incompatible model output spaces hinder fair model comparison. Second, flawed data splitting strategies can permit hidden sequence leakage and inflate reported performance. Third, existing evaluations typically lack comprehensive cross-species benchmarking and systematic assessment of model robustness to influential experimental conditions. To address these challenges, we propose PepSpecBench, a unified benchmark for peptide MS/MS spectrum prediction. PepSpecBench standardizes data preprocessing across complementary public datasets, enforces a strict backbone-disjoint splitting strategy to eliminate sequence leakage, and evaluates diverse architectures within a shared fragment-ion representation space. It further introduces a comprehensive multi-species evaluation suite and physically grounded metadata perturbation probes to assess model robustness and instrument awareness. We uncover previously unrecognized performance discrepancies and robustness limitations across six representative models, providing actionable insights for future model design, evaluation and practical deployment.

Tandem mass spectrometry (MS/MS) is central to small molecule identification, but current deep learning systems for spectrum prediction still remain difficult to evaluate and deploy in practice. While novel architectures constantly claim state-of-the-art performance, inconsistent metadata conditioning and entangled preprocessing pipelines hinder fair architectural comparisons. Besides, existing evaluations are often restricted to curated datasets, failing to capture the heterogeneity and cross-domain shifts of real-world metabolomics. Furthermore, current benchmarks lack difficulty-aware diagnostics and leave blind to how models behave under specific compute or data constraints. To address this, we present FlexMS, a modular public-data benchmark framework that standardizes MS/MS prediction across public resources while keeping molecular encoders, metadata conditioning, predictor heads, and downstream retrieval under one protocol. FlexMS establishes a fair evaluation playground which significantly lowers the barrier for integrating new predictive tools. Rather than solely optimizing for average scores, FlexMS augments aggregate accuracy with difficulty-aware diagnostics, providing actionable guidance on model selection across different compute constraints, data scales, and downstream retrieval objectives. Ultimately, FlexMS provides the community with a reproducible standard to identify which algorithmic conclusions are stable and which operating points are most viable in practice.