Cell membrane chromatography (CMC) is a unique biomimetic technique that overcomes the limitations of conventional physical property-based separation methods by enabling direct, activity-based identification of target components through interactions between ligands and receptors. It greatly simplifies analytical workflows while improving the specificity and efficiency of the screening of active components, thereby providing more efficient and precise analytical solutions for drug discovery, modernization of traditional Chinese medicines (TCMs), food and environmental testing. CMC drives analytical technologies toward a “directly targeted, activity-oriented” paradigm. The CMC-dependent development of analytical instruments represents a characteristic innovation at the intersection of medical and engineering disciplines. By deeply integrating chromatographic engineering, cell biology, and pharmaceutical analysis, CMC translates in vivo ligand–receptor mechanisms into standardized in vitro analytical devices. This approach supports the development of medicine using precise, activity-oriented analytical tools, and also steers engineering technologies toward biomedical needs characterized by biomimetic simulation and target-specific adaptation. Overall, CMC helps overcome key challenges in screening active components from complex systems and evaluating drug bioactivity. It also serves as a bridge between basic medical research and engineering translation and provides a model for medical–engineering integration, thereby facilitating the development of the biopharmaceutical industry. In this paper, we systematically review the development of CMC technology and CMC-based instruments. Furthermore, we analyze the instrument configuration and typical applications in screening of active components from complex systems.

Metabolites can regulate protein structure and function via covalent modifications, with different isomers exerting distinct effects. Now, stereospecific O-2-hydroxyglutarylation by oncometabolites D2HG and L2HG has been uncovered on distinct proteomes, revealing a chiral layer of post-translational regulation that tunes kinase signalling.

Prostate cancer (PC) is one of the most common malignancies in men, and the emergence of androgen receptor-low/negative castration-resistant PC (ARL/- CRPC) following androgen receptor signaling inhibitor (ARSI) therapy remains a critical clinical challenge. The RNA-binding protein DEAD-box helicase 3 X-linked (DDX3X) has been implicated in the translational regulation of androgen receptor (AR) mRNA; however, the underlying binding mechanisms are not well defined. Here, we show that DDX3X colocalizes with AR mRNA in ARL/- CRPC cells and selectively recognizes non-canonical RNA G-quadruplex (rG4) motifs within the sequence of AR mRNA. RNA immunoprecipitation sequencing (RIP-seq) revealed enrichment of DDX3X-AR mRNA interactions in ARL/- CRPC cells. Fluorescence imaging confirmed the colocalization of DDX3X and AR mRNA within cytoplasmic granules, and biochemical assays confirmed the ability of selected AR mRNA fragments to form rG4 structures bound by DDX3X. Proteomic profiling of DDX3X-Ras GTPase-activating protein-binding protein 1 (G3BP1) complexes identified several RNA-binding proteins, including IGF2BP1, PUM2, and UBAP2, which may act as candidate cofactors. Together, these findings shed light on the interaction between AR mRNA and DDX3X and identify putative protein partners, offering insights into future therapeutic strategies.

Neuropeptides are chemically diverse signaling molecules that regulate physiology and behavior, yet many species lack neuropeptidomic characterization due to sparse genomic annotation. Here, we integrate genomic sequence information with neuropeptidomics to define the neuropeptidomes of two widely used crustacean model organisms, Callinectes sapidus and Cancer borealis. Using a curated multispecies precursor database, tBLASTn alignment, signal peptide detection, and in silico processing, we predicted more than 23,000 putative peptides across both genomes, including numerous sequences bearing hallmarks of mature neuropeptides. Mass spectrometry-based profiling provided experimental support for many predicted peptides and revealed substantial chemical diversity, including novel allatostatin B and C isoforms, an insulin-like peptide B-chain-like isoform in C. sapidus, and the first report of natalisin peptides in C. borealis. Notably, we observed an atypical precursor architecture in which a single prohormone encoded two distinct neuropeptide families, suggesting previously unrecognized modes of neuropeptide copackaging and signaling. Finally, leveraging a feeding perturbation model, we observed tissue-specific differences in the abundance of newly identified peptides in the thoracic ganglion, commissural ganglion, and pericardial organs, consistent with functional neuroendocrine roles. Together, this work expands the known repertoire of crustacean neuropeptides, provides a resource for comparative peptide biology, and establishes a genome-enabled framework for discovery of endogenous peptide signaling molecules in newly sequenced species.

Global warming and rising ocean temperatures pose substantial challenges to marine ecosystems and crustacean populations. As an ectothermic species, the American lobster ( Homarus americanus ) relies on physiological and neurochemical mechanisms to maintain homeostasis under varying environmental conditions. To elucidate the role of neuropeptides in neuronal plasticity and systemic adaptation to temperature fluctuations, we employed a quantitative mass spectrometry-based approach to probe key neuropeptides involving thermal adaptation in four lobster neural tissues at three temperatures: 4 °C (cold), 11 °C (control), and 18 °C (warm). Peptidomic profiling revealed a global reduction in peptide abundance during cold exposure, alongside coordinated, tissue-specific reconfigurations of the neuropeptidome between experimental groups. Cold exposure led to a significant downregulation of RFamide, leucokinin, and pyrokinin peptides in the commissural ganglia, whereas B-type allatostatin (AST-B), natalisin, and RYamide peptides were drastically elevated in the brain of warm-acclimated animals, with comparatively fewer detectable peptide abundance changes in the sinus gland and the stomatogastric ganglion. Collectively, our findings elucidate neuropeptide signaling pathways underlying thermal tolerance and adaptive resilience in Homarus americanus , offering insights into the survival mechanism and neurochemical basis of neural circuits in response to thermal acclimation. Abstract Figure:

A global exploration of the complex interplay between protein conformational changes and phosphorylation events in cell-cell interactions is crucial for understanding the dynamic nature of protein modifications. This understanding is essential for developing novel targeted therapies for pancreatic cancer, particularly in the context of interactions between pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs). However, protein conformational changes that occur during PCCs-PSCs interactions remain poorly studied, and the relationship between these changes and phosphorylation is not well understood. Here, we present a comprehensive mass spectrometry-based study investigating the interplay between protein conformational alterations and phosphorylation in a coculture system of pancreatic ductal adenocarcinoma cells (PANC-1) and PSCs. Our results demonstrate that 435 proteins exhibiting conformational changes were detected during the coculture of PANC-1 with PSCs, primarily involving proteins associated with the tricarboxylic acid (TCA) cycle, glycolysis/gluconeogenesis, and carbon metabolism. Our findings also highlight a potential association between phosphorylation and protein conformational changes. Moreover, we identified five potential conformational targets, including ACLY, ACO1, ACO2, IDH1, and OGDH, which may provide valuable insights into the molecular pathways underlying gemcitabine resistance in pancreatic cancer. Overall, these results offer insights into protein conformational changes and their potential link to phosphorylation in the context of cancer-stromal cell interactions, paving the way for structure-based, targeted therapeutic strategies for pancreatic cancer treatment.

Neuropeptides modulate a diverse range of physiological functions, including those associated with feeding. Post-translational modifications (PTMs) contribute significantly to the dynamic nature of neuropeptide isoforms, influencing their functional diversity. Mass spectrometry is the gold-standard analytical technique for peptidomic analyses and is complemented by computational methods for peptide identification; however, the computational search space becomes increasingly difficult to manage as more potential modifications are considered. Using innovative approaches capable of addressing the vast combinations of possible modifications, such as the PEAKS PTM search algorithm, we globally profiled the neuropeptidome ofCancer borealis(Jonah crab) to investigate the role of PTMs in feeding- and appetite-related processes over time. Through an in-depth examination of several notable modifications, we proposed PTM-associated motifs for neuropeptides, which may enhance future identification capabilities. Furthermore, this work revealed neuropeptides that were characteristically modified depending on the crab's feeding status and time post-feeding, suggesting potential biological significance. This study represents the first large-scale investigation of the modified crustacean neuropeptidome, providing new insights into the regulatory implications of PTMs in biological systems.

BACKGROUND:Protein glycosylation and phosphorylation are critical post-translational modifications (PTMs) that regulate diverse physiological and pathological processes, yet their comprehensive characterization remains challenging due to low abundance and poor ionization efficiency. Recent advances using epoxy-ATP-Ti4+-IMAC materials have enabled simultaneous enrichment of N-glycopeptides, phosphopeptides, and mannose-6-phosphate glycopeptides. However, high-throughput, multiplexed quantification of these PTMs is still lacking. This work addresses the need for an efficient strategy capable of simultaneously enriching, identifying, and quantitatively comparing glycosylation and phosphorylation across multiple biological samples. RESULTS:We developed a high-throughput workflow integrating epoxy-Ti4+-IMAC enrichment with custom N,N-dimethyl leucine (DiLeu) isobaric tags to achieve 12-plex quantitative analysis of N-glycosylation and phosphorylation for the first time. This streamlined one-tube sample preparation protocol enabled robust, simultaneous enrichment and quantification of PTMs from complex mouse brain samples. Application to APP/PS1 transgenic mice versus wild-type controls produced quantitative identification of 1975 N-glycopeptides and 1181 phosphopeptides. Comparative profiling revealed substantial PTM alterations associated with Alzheimer's disease (AD)-related pathology. Differentially modified proteins mapped to key biological pathways, including synapse organization, synaptic membrane regulation, and cell adhesion. The abundance patterns highlighted broad disruptions in PTM-mediated signaling and provided molecular insights into synaptic dysfunction in the APP/PS1 model. SIGNIFICANCE AND NOVELTY:This integrated DiLeu isobaric labeling-epoxy-Ti4+-IMAC platform provides a powerful, high-throughput solution for the simultaneous quantification of glycosylation and phosphorylation, enabling detailed investigation of PTM interplay. By uncovering disease-associated modifications in AD mouse models, this method offers new opportunities to identify mechanistic biomarkers and therapeutic targets. Its versatility and scalability make it broadly applicable to PTM-centric studies across diverse biological systems, including biofluids, cell lysates and tissues.

Ocean acidification (OA) refers to the ongoing decline in ocean pH caused by the cascading effects of increased atmospheric CO2, which has significant negative impacts on various marine organisms, particularly crustaceans with calcified shells. However, research on the metabolic responses of crustaceans remains limited. In this study, we performed untargeted metabolomics on hemolymph samples from Cancer borealis (Jonah crab), a crustacean species well known for its tolerance to temperature and pH changes, to investigate its metabolic responses to OA. Two extraction methods-isopropanol (IPA) and acidified methanol (AcMeOH)-were employed to capture a broad range of metabolites and small peptides. Both methods enabled comprehensive detection; however, IPA extraction yielded more consistent and extensive metabolite coverage, identifying 43 lipids compared to only 15 with AcMeOH. We identified 15 metabolites that responded significantly to OA. Several metabolites, including the potential neuropeptide cycloprolylglycine and the exogenous compound curcumin, exhibited concentration changes under OA exposure, suggesting their potential relevance in stress response pathways triggered by environmental stress. Overall, we highlight IPA as a more effective extraction method for untargeted metabolomics of crustacean hemolymph. Our study elucidates metabolic dynamics that enhance our understanding of the physiological adaptability of marine crustaceans under environmental stress and provides a comprehensive dataset for future OA research.

Alzheimer's disease (AD) is characterized by progressive neurodegeneration and protein misfolding, yet the structural dynamics of proteins and their post-translational modifications during disease progression remain poorly understood. Here, we present an integrated structural and glycoproteomic analysis of paired serum and cerebrospinal fluid (CSF) samples from individuals across three clinical stages: normal cognition, mild cognitive impairment, and AD. Using limited proteolysis mass spectrometry (LiP-MS) combined with high-field asymmetric waveform ion mobility spectrometry and data-independent acquisition, we identified 54 proteins exhibiting structural alterations, two of which (clusterin and ceruloplasmin) showed structural changes in both serum and CSF. Furthermore, our findings reveal potential crosstalk between protein structural changes and N-glycosylation, supported by correlations between LiP-derived structural features and glycosylation patterns in key proteins, such as haptoglobin and kininogen-1. This study demonstrates that integrating structural proteomics with glycoproteomics in matched serum and CSF samples enhances biomarker discovery and provides novel insights into the molecular mechanisms of AD. Our approach offers a powerful platform for identifying robust, minimally invasive biomarkers and for understanding post-translational modification-induced protein remodeling in neurodegenerative diseases.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a transformative molecular imaging technology capable of mapping diverse chemical classes, from small metabolites, neurotransmitters and lipids to N-glycans and proteins, at cellular resolution. The field has rapidly matured, offering a range of mass analysers, including axial time-of-flight, quadrupole (or orthogonal) time-of-flight, and high-resolution Orbitrap and Fourier-transform ion cyclotron resonance systems, each with distinct characteristics in terms of spatial resolution, chemical specificity and throughput. In practice, MALDI IMS involves applying a light-absorbing chemical matrix onto a tissue section, followed by automated laser irradiation at discrete coordinates (like pixels) to desorb and ionize endogenous molecules for mass analysis. This label-free approach preserves spatial context, providing a molecular map that links highly multiplexed molecular distributions to distinct anatomical regions, functional tissue units and cell types in situ. This Primer includes an overview of basic imaging MALDI IMS concepts, instrumentation, data processing approaches and advanced applications. We also address key challenges and considerations, with an eye towards optimizing instrumentation and methods to overcome issues in spatial resolution, sensitivity and specificity. Finally, we look towards the future of the technology, including its integration with other spatial omics modalities and its potential as a tool for precision medicine. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a label-free molecular imaging approach mapping diverse biomolecules at cellular resolution. In this Primer, Spraggins et al. outline MALDI IMS concepts, instrumentation, data processing and integration with spatial omics and medicine.

Benign prostatic hyperplasia (BPH) is a disease affecting the majority of aging men; 90% of men develop histological BPH by the time they reach their eighties. BPH can lead to bothersome lower urinary tract symptoms (LUTS), which may reduce quality of life. Many patients fail current treatment options and may progress to surgical intervention. Furthermore, diagnosis is reliant on symptom questionnaires and the cause of LUTS can be difficult to distinguish. Currently, BPH can only be definitively diagnosed through histological analysis of prostate tissue, which is not the standard of care. The resulting lack of clinical tissue samples is a major limitation in investigating disease pathology. Improved understanding of disease development and progression, along with objective biomarkers of disease, is needed for BPH. This investigation uses mass spectrometry (MS)-based proteomics and glycoproteomics to compare healthy prostate tissue with prostate tissue affected by BPH to address this gap in knowledge. By integrating proteomics and glycoproteomics, we identified 206 proteins and 44 glycopeptides that were significantly altered between BPH and control samples. These findings provide deeper insight into disease-associated pathways and may facilitate the identification of clinically relevant targets for further investigation.

Human pancreatic islets are highly heterogeneous; thus, understanding their biological organization is crucial for elucidating metabolic function and diabetes pathogenesis. High-resolution mass spectrometry imaging of intact human pancreas is challenging due to the small size and dispersed distribution of individual islets within dense exocrine tissue. Here, we establish an ultra-low-flow-rate DESI mass spectrometry imaging (u-DESI-MSI) platform that enables lipidomic analysis of individual islets in human pancreatic tissue. Optimization of raster step size and scan rate parameters for u-DESI resulted in lipid ion images with enhanced spatial fidelity. The analysis demonstrates a highly reproducible central-peripheral spatial lipid distribution within pancreatic tissue. Diacyl phosphatidylcholines (diacyl-PCs), ether-linked phosphatidylcholines (ether-linked PCs) and sphingomyelins (SMs) are predominantly localized to the central endocrine region and co-register with intact insulin distributions, as further validated by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). In contrast, specific lysophosphatidylcholines (LPCs) delineate the endocrine-exocrine interface. These results indicate the existence of position-dependent lipid organization within human islets. This platform provides a robust spatial-mapping foundation for future studies investigating structural and metabolic alterations in human islets under disease conditions.

Vocal fold epithelial cells (VFEs) serve critical physiologic and immunologic functions at the boundary between the upper and lower airways but are difficult to maintain and expand in primary cultures. This technical challenge has impeded progress in VFE biology as well as cell banking for translational applications. Here, using primary human VFEs, we show that simultaneous inhibition of transforming growth factor β (TGF-β), Rho-associated protein kinase (ROCK), and Notch signaling with a small-molecule inhibitor cocktail enables rapid proliferation, successful passaging, and long-term expansion while preserving the core epithelial phenotype. Under anchorage-independent culture conditions, VFE progenitors generate clonal spheres that can be expanded over multiple generations; sphere-dissociated VFEs then revert toward their original phenotype, which includes the ability to form stratified squamous epithelium in organotypic cocultures. Both pathway-inhibited and sphere-cultured VFEs exhibit mechanistically appropriate remodeling of the cellular proteome. These advances offer a robust toolkit for upper airway mucosal biology and regenerative medicine.

Endogenous peptides are critical regulators of signaling and immunity but remain difficult to characterize in organisms with incomplete genomic annotation. We developed a hybrid discovery platform that integrates transformer-based de novo sequencing (Casanovo), neuropeptide-focused database searching (EndoGenius), and empirical false discovery rate estimation via NovoBoard. This pipeline enables confident identification of endogenous peptides while expanding coverage beyond conventional database-only or de novo -only approaches. Applied to neuroendocrine tissues from Callinectes sapidus and Cancer borealis , the workflow revealed numerous high-abundance novel peptides and provided structural and genomic support for their biological relevance. Notably, we report the first histone-2A-derived antimicrobial peptide in the C. sapidus and characterize naturally occurring sequence variants. We also identified unexpected peptide homologies between crustaceans and Rattus norvegicus , enabling annotation of conserved housekeeping proteins in sparsely annotated genomes. This hybrid platform establishes a scalable, open-source strategy for advancing neuropeptidomics and endogenous peptide discovery in emerging model organisms.

Lysosome targeting chimeras (LYTACs) represent a promising strategy to harness lysosomal degradation for eliminating extracellular and membrane disease-causing proteins. These bifunctional molecules link a target protein to a lysosome targeting receptor (LTR), forming a ternary complex that drives internalization and degradation. The first generation of LYTAC used cation-independent mannose-6-phosphate receptor (CI-M6PR), also known as Type II insulin-like growth factor receptor (IGF-IIR), as the LTR, with polymeric glycopeptides as the ligands. However, their complex and heterogeneous composition limits therapeutic potential. To improve specificity and efficacy, natural IGF-II has been explored as an alternative ligand. However, wild-type IGF-II activates both Type I insulin-like growth factor receptor (IGF-IR) and insulin receptor isoform A (IR-A), posing off-target risks. In this study, we engineered a novel IGF-II mutant (mutIGF-II) with two mutations (Del1-7 and Y27L), which confer high affinity for IGF-IIR while minimizing binding to IGF-IR and IR-A. The mutIGF-II-based bifunctional degraders significantly enhanced internalization and degradation of both secreted and membrane-bound proteins. Additionally, we developed a practical all-protein mutIGF-II LYTAC by genetically encoding mutIGF-II into a mammalian expression vector and transfecting it into cancer-relevant cell lines. The secreted mutIGF-II-based PD-L1 degrader effectively induced PD-L1 degradation.

Site-specific antibody conjugation through glycoengineering offers a promising route to generate homogeneous glycosite-specific antibody‒drug conjugates (gsADCs) with improved therapeutic indices. Dozens of gsADCs are advancing from preclinical studies to clinical trials. However, current methods involve either multiple enzymes or lengthy preparation of substrates. Herein, we report a novel and synthetically streamlined platform utilizing LacNAc-derived 4,6-acetal glycosyl donors for glycosite-specific transglycosylation mediated by a single enzyme. These glycosyl donors can be synthesized in as few as two steps, representing a major advancement in synthetic accessibility compared to previously reported glycosyl donors, which often require more than 15 steps. Computational analysis showed that the acetal ring restricts conformation, directing donor 7 to a π–π-stabilized groove of the enzyme. Donor 7 , along with a positive control, was evaluated in the context of gsADCs, consistently demonstrating potent and selective cytotoxicity toward HER2-positive cancer cells, while sparing HER2-negative cells. Furthermore, donor 7 was successfully adapted to generate glycosite-specific degrader-antibody conjugates (gsDACs), highlighting its broad utility. Additional studies revealed that donor 7 produces antibodies with markedly enhanced resistance to Endo S2 mediated hydrolysis. Together, these findings establish a practical and broadly applicable platform for glycosite-specific antibody conjugation, paving the way for next-generation antibody-based therapeutics.