MetaboLightsapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14425/m_MTBLS14425_LC-MS_negative_reverse-phase_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14425/m_MTBLS14425_LC-MS_positive_reverse-phase_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14425/s_MTBLS14425.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14425/a_MTBLS14425_LC-MS_negative_reverse-phase.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14425/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14425/a_MTBLS14425_LC-MS_positive_reverse-phase.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14425<p>Metabolites were annotated by using the METLIN database (https://metlin.scripps.edu). The quantification of identified metabolites was based on the primary ion heights.</p>MetaboLightsPublicLiquid Chromatography MS - negative - reverse-phaseLiquid Chromatography MS - positive - reverse-phase<p>Each sample (5 μl) was injected onto a reverse-phase Kinetex core-shell C18 column (1.7 μm, 2.4 × 100 mm, Phenomenex, Switzerland) using a 1290 Infinity II UHPLC system (Agilent, US). The gradient mobile phase consisted of water containing 0.1% formic acid (Solvent A) and acetonitrile containing 0.1% formic acid (Solvent B). Each sample was resolved at a flow rate of 0.3 ml/min for a total run time of 14 min. The UHPLC gradient was as follows: 95% A and 5% B for 0.5 min, ramping to 10% A and 90% B from 0.5 to 12.0 min, and then returning to 95% A and 5% B from 12.0 to 14.0 min.</p>Divalent Aptamer-mediated Clustering for Extracellular Vesicle Separation.Duo KeaiPeking Universityurinemass spectrometry assay<p>Urine-derived EVs isolated using UC, SEC, or DAC, with equal particle numbers (1E9 particles), were diluted to 100 μl in PBS and mixed with 400 μl of extraction buffer (50% acetonitrile and 50% methanol). The samples were vortexed for 30 s and sonicated for 30 min at 4°C. Then the samples were incubated at -20°C for 30 min and centrifuged at 13,000 × g for 15 min at 4°C. The supernatant was transferred to a fresh tube and dried under vacuum. The dried samples were reconstituted in 100 μl of buffer containing 50% acetonitrile and 50% water and sonicated for 5 min at 4°C. Afterward, the samples were centrifuged at 13,000 × g for 10 min at 4°C to remove fine particulates. The supernatant was transferred to a glass vial for ultrahigh-performance liquid chromatography-coupled quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF MS) analysis.</p>Homo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS14425yaxuan liang. Beijing Normal University. yaxuan.liang9@gmail.com.xiaoling liu. Beijing Normal University. 1451233915@qq.com.<p>Data were acquired in centroid MS mode over a mass range of 100 to 1,200 m/z, with a single injection per sample. The batch acquisition was repeated to ensure experimental reproducibility, and the sample queue was randomized to minimize bias. Raw data were processed using MassHunter Profinder (v10.0, Agilent, US) and Mass Profiler Professional (v15.1, Agilent, US) for peak detection, extraction, alignment, and integration.</p>Groupkeaiduoduo998@126.com<p>Urine samples (20-50 ml) were collected from 9 healthy volunteers for DAC optimization and method comparisons. Urine samples were transported in sealed containers on ice, and immediately pre-processed by centrifuging at 2,000 × g for 10 min at 4°C. The supernatant was aliquoted and stored at -80°C.</p><p><br></p>Metabolomics100-1200Agilent 6546 LC/Q-TOFmetabolomicsurineuntargeted analysisHomo sapiensAgilent 1290 Infinity LChuman urineLCMS100-1200Agilent 6546 LC/Q-TOFurineuntargeted analysismetabolomicsHomo sapiensAgilent 1290 Infinity LChuman urineLCMS<p>The column effluent was introduced directly into the mass spectrometer via electrospray ionization (AJS ESI, Agilent, US). Mass spectrometry was performed on a quadrupole time-of-flight (Q-TOF) instrument (6546 LC/Q-TOF, Agilent, US) operating in either negative (ESI-) or positive (ESI+) electrospray ionization mode with a capillary voltage of 3,500 V in positive mode and 2,800 V in negative mode.</p>falseDivalent Aptamer-mediated Clustering for Extracellular Vesicle SeparationExtracellular vesicles (EVs) are important mediators of intercellular communication and promising sources of diagnostic and therapeutic biomarkers, yet effective EV isolation remains challenging due to trade-offs among yield, purity, and adaptability across biofluids. Here, we introduce divalent aptamer-mediated clustering (DAC), a streamlined affinity-based EV isolation strategy that induces controllable vesicle clustering and enables recovery by standard filtration. By exploiting multivalent aptamer binding to EV surface markers, DAC converts nanoscale vesicles into micron-scale clusters while preserving EV integrity and biological activity. We demonstrate robust EV isolation from plasma, urine, and cell culture media, and benchmark DAC against ultracentrifugation, density gradient ultracentrifugation, and size-exclusion chromatography. DAC achieves comparable or improved EV yield and purity with reduced processing time, cost, and operational complexity. Proteomic and metabolomic analyses show that DAC isolates affinity-defined EV subpopulations with cargo profiles distinct from those obtained using conventional methods. Moreover, DAC is readily adapted to alternative EV targets, exemplified by enrichment of EpCAM-positive EVs. Together, DAC provides a versatile and accessible platform for studying EV heterogeneity, function, and molecular composition.2026-05-042026-05-04MTBLS14425