<p><strong>🎤Kelly Broster McMahon (Senior Manager of Market Development and Collaborations at Thermo Fisher Scientific).&nbsp;</strong></p><p>Kelly has over 15 years of experience in LC-MS and protein mass spectrometry. Her work focuses on translating complex analytical challenges into scalable, compliant workflows for the biopharmaceutical industry.</p><p><strong>In this episode of Concentrating on Chromatography, we sit down with Kelly Broster McMahon to explore the analytical science behind one of the most important pharmaceutical trends today: GLP-1 therapeutics.</strong></p><p>Drugs like Semaglutide have transformed the treatment of diabetes and obesity—but ensuring their safety, efficacy, and quality requires cutting-edge analytical workflows.</p><p>In this conversation, we break down how chromatography and high-resolution mass spectrometry (LC-MS) work together to characterize complex peptide therapeutics, detect trace-level impurities, and support drug development from early research through regulated manufacturing.</p><h4><strong>What You’ll Learn</strong></h4><ul><li>Why GLP-1 drugs are uniquely challenging to analyze</li><li>How LC-MS enables detailed peptide characterization</li><li>The importance of detecting low-level impurities for patient safety</li><li>How analytical workflows scale from R&amp;D to manufacturing</li><li>The role of software and data platforms in modern labs</li><li>Where chromatography and mass spectrometry are heading next</li></ul><h4><strong>Key Takeaways</strong></h4><ul><li>GLP-1 therapeutics are a “proof point” for the future of analytical chemistry—where success depends not on measuring more, but on measuring the right things earlier, with confidence.</li><li>As molecules become more complex, integrated workflows combining chromatography, high-resolution mass spectrometry, and advanced informatics are becoming essential to ensure data integrity, regulatory compliance, and ultimately, patient safety.</li></ul><h3><strong>Video Transcription</strong></h3><p>In this episode, Kelly, Senior Manager of Market Development and Collaborations at Thermo Fisher Scientific, discusses the analytical challenges associated with GLP-1 peptide therapeutics and explains how modern chromatography, high-resolution mass spectrometry, software, and data platforms are shaping the future of biopharmaceutical development. Drawing on more than 15 years of experience in LC-MS and protein mass spectrometry, she shares insights into peptide characterization, impurity profiling, workflow continuity, and the growing role of AI in analytical science.</p><h4><strong>Kelly's Background in Biopharmaceutical Analysis</strong></h4><p>Kelly currently focuses on biopharmaceutical development at Thermo Fisher Scientific, working closely with industry partners to transform complex analytical challenges into scalable and compliant workflows. Her scientific background includes cancer biology, quantitative proteomics, biomarker assays, systems oncology, and mass spectrometry-based drug discovery. Throughout her academic and industrial career, she has applied mass spectrometry to support patient stratification, treatment monitoring, and therapeutic development.</p><h4><strong>Why GLP-1 Therapeutics Are So Important Right Now</strong></h4><p>GLP-1 drugs have become one of the most discussed therapeutic classes because they occupy a unique space between traditional small molecules and large biologics. Their structural complexity, sensitivity to degradation, and global manufacturing scale make them an ideal example of how analytical science must evolve.</p><p>According to Kelly, these molecules require greater analytical sensitivity, deeper structural characterization, and seamless workflows that support everything from discovery through manufacturing and regulatory submission.</p><h4><strong>Analytical Challenges of Peptide Therapeutics</strong></h4><p>Unlike conventional small molecules, peptide drugs such as semaglutide are larger, conformationally dynamic, and often chemically modified to improve stability, half-life, and absorption.</p><p>These therapeutics may contain:</p><ul><li>Sequence variants</li><li>Oxidation products</li><li>Deamidation products</li><li>Aggregates</li><li>Trace-level impurities</li></ul><p>Many of these species are difficult to separate chromatographically and challenging to identify confidently. As a result, peptide characterization requires both highly selective chromatography and high-resolution accurate-mass spectrometry.</p><h4><strong>What GLP-1 Drugs Teach Us About the Future of Analytical Science</strong></h4><p>Kelly emphasized that the future of analytical science is not necessarily about generating more data but about measuring the right attributes earlier and with greater confidence.</p><p>GLP-1 developers increasingly require integrated analytical workflows that generate actionable insights during discovery while remaining robust enough to support late-stage development, manufacturing, and global regulatory filings. This trend highlights the growing importance of scalable and harmonized analytical ecosystems.</p><h4><strong>Why Small Molecular Changes Matter</strong></h4><p>Even a single amino acid substitution or post-translational modification can dramatically alter a peptide's biological behavior.</p><p>Minor structural changes may affect:</p><ul><li>Receptor binding</li><li>Potency</li><li>Stability</li><li>Immunogenicity</li></ul><p>Because these changes can directly influence safety and efficacy, regulatory agencies require precise structural characterization and control throughout development and manufacturing.</p><h4><strong>The Power of Combining Chromatography and Mass Spectrometry</strong></h4><p>For complex peptide therapeutics, chromatography and mass spectrometry work together to provide comprehensive characterization.</p><p>Chromatography separates complex mixtures and resolves closely related species, while high-resolution mass spectrometry provides molecular specificity and structural confirmation. Together, advanced UHPLC and HRAM MS deliver the selectivity, sensitivity, and confidence required for modern peptide analysis.</p><h4><strong>The Value of High-Resolution Accurate-Mass Spectrometry</strong></h4><p>Kelly described high-resolution accurate-mass (HRAM) mass spectrometry as an indispensable technology for drug development.</p><p>Compared with older analytical approaches, HRAM provides:</p><ul><li>Exact mass measurements</li><li>Isotopic resolution</li><li>Structural confirmation</li><li>Trace impurity detection</li><li>Identification of sequence variants</li><li>Characterization of post-translational modifications</li></ul><p>These capabilities enable confident data generation that can withstand regulatory scrutiny, audits, and global submissions.</p><h4><strong>Why Sensitivity Is Critical</strong></h4><p>The distinction between a safe and unsafe therapeutic can exist at extremely low concentration levels.</p><p>Detecting impurities at parts-per-billion levels allows developers to identify risks early in development and address them before manufacturing scale-up. Analytical sensitivity therefore supports both regulatory confidence and patient safety.</p><h4><strong>Resolving Co-Eluting Impurities</strong></h4><p>One of the greatest challenges in peptide analysis is distinguishing nearly identical molecular species.</p><p>By combining ultra-high-resolution mass spectrometry with optimized chromatographic separations, scientists can accurately differentiate closely related compounds and remove uncertainty from impurity profiling. This capability is essential for both regulatory submissions and therapeutic safety assessments.</p><h4><strong>Workflow Continuity Across Drug Development</strong></h4><p>Kelly stressed that analytical methods should not be reinvented at every stage of development.</p><p>Instead, workflows should transition smoothly from:</p><ul><li>Research and discovery</li><li>Development</li><li>Quality control</li><li>Manufacturing</li></ul><p>Maintaining continuity improves reproducibility, preserves data integrity, and enables scalable development without disrupting analytical performance.</p><h4><strong>The Evolution of Instruments, Software, and Data Platforms</strong></h4><p>Modern analytical ecosystems extend beyond LC-MS instrumentation alone.</p><p>Today's workflows integrate:</p><ul><li>Consumables</li><li>Chromatography systems</li><li>Mass spectrometers</li><li>Informatics platforms</li></ul><p>Instrumentation continues to become faster, more sensitive, and capable of higher throughput, while advanced software transforms complex datasets into actionable insights. These integrated platforms support compliance, traceability, and harmonized global operations.</p><h4><strong>Data Integrity and Regulatory Compliance</strong></h4><p>As drug development becomes increasingly global, maintaining data integrity is more important than ever.</p><p>Validated software systems, secure data management tools, and comprehensive audit trails help organizations harmonize workflows across multiple sites while ensuring that generated data can withstand regulatory review worldwide.</p><h4><strong>Lessons for Next-Generation Biologics</strong></h4><p>Although GLP-1 therapeutics are currently in the spotlight, Kelly believes the broader lesson is that molecular complexity is increasing across all therapeutic classes.</p><p>This includes:</p><ul><li>Peptides</li><li>Monoclonal antibodies</li><li>Fusion proteins</li><li>Advanced biologics</li></ul><p>Across all these modalities, the need for deeper molecular understanding, early risk detection, and scalable analytical control continues to grow.</p><h4><strong>Biosimilars, Generics, and Equivalence Testing</strong></h4><p>As GLP-1 patents expire, analytical science will play a crucial role in establishing biosimilarity and equivalence.</p><p>Developers must demonstrate rigorous structural and functional comparability between original products and follow-on versions. High-resolution analytical platforms provide the depth of characterization required to prove equivalence while protecting patient safety and therapeutic efficacy.</p><h4><strong>Future Opportunities in Chromatography and Mass Spectrometry</strong></h4><p>Looking ahead, Kelly sees significant innovation opportunities in:</p><ul><li>Greater integration of analytical platforms</li><li>Faster chromatographic separations</li><li>Enhanced multiplexing</li><li>Smarter data analytics</li><li>Increased automation</li><li>Real-time process monitoring</li><li>Process analytical technologies (PAT)</li></ul><p>These developments will bring analytical insights closer to manufacturing environments and improve efficiency across the drug development lifecycle.</p><h4><strong>The Next 5–10 Years</strong></h4><p>Over the next decade, Kelly expects analytical workflows to become:</p><ul><li>More predictive</li><li>More automated</li><li>More interconnected</li><li>Increasingly supported by AI-driven interpretation</li></ul><p>However, she emphasized that these advances must remain grounded in high-quality analytical data generated by ultra-high-resolution instrumentation.</p><h4><strong>Final Thoughts</strong></h4><p>Kelly concluded by highlighting the critical role analytical scientists play in enabling patient access to safe and effective medicines. By identifying hidden risks, characterizing complex molecules, and supporting innovation without compromising scientific rigor, analytical science remains a cornerstone of modern drug development and aligns directly with Thermo Fisher Scientific's mission of making the world healthier, cleaner, and safer.</p><blockquote><p><strong>This text has been automatically transcribed from a video presentation using AI technology. It may contain inaccuracies and is not guaranteed to be 100% correct.</strong></p></blockquote><blockquote><h4><strong>Concentrating on Chromatography Podcast</strong></h4><p>Dive into the frontiers of chromatography, mass spectrometry, and sample preparation with host David Oliva. Each episode features candid conversations with leading researchers, industry innovators, and passionate scientists who are shaping the future of analytical chemistry. From decoding PFAS detection challenges to exploring the latest in AI-assisted liquid chromatography, this show uncovers practical workflows, sustainability breakthroughs, and the real-world impact of separation science. Whether you’re a chromatographer, lab professional, or researcher you'll discover inspiring content!</p><p>You can find <strong>Concentrating on Chromatography Podcast </strong>in podcast apps:</p><ul><li><a target="_blank" rel="noopener noreferrer" href="https://open.spotify.com/show/1wZg67rCjoGju4yhqxI3tL"><strong>Spotify</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podcasts.apple.com/tz/podcast/concentrating-on-chromatography/id1788429185"><strong>Apple podcast</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podscan.fm/podcasts/concentrating-on-chromatography"><strong>podscan</strong></a></li></ul><p>and on <a target="_blank" rel="noopener noreferrer" href="https://www.youtube.com/@ChromatographyTalk/videos"><strong>YouTube channel</strong></a></p></blockquote>

How GLP-1 Drugs Are Analyzed: Chromatography & Mass Spectrometry Explained

<p><strong>In the week of June 15, the following webinars await you in the field of liquid phase and especially HPLC and LC/MS.</strong></p><blockquote><h4><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinars"><strong>👉 DETAILS AND REGISTRATION IN THE WEBINARS SECTION</strong></a></h4><p><strong>👉 Search and filter in the largest global database</strong> with over <strong>5 500 GC, LC, MS and spectroscopy webinars</strong>.</p></blockquote><h5><strong>1. Agilent Technologies: Innovations and Emerging Tools in Peptide Science</strong></h5><ul><li>Tu, 16.6.2026 11:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6252">Registration</a></li></ul><p>Master peptide characterization and manufacturing. Discover Agilent solutions for GLP-1 API scale-up, xCELLigence cell-based bioassays, non-destructive Raman RMID, and inert LC/MS impurity profiling workflows.</p><h5><strong>2. Waters Corporation: DMPK: From Sample to Result</strong></h5><ul><li>Tu, 16.6.2026 15:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6227">Registration</a></li></ul><p>Discover practical DMPK bioanalysis workflows in action and learn how modern software and MS systems improve efficiency, consistency, and confidence from sample to result.</p><h5><strong>3. Waters Corporation: Which Detector Do You Need for GLP-1RAs? Choosing Between MALS and Mass Spec</strong></h5><ul><li>Tu, 16.6.2026 17:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6250">Registration</a></li></ul><p>This webinar explains how multi-angle light scattering (MALS) and mass spectrometry (MS) provide complementary insights into GLP-1RA quality.</p><h5><strong>4. Phenomenex: Enabling Robust HILIC LC–MS Workflows for Highly Polar Compound Analysis</strong></h5><ul><li>We, 17.6.2026 10:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6254">Registration</a></li></ul><p>Enable robust HILIC LC-MS workflows for highly polar compounds. Learn how Kinetex Amide columns provide consistent retention, high selectivity, and excellent reproducibility in complex matrices.</p><h5><strong>5. Waters Corporation: Development and Implementation of an Efficient LC-TOF MS Screening Workflow in Seized Drug Analysis</strong></h5><ul><li>We, 17.6.2026 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6272">Registration</a></li></ul><p>The upcoming webinar will address how the Acquity UPLC with the RDa detector has been employed to develop a workflow for screening seized drug cases.</p><h5><strong>6. Waters Corporation: Charge! Next-Generation LNP Assays with Waters Reversed Phase Charge Aerosol Detection</strong></h5><ul><li>We, 17.6.2026 17:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6258">Registration</a></li></ul><p>Optimize lipid nanoparticle separations. Discover new lipid stationary phases and method development strategies for LNP composition and impurity analysis using UV, ELSD, CAD, and MS detection.</p><h5><strong>7. Waters Corporation: Advancing Size Exclusion Chromatography (SEC) for Characterization of Genetic Therapeutics</strong></h5><ul><li>Th, 18.6.2026 11:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6259">Registration</a></li></ul><p>Optimize size exclusion chromatography for genetic therapeutics. Learn how wide-pore SEC columns enable high-resolution profiling of mRNA, LNPs, and viral vectors while preventing shear degradation.</p><h5><strong>8. Metrohm: From Challenge to Confidence: PFAS Analysis with Ion Chromatography-Based Techniques</strong></h5><ul><li>Th, 18.6.2026 15:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6264">Registration</a></li></ul><p>Master comprehensive PFAS water analysis. Learn how Combustion IC (CIC) determines total AOF, and discover how IC-MS/MS quantifies highly hydrophilic ultra-short chain PFAS like trifluoroacetic acid (TFA).</p><h5><strong>9. Evosep Biosystems: Assay Development for translational and research applications</strong></h5><ul><li>Th, 18.6.2026 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6265">Registration</a></li></ul><p>Discover how the Evosep Eno system, when combined with the Orbitrap Exploris 480 instrument, enables high-throughput proteomic analysis of plasma for machine learning models.</p><h5><strong>10. Unchained Labs: Know the ins and outs of your lentivirus with Leprechaun</strong></h5><ul><li>Th, 18.6.2026 17:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6266">Registration</a></li></ul><p>Optimize lentivirus characterization for in vivo CAR-T therapies. Learn how Leprechaun combines interferometry and fluorescence to deliver complete size and structural data on individual viral particles.</p><h5><strong>11. Technology Networks: Selecting Biomarker Methods for Evolving Oncology Research Workflows</strong></h5><ul><li>Th, 18.6.2026 17:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6260">Registration</a></li></ul><p>Select the right biomarker methods for precision oncology. Balance sensitivity with turnaround time.</p><h5><strong>12. SelectScience: Native fluorescence detection for high-throughput CE-SDS and AAV characterization in drug development</strong></h5><ul><li>Fr, 19.6.2026 17:30 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6268">Registration</a></li></ul><p>Enhance CE-SDS and AAV characterization. Learn how native fluorescence detection (NFD) on the BioPhase 8800 system improves signal-to-noise ratios without protein labeling or workflow complexity.</p>

Webinars LabRulezLCMS Week 25/2026

<p><strong>This study systematically compares four widely used lysosome enrichment strategies, including density gradient separation, magnetic bead isolation, filter-based enrichment, and subcellular fractionation. Using the THP-1 monocytic cell line, the methods were evaluated for lysosome yield, purity, membrane integrity, reproducibility, scalability, and contamination through complementary analytical techniques and proteomics.</strong></p><p>The results showed that gradient- and bead-based methods achieved the highest lysosomal purity, while subcellular fractionation yielded larger numbers of lysosomes but with greater contamination and variability. Filter-based enrichment enabled rapid processing but recovered mainly nonintact lysosomes. The findings provide practical guidance for selecting lysosome isolation methods in both research and clinical applications.</p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.5c05792"><strong>Benchmarking Lysosome Enrichment Methods: A Guide for Research and Clinical Translation</strong></a></h4><p>Anniek L. de Jager, Sara Kassem, Louis Alesha, Brigitta A.E. Naber, Inge F. de Laat, Bas de Mooij, Kyra van der Pan, Erik Bos, Roman I. Koning, Jacques J.M. van Dongen*, Cristina Teodosio*, and Paula Díez*</p><p>Anal. Chem. 2026, 98, 6, 4585–4600</p><p><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acs.analchem.5c05792">https://doi.org/10.1021/acs.analchem.5c05792</a></p><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><p><strong>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.</strong></p><div><p>Since their discovery by De Duve et al. <a href="javascript:void(0);">(1)</a> in 1955, lysosomes have emerged as dynamic organelles critical to cellular homeostasis, regulating processes from nutrient sensing to plasma membrane repair. <a href="javascript:void(0);">(2−5)</a> These acidic, enzyme-packed vesicles <a href="javascript:void(0);">(6,7)</a> are now acknowledged as central players in pathologies far beyond their classical association with lysosomal storage disorders (LSDs). Dysfunctional lysosomes are involved in neurodegenerative diseases (e.g., Alzheimer’s, Parkinson’s), <a href="javascript:void(0);">(8−10)</a> cancer metastasis, <a href="javascript:void(0);">(11−13)</a> and cardiovascular disorders. <a href="javascript:void(0);">(14,15)</a> At the same time, lysosomal proteins like GBA1 and LAMP2 serve as biomarkers and therapeutic targets. <a href="javascript:void(0);">(16−19)</a> Despite their clinical significance, a major challenge in lysosomal research persists since lysosomes constitute less than 5% of cellular volume, <a href="javascript:void(0);">(20,21)</a> requiring precise enrichment for accurate analysis in diagnostic and therapeutic development. <a href="javascript:void(0);">(22,23)</a></p><p>Current lysosome isolation methods, including density gradient-based separation, <a href="javascript:void(0);">(24−28)</a> filter-based size exclusion approaches, <a href="javascript:void(0);">(29)</a> magnetic particle phagocytosis, <a href="javascript:void(0);">(30−33)</a> subcellular fractionation (SCF), <a href="javascript:void(0);">(34)</a> and immunoprecipitation strategies, <a href="javascript:void(0);">(35,36)</a> vary widely in yield, purity, structural integrity, and practicality. For instance, density gradient-based methods, which often use Percoll <a href="javascript:void(0);">(37)</a> or sucrose, <a href="javascript:void(0);">(38)</a> while well-established, user-friendly, and cost-effective, coisolate organelles with similar densities, such as endosomes, peroxisomes, and mitochondria, <a href="javascript:void(0);">(39,40)</a> and can be time-consuming, potentially affecting lysosomal protein integrity. Conversely, filter-based size exclusion methods isolate lysosomes based on their size, <a href="javascript:void(0);">(29)</a> offering shorter processing times and straightforward protocols. Nevertheless, these approaches may result in cross-contamination, as various cellular vesicles share similar sizes. <a href="javascript:void(0);">(20,21)</a> Magnetic bead-based methods (e.g., superparamagnetic iron oxide nanoparticles, SPION) involve the endocytic uptake of magnetic beads, <a href="javascript:void(0);">(35,41)</a> offering specificity but altering lysosomal physiology through forced phagocytosis, which hampers functional assays, such as in cancer drug screening. <a href="javascript:void(0);">(42,43)</a> Finally, SCF, which employs sequential lysis steps to enrich specific organelles, including lysosomes, <a href="javascript:void(0);">(34,44)</a> is a rapid, easy-to-use, and cost-effective method; however, significant cross-contamination is linked to this method. <a href="javascript:void(0);">(34,44)</a> Such trade-offs are rarely addressed in single-modality studies, leaving clinicians and researchers without clear guidance for protocol selection. This gap is particularly critical in translational contexts, where diagnostic laboratories that prioritize high-purity lysosomes for biomarker discovery require protocols different from those of therapeutic teams that need lysosomes for drug efficacy testing.</p><p>Previous comparative studies, such as the proteomic analysis by Singh et al., <a href="javascript:void(0);">(35)</a> have provided valuable insights but primarily focused on yield and purity assessments via proteomic markers, overlooking several key parameters with potentially relevant impacts in translational and clinical applications. Critically, these studies often omit functional and practical metrics: for instance, they typically do not evaluate lysosomal membrane integrity, which is vital for enzyme activity and therapeutic testing, nor do they consider the scalability and feasibility of protocols for contexts where only limited patient samples are available or the potential presence of isolation artifacts. This omission of practical, multimodal assessment represents a critical gap that prevents researchers from selecting a protocol optimized for specific clinical goals.</p><p>Our study seeks to address these limitations by employing a multimodal framework that evaluates four widely used lysosome isolation methods across several metrics that are critical for clinical applications. We systematically assessed the yield and reproducibility of each method, factors that are essential for biobanking and high-throughput diagnostic workflows. We also examined organelle purity, crucial for avoiding false positives in proteomic biomarker screenings, together with lysosomal membrane integrity, an important consideration for functional studies and drug testing. Finally, we considered the scalability of each method, ensuring that our findings are relevant for studies involving rare or precious patient-derived cells together with the cost and processing time of each technique. Thus, by integrating nanoparticle tracking analysis (NTA), spectral flow cytometry, transmission electron microscopy (TEM), and mass spectrometry (MS)-based proteomics, our study revealed distinct advantages for each method depending on the intended application. With this comprehensive benchmark, we bridged a critical gap between fundamental lysosome biology and clinical implementation, guiding researchers and clinicians in the selection of the most appropriate isolation strategies tailored to their diagnostic or therapeutic goals.</p><h3><strong>Experimental Section</strong></h3><h4><strong>Liquid Chromatography–Tandem Mass Spectrometry Analysis (LC–MS/MS)</strong></h4><p>For liquid chromatography–tandem mass spectrometry (LC–MS/MS) studies, a total of 15 μg of proteins were reduced with 10 m<span>M</span> DTT at 45 °C for 30 min, followed by alkylation with 40 m<span>M</span> 2-iodoacetamide (IAA, Sigma-Aldrich) at RT for 30 min. This last reaction was quenched with 20 m<span>M</span> DTT, and protein digestion and peptide recovery were performed using hydrophilic and hydrophobic beads (Sera-Mag Carboxylate-Modified Magnetic Particles, Cytiva, Marlborough, MA/USA) according to the Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) method. <a href="javascript:void(0);">(46,47)</a> In short, samples were incubated twice with beads (1:10 (w/w) protein:bead ratio) in 70% acetonitrile (ACN) for 18 min at RT. After incubation, bead-bound proteins were retained by using a DynaMag-PCR Magnet (Thermo Fisher), and contaminants were washed away three times with 80% ethanol (Sigma-Aldrich). Proteins were digested using a Trypsin/LysC mix (1:25 (w/w) enzyme:protein ratio, Promega, Madison, WI/USA) in 50 m<span>M</span> ammonium bicarbonate at RT overnight. Beads were then washed twice with ACN, after which peptides were eluted in 2% (v/v) dimethyl sulfoxide (DMSO, Thermo Fisher), lyophilized in a freeze-dryer, and stored at −20 °C. Samples were then tagged using the tandem mass tag (TMT) labeling kit (TMTpro 16plex tags, Thermo Fisher) by resuspending the lyophilized peptides in 40 m<span>M</span> HEPES pH 8.4 and labeling with the TMT tags at a 1:5 (w/w) sample:tag ratio for 1 h at RT. The labeling reaction was quenched with 5% hydroxylamine. TMT-labeled lysosome samples were pooled, lyophilized in a freeze-dryer, and stored at −20 °C until LC–MS/MS analysis.</p><h4><strong>Mass Spectrometry Data Acquisition and Analysis</strong></h4><p>MT-labeled peptides were dissolved in solvent A (water/formic acid (FA), 100/0.1 (v/v)) and analyzed by online C18 nano-HPLC MS/MS, coupling an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=3&amp;search=UltiMate+3000"><strong>UltiMate 3000 gradient HPLC system</strong></a> (Thermo Fisher) and an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/401"><strong>Orbitrap Exploris 480 mass spectrometer</strong></a> (Thermo Fisher). A total of 12 fractions were injected onto a precolumn (300 μm × 5 mm; PepMap100 C18 5 μm, Thermo Fisher) equilibrated with solvent A and eluted via a homemade analytical nano-HPLC column (50 cm × 75 μm; ReproSil-Pur C18-AQ 1.9 μm) using solvent B (80/20/0.1 (v/v/v) ACN/water/FA) in a gradient from 5 to 30% in 160 min. The nano-HPLC column was drawn to a tip of ∼5 μm and functioned as an electrospray needle. A Sonation PRSO-V2 column oven was used to maintain the analytical column temperature at 50 °C. The MS1 spectra were recorded in the Orbitrap at a mass-to-charge ratio (<i>m</i>/<i>z</i>) range of 350–1,600, a resolution of 120,000, a maximum injection time of 50 ms, and automatic gain control (AGC) at standard. Dynamic exclusion was applied after <i>n</i> = 1 with an exclusion duration of 45 s and a mass tolerance of 10 ppm. Charge states 2–5 were included. Precursors for MS2 were selected using a TopSpeed method of 3 s at a resolution of 45,000 and fragmented by high-energy collision-induced dissociation (HCD) at a normalized collision energy (NCE) of 36%, with an AGC at 200 and the maximum injection time set to auto. The isolation window for MS/MS was 1.2 Da.</p><p>Raw data files were converted to the peak list by using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1782"><strong>Proteome Discoverer v2.4</strong></a> (Thermo Fisher), followed by protein identification employing the UniProtKB database (<i>Homo sapiens</i>, 20,596 entries) using Mascot v2.2.07 (Matrix Science Inc., Boston, MA/USA) with the following parameters: precursor and fragment mass tolerances of 10 ppm and 0.02 Da, respectively; TMT16plex on N-term and Lys as fixed modifications; oxidation on Met and acetylation of N-term as variable modifications; and full tryptic digestion (no P rule) with up to 2 missed cleavages. A false discovery rate (FDR) of 1% was set for peptide spectrum matches (PSMs), peptides, and proteins.</p><p>The MS data have been deposited via the PRIDE partner repository to the ProteomeXchange Consortium with the identifier PXD040003.</p><h3><strong>Results</strong></h3><h4><strong>Quantification and Size Distribution of Isolated Vesicle Fractions</strong></h4><p>To assess the yield and size distribution of vesicles isolated by different methods, nanoparticle tracking analysis (NTA) was employed. The SCF procedure yielded the highest median number of vesicles per cell (2,356.3 ± 7,461.7; median ± range), followed by gradient-based (1,630.2 ± 1632.3), filter-based (228.2 ± 265.6), and bead-based isolation (27.8 ± 35.2). Both filter- and bead-based approaches resulted in significantly lower yields compared to SCF (<i>p</i>-value = 0.002 and <i>p</i>-value &lt;0.0001, respectively), and the gradient-based method yielded significantly more vesicles than the bead-based approach (<i>p</i>-value = 0.002; <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.5c05792#fig1">Figure 1</a>A).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Anal_Chem_2026_98_6_4585_4600_Figure_1_Quantification_and_size_analyses_of_isolated_vesicles_2d5c120f05.jpeg" alt="Anal. Chem. 2026, 98, 6, 4585–4600: Figure 1. Quantification and size analyses of isolated vesicles. A) Quantification of isolated vesicles per cell, shown as median with range. Circles represent technical replicates. B) Size distribution of isolated vesicles (in nm), displaying average D10 (red), D50 (blue), and D90 (gray), depicted as median with range. Circles represent technical replicates. C–F) Particle distribution plots showing mean concentration at specific sizes; each line represents a technical replicate. For each method, six technical replicates are shown, except for SCF, which includes 12 replicates. SCF, subcellular fractionation. Statistical significance: p &lt; 0.05 and FDR 5%. For statistical analysis, the nonparametric Kruskal–Wallis test was used and corrected for multiple comparisons using the method of Benjamini and Hochberg." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Anal_Chem_2026_98_6_4585_4600_Figure_1_Quantification_and_size_analyses_of_isolated_vesicles_2d5c120f05.jpeg 126w," sizes="100vw" width="1383" height="1708"></p><p>Median vesicle size (D50) was comparable across all methods: 159.5 ± 17.2 nm (gradient-based), 169.9 ± 82.7 nm (filter-based), 168.9 ± 42.4 nm (bead-based), and 150.0 ± 55.6 nm (SCF). Similarly, decile 10 (D10) values were consistent, ranging from 87.1 ± 87.3 nm (SCF) to 108.2 ± 3.1 nm (gradient-based). However, filter-based isolation led to higher size heterogeneity of the isolated particles, capturing significantly larger vesicles at the upper end (D90:308.5 ± 110.7 nm) compared to SCF (244.9 ± 112.6 nm, <i>p</i>-value = 0.005), while gradient- and bead-based methods showed intermediate D90 values (250.8 ± 31.7 nm and 273.0 ± 45.9 nm, respectively; <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.5c05792#fig1">Figure 1</a>B). Notably, greater intra-assay variation in concentration and size distribution was observed with filter-based and SCF methods compared to the other approaches (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.5c05792#fig1">Figure 1</a>C–F).</p><h4><strong>Morphology of the Isolated Fractions</strong></h4><p>Transmission electron microscopy (TEM) of the isolated fractions confirmed the presence of heterogeneously sized vesicles, in line with the size distributions obtained by NTA. Notably, only the gradient-based method yielded relatively pure vesicle preparations. In contrast, filter-based and SCF methods displayed contamination with cellular debris or protein aggregates, while the bead-based approach was associated with the presence of lipid structures (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.5c05792#fig2">Figure 2</a>).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Anal_Chem_2026_98_6_4585_4600_Figure_2_Transmission_electron_microscopy_TEM_of_isolated_fractions_f14ddbe180.jpeg" alt="Anal. Chem. 2026, 98, 6, 4585–4600: Figure 2. Transmission electron microscopy (TEM) of isolated fractions. Panels A–D show TEM images of the four isolated fractions. Insets display magnified views of the regions outlined in black squares. Black arrows indicate identified representative isolated vesicles, and white arrows denote areas of contamination." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Anal_Chem_2026_98_6_4585_4600_Figure_2_Transmission_electron_microscopy_TEM_of_isolated_fractions_f14ddbe180.jpeg 145w," sizes="100vw" width="1263" height="1361"></p><h3><strong>Conclusion</strong></h3><p>This study provides a systematic benchmarking of four lysosome isolation strategies, revealing inherent trade-offs among yield, integrity, purity, and reproducibility driven by their underlying separation principles. No single method is universally optimal; instead, the method selection must be guided by the intended downstream application. By integrating complementary analytical approaches in a clinically relevant and challenging monocytic model, our work offers a practical framework to support the informed selection of lysosome enrichment strategies, facilitating reliable lysosomal analyses in both research and translational settings.</p></div>

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