Join us for a fascinating conversation with Dr. Subhoja Chakraborty from the University of Central Florida, whose cutting-edge research targets the global challenge of malaria drug resistance. Dr. Chakraborty shares her journey from structure-based drug design during her PhD to her recent collaborative work repurposing human kinase inhibitors as rapid, selective antimalarials against drug-resistant Plasmodium falciparum strains.<h4>In this episode, you'll learn:</h4><ul><li>How structure-guided approaches reveal potential drug targets, including cysteine proteases like falcipain-2, and kinases implicated in malaria's complex life cycle.</li><li>The power of repurposing kinase inhibitors originally developed for cancer and other diseases, focusing on compound 12, which blocks hemozoin formation and protein kinase 6—key steps in parasite survival.</li><li>Lab techniques such as affinity, gel filtration, and size exclusion chromatography for recombinant protein purification—and how troubleshooting is crucial for high-yield, stable complexes.</li><li>Advances in mass spectrometry for characterizing protein interactions, validating drug targets, and mapping molecular pathways pivotal to malaria therapeutics.</li><li>Tips and lessons for new researchers facing the challenges of sample preparation, complex protocols, and working as part of a collaborative team.</li></ul>Dr. Chakraborty’s insights bridge the gap between basic science and translational drug discovery, offering hope for more effective, targeted malaria treatments in a world where resistance to frontline therapies threatens millions.<h4>Referenced Papers:</h4><ul><li>"Plasmodium falciparum protein kinase 6 and hemozoin formation are inhibited by a type II human kinase inhibitor exhibiting antimalarial activity" (Cell Chemical Biology, 2025)</li><li>"New insights of falcipain 2 structure from Plasmodium falciparum 3D7 strain" (BBRC, 2022)</li><li>"Structure-Based Optimization of Protease−Inhibitor Interactions to Enhance Specificity of Human Stefin‑A against Falcipain‑2 from the Plasmodium falciparum 3D7 Strain" (Biochemistry, 2023)</li></ul>If you’re a student, scientist, or simply passionate about advances in infectious disease research, this episode offers actionable insights, inspiring advice, and a front-row seat to innovation in combating malaria.<h3>Video Transcription</h3><h4>From Structural Biology to Drug Discovery: Chromatography and Mass Spectrometry in Malaria Research</h4><h5>Introduction</h5>In this discussion, Subhoja shares insights into her research journey, focusing on malaria drug discovery and the role of modern analytical techniques in biological and biomedical research. The conversation is aimed primarily at undergraduate students and early-career researchers interested in chemistry, biotechnology, and life sciences.<h5>Research Focus: Malaria Drug Discovery and Kinase Targets</h5>Subhoja is currently involved in malaria drug discovery research at the University of Central Florida, where she has been working since beginning her postdoctoral position two years ago. Her earlier PhD research focused on structure-based drug design in malaria, using a combination of in silico approaches, recombinant protein expression, and X-ray crystallography to determine precise three-dimensional structures of target proteins and their inhibitors.This structural perspective provided a foundation for her current work, which centers on drug discovery through compound screening and repurposing. A recent publication from her laboratory explores the use of repurposed mammalian CDK-like kinase inhibitors as potential antimalarial agents. While such kinase inhibitors are well known in cancer and cardiovascular research, their application to malaria targets represents a novel and promising approach.Kinases are attractive drug targets because of their central role in cell signaling and growth pathways. By targeting specific kinases involved in different stages of the malaria parasite life cycle—whether asexual growth, gametocytogenesis, or differentiation—it may be possible to significantly inhibit parasite growth and progression.<h5>Chromatography in Protein Expression and Purification Workflows</h5>Chromatography plays a critical role throughout Subhoja’s research. Her experience spans from basic chromatography techniques during her early training to advanced protein purification workflows using affinity chromatography, gel filtration (size exclusion chromatography), and ion exchange chromatography.Recombinant proteins are routinely purified using affinity resins such as nickel or cobalt, followed by further purification to remove contaminants. Fast Protein Liquid Chromatography (FPLC) enables compact, stepwise purification workflows suitable for producing proteins for in vitro assays.A recurring challenge in this process is the expression of high-molecular-weight proteins, which often accumulate in insoluble fractions or inclusion bodies. Careful selection of expression vectors, host systems, and induction conditions is therefore essential to maximize yield and solubility.<h5>Integrating Size Exclusion Chromatography into the Workflow</h5>Size exclusion chromatography (SEC) is typically used after affinity purification to further improve protein purity. Following elution with imidazole or glutathione, desalting is required to prevent buffer components from interfering with downstream assays. This is often achieved using desalting columns such as G-25 or PD-10.To remove lower-molecular-weight contaminants, finer SEC media—such as Superdex or Sephacryl columns—are employed. Concentration steps using molecular-weight cut-off filters are then applied to obtain the desired protein concentration for functional studies.These iterative purification and troubleshooting steps are a routine part of protein biochemistry and reflect the importance of methodical optimization and careful validation.<h5>The Role of Mass Spectrometry in Biomolecular Characterization</h5>Mass spectrometry is a key analytical tool in Subhoja’s research pipeline, particularly for target identification and validation. In both malaria and cancer research, MS is used alongside chromatography, immunoprecipitation, and pull-down assays to study protein interactions and molecular recognition events.Affinity-based mass spectrometry enables the identification of binding partners and interactors, whether studying proteins, long non-coding RNAs, or protein complexes. Techniques such as native MS, tandem affinity purification, and proximity labeling (e.g., TurboID-based workflows) provide deeper insights into molecular networks and signaling pathways.These approaches are essential for understanding how candidate drug targets function within complex biological systems.<h5>Sample Preparation Challenges in Biological Research</h5>Sample preparation remains one of the most challenging aspects of biological experimentation. Whole-cell lysates contain proteases, nucleases, and other components that can rapidly degrade target molecules. Preventing degradation requires minimizing preparation time, maintaining low temperatures, and using appropriate inhibitor cocktails.Reproducibility is also critical: biological experiments require multiple replicates, and variability is often unavoidable. Careful handling, protocol optimization, and continuous troubleshooting are essential to ensure reliable results, whether preparing samples for chromatography, mass spectrometry, sequencing, or immunoprecipitation.<h5>Advances in Chromatography and MS for Malaria Research</h5>Modern advances in chromatography and mass spectrometry significantly enhance malaria research by enabling precise target identification, quantification, and functional characterization. Drug discovery workflows begin with construct design and recombinant expression, followed by purification and interaction studies.In malaria research, stage-specific parasite biology requires careful extraction and analysis of proteins from different life cycle stages. Tandem affinity MS, immunoprecipitation, and in vivo validation studies help confirm target relevance and therapeutic potential.Recent work on repurposed kinase inhibitors has shown promising results, including inhibition of hemozoin formation—a key detoxification pathway for the malaria parasite—highlighting the potential of such compounds for further development.<h5>Malaria, Drug Resistance, and Global Impact</h5>Malaria remains a major global health burden, with over 600,000 deaths annually and widespread drug resistance complicating treatment. Artemisinin resistance, particularly associated with mutations in the K13 propeller domain, has driven the search for new targets and therapies.During her PhD, Subhoja focused on falcipain-2, a cysteine protease involved in hemoglobin degradation within the parasite. This pathway is critical for parasite survival and has emerged as a promising drug target. Her work revealed that falcipain-2 operates within a larger protein complex responsible for hemoglobin processing and amino acid release.Understanding these molecular mechanisms is essential for developing robust, stage-specific antimalarial strategies.<h5>Advice for Early-Career Researchers</h5>For researchers just starting out, Subhoja emphasizes the importance of hands-on experience. Working directly with cells, lysates, and analytical tools is essential for truly understanding experimental systems. Failure is an inherent part of research, and perseverance is key.Motivation, teamwork, careful sample handling, and thorough preparation all contribute to long-term success. Reading extensively, critically evaluating one’s own work, and appreciating incremental progress are vital aspects of a sustainable research career.Every successful experiment—no matter how small—represents meaningful progress and contributes to the broader goals of science and medicine.<h5>Closing Remarks</h5>The discussion highlights how interdisciplinary approaches combining chromatography, mass spectrometry, structural biology, and biochemistry are driving advances in malaria drug discovery. Through careful experimental design, analytical rigor, and collaborative teamwork, researchers can address complex biological challenges and contribute to global health solutions.<blockquote>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.</blockquote><blockquote><h4>Concentrating on Chromatography Podcast</h4>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!You can find Concentrating on Chromatography Podcast in podcast apps:<ul><li><a target="_blank" rel="noopener noreferrer" href="https://open.spotify.com/show/1wZg67rCjoGju4yhqxI3tL">Spotify</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podcasts.apple.com/tz/podcast/concentrating-on-chromatography/id1788429185">Apple podcast</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://podscan.fm/podcasts/concentrating-on-chromatography">podscan</a></li></ul>and on <a target="_blank" rel="noopener noreferrer" href="https://www.youtube.com/@ChromatographyTalk/videos">YouTube channel</a></blockquote>

Affinity, Gel Filtration, Size Exclusion Chromatography: Repurposing Kinase Inhibitors for Malaria

In the week of February 9, the following webinars await you in the field of liquid phase and especially HPLC and LC/MS.<blockquote><h4><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinars">👉 DETAILS AND REGISTRATION IN THE WEBINARS SECTION</a></h4>👉 Search and filter in the largest global database with over 5 000 GC, LC, MS and spectroscopy webinars.</blockquote><h5>1. SelectScience: High-Throughput Stability Screening Strategies for Antibody Formulation</h5><ul><li>Tu, 10.2.2026 17:00 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5807">Registration</a></li></ul>Learn how high-throughput DLS/SLS and rapid-screening DSC accelerate stability assessment of high-concentration antibody formulations, enabling faster, data-driven biologics development.<h5>2. Phenomenex: Big Scale, Big Wins – Prep Chromatography Explained</h5><ul><li>We, 11.2.2026 10:00 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5793">Registration</a></li></ul>Learn how to scale analytical methods to preparative HPLC, optimize loading capacity, and understand the limits of preparative columns for pharmaceutical applications.<h5>3. ALS Czech Republic: PFAS in Food &amp; Food Contact Material</h5><ul><li>We, 11.2.2026 15:00 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5816">Registration</a></li></ul>Get a practical overview of PFAS in food and food contact materials, including EU and US regulations, key analytical challenges, and reliable PFAS testing strategies.<h5>4. New Food Magazine: The Science of Next-Gen Ingredients – Fermentation, Formulation and Function</h5><ul><li>We, 11.2.2026 16:00 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5720">Registration</a></li></ul>Explore how fermentation-driven innovation is transforming functional food ingredients and how advanced analytics optimize flavour, functionality, and stability.<h5>5. SCIEX: Accurate Mass Fundamentals – Interpreting and Applying Your Data</h5><ul><li>We, 11.2.2026 19:00 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5772">Registration</a></li></ul>Session 3 focuses on interpreting and applying accurate mass data to confirm compound identity, move beyond basic quantitation, and troubleshoot results for confident reporting.<h5>6. Agilent Technologies: Process Analytical Techniques – Online HPLC for Real-Time Monitoring and Control of Bioprocesses</h5><ul><li>Th, 12.2.2026 06:30 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5834">Registration</a></li></ul>Discover how PAT HPLC enables near-real-time monitoring of critical quality attributes in bioprocessing, with a case study on charge-variant control by cation-exchange chromatography.<h5>7. Thermo Fisher Scientific: Introducing the Orbitrap Excedion Pro Hybrid MS</h5><ul><li>Th, 12.2.2026 11:00 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5889">Registration</a></li></ul>Discover the new Orbitrap Exploris Pro with expanded ETD/EThcD capabilities for confident biopharmaceutical characterization, including intact proteins, PTMs, ADCs, and large biomolecules.<h5>8. Waters Corporation: Planning Your NuGenesis Upgrade – Insights and Best Practices for a Smooth Transition</h5><ul><li>Th, 12.2.2026 19:00 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5833">Registration</a></li></ul>Learn how to plan a successful NuGenesis upgrade, transition to Windows 11, and unlock new capabilities such as dashboards for real-time operational insights.<h5>9. Thermo Fisher Scientific: Comprehensive Analytical Strategies for Peptide Therapeutics and GLP-1 Agonists with Agilent</h5><ul><li>Th, 12.2.2026 19:00 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5814">Registration</a></li></ul>Explore analytical workflows for peptide therapeutics, including method development, peptide mapping, GLP-1 QC, and LC-MS strategies across Agilent platforms.<h5>10. Agilent Technologies: Moving from R&amp;D and Limited QC on a UV-Vis-NIR Instrument to Large-Scale QC Using an Autosampler</h5><ul><li>Th, 12.2.2026 20:00 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/2104">Registration</a></li></ul>This webinar covers practical aspects of adding and using an autosampler to maximize UV-Vis-NIR QC workflows at scale.<h5>11. Agilent Technologies: LC Method Transfer – Hidden Challenges &amp; Absolute Solution</h5><ul><li>Fr, 13.2.2026 06:30 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5787">Registration</a></li></ul>Discover why LC method transfer between legacy and modern systems is challenging, which hidden parameters affect retention and peak shape, and how to achieve robust, reliable results.

Webinars LabRulezLCMS Week 07/2026

Charge detection mass spectrometry (CDMS) allows direct mass determination of individual ions and is increasingly used to study heterogeneous biological samples. This work demonstrates the use of Orbitrap-based CDMS/Direct Mass Technology (DMT) for quantitative analysis of transthyretin (TTR) tetramer samples below 100 kDa, extending CDMS beyond its traditional qualitative role.DMT resolved wild-type and C-terminally modified TTR homotetramers, as well as hybrid tetramers, enabling kinetic analysis of subunit exchange and stability. The approach also quantified thyroxine binding to TTR variants, revealing differences in binding affinity linked to C-terminal modification. By separating overlapping m/z signals through charge measurement, CDMS enables accurate mass and abundance determination, highlighting its potential for quantitative analysis of complex protein systems.<blockquote><h4>The original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.5c05771">Dissecting Heterogeneous Populations of Protein-Complex Samples Using Direct Mass Technology</a></h4>Robert L. Rider, Jared Hampton, Zhenyu Xi, Carter Lantz, Sangho D. Yun, Weijing Liu, Rosa Viner, Arthur Laganowsky, and David H. Russell*Anal. Chem. 2025, 97, 49, 27057–27063<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acs.analchem.5c05771">https://doi.org/10.1021/acs.analchem.5c05771</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.Transthyretin (TTR) is a homotetrameric transport protein that circulates in plasma and cerebrospinal fluid, delivering thyroxine (T4) and retinol-binding protein–vitamin A complexes. <a href="javascript:void(0);">(1)</a> The stability of the TTR tetramer is central to its physiological role because dissociation into monomers can initiate misfolding and amyloid formation, leading to systemic or hereditary transthyretin amyloidosis. <a href="javascript:void(0);">(2)</a> Subunit exchange between wild-type (WT) and variant TTR proteoforms, including disease-associated mutations such as V30M and V122I, have been implicated in amyloid progression by generating hybrid tetramers (tetramers containing WT and mutant subunits) with altered stabilities. <a href="javascript:void(0);">(3,4)</a> Ligand binding, particularly at the two thyroxine-binding sites located at the dimer–dimer interface, can kinetically stabilize the tetramer and modulate amyloidogenicity. <a href="javascript:void(0);">(5,6)</a> Accurate measurement of both subunit exchange kinetics and ligand binding stoichiometry is therefore critical for elucidating the TTR dynamics and stability.Affinity or sequence tags are widely employed in protein expression, purification, or detection. <a href="javascript:void(0);">(7,8)</a> While these tags are often assumed to be inert, their influence on protein structure and dynamics are rarely examined in detail. <a href="javascript:void(0);">(9,10)</a> Protein tags at the N- and C-terminus of TTR have been shown to have differing gas phase stabilities from those of WT-TTR. <a href="javascript:void(0);">(11)</a> The C-terminus is of particular interest because this region participates in the dimer–dimer interface that governs tetramer stability. <a href="javascript:void(0);">(11,12)</a> Characterizing how the C-terminal tag (CT) (-ASGENLFYQ) affects TTR’s solution stability and ligand binding is not only relevant for understanding TTR dynamics but also provides an example of how expression tags can alter protein dynamics. <a href="javascript:void(0);">(13)</a>Native mass spectrometry (nMS) has been invaluable for characterizing protein complexes, <a href="javascript:void(0);">(14)</a> yet conventional nMS often struggles with heterogeneous samples where overlapping charge state distributions and adduct heterogeneity limit resolution and quantitation. <a href="javascript:void(0);">(15)</a> Charge detection mass spectrometry (CDMS) addresses these limitations by independently measuring the charge and m/z of individual ions, enabling the direct determination of molecular mass and abundance. Development of CDMS has largely focused on achieving higher mass resolution <a href="javascript:void(0);">(16)</a> and extending measurements toward assemblies into the GDa range. <a href="javascript:void(0);">(17,18)</a> However, there is untapped utility for CDMS experiments other than mass and proteoform determination. Jordan et al. showed the capability to assess conformational heterogeneity of mAb aggregates using the added charge dimension that CDMS provides. <a href="javascript:void(0);">(19)</a> The current work demonstrates that CDMS can separate proteoforms with overlapping m/z values based on charge and quantify their relative abundances in complex mixtures. While nMS is already a highly quantitative technique, our results establish CDMS as an equally powerful tool for quantitative analysis in heterogeneous systems. <a href="javascript:void(0);">(20,21)</a>Here, we apply Orbitrap-based CDMS/Direct Mass Technology (DMT) using the selective temporal overview of resonant ions (STORI) approach <a href="javascript:void(0);">(22,23)</a> to quantitatively compare WT-TTR and CT-TTR, focusing on two biologically relevant properties: tetramer stability assessed via subunit exchange kinetics and ligand-binding affinity. Conventional nMS cannot fully resolve the overlapping charge states of WT- and CT-TTR or their ligand-bound states. However, DMT can resolve these species by directly measuring the mass (m/z × charge), enabling quantitation of all proteoforms. The data reveal that the CT tag alters tetramer stability and thyroxine-binding affinity, demonstrating the structural sensitivity of TTR to modifications at its C-terminus. More broadly, this work demonstrates the impact that protein tags can have on native protein interactions and highlights the broader utility of quantitative CDMS for probing heterogeneous biological samples.<h4>Experimental Section</h4><h5>Data Collection</h5>All experiments were conducted on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/560">Thermo Scientific Q-Exactive UHMR</a> with added DMT capabilities (Bremen, Germany). Before DMT, mass spectra were collected at various resolving powers (6–200k) for each sample, with the instrument parameters reported in the Supporting Information (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.analchem.5c05771/suppl_file/ac5c05771_si_001.pdf">Table S1</a>). For DMT experiments, the trapping gas pressure in the HCD cell was reduced to 0.3–0.8 to attenuate the signal intensity to a single ion level, as well as decreasing the UHV pressure to assist ion retention through the entire transient of Orbitrap analysis. We observed no significant difference between mass spectra collected at various HCD cell pressures (Supporting Information, <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.analchem.5c05771/suppl_file/ac5c05771_si_001.pdf">Figure S1</a>). For DMT collection, automated ion control (AIC) and 100% target density were used with 200k resolving power.<h4>Results and Discussion</h4>CDMS has emerged as a powerful tool for analyzing heterogeneous biomolecular samples, enabling accurate, direct mass determination by simultaneously measuring the charge and m/z for individual ions. While CDMS has been widely applied for qualitative analyses, its potential for quantitative and less than MDa applications remains underexplored. <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.5c05771#fig1">Figure 1</a>A shows the theoretical mass spectrum of WT-TTR (14+ to 16+) and CT-TTR (15+ to 17+) homotetramers, where the two proteoforms become increasingly resolved as the charge states increase (&lt;3900 m/z). <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.5c05771#fig1">Figure 1</a>B zooms in on the 14+ (WT) and 15+ (CT) charge states, highlighting their strong overlap, which prevents accurate deconvolution of the proteoforms by conventional methods. <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.5c05771#fig1">Figure 1</a>C displays theoretical distributions for 0, 1, and 2 T4 bound states of both WT- and CT-TTR tetramers, where &lt;3900 m/z has observable separation between all proteoforms. However, for the 14+ and 15+ charge states of WT- and CT-TTR, respectively, the 0 T4- and 1 T4-bound populations overlap (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.5c05771#fig1">Figure 1</a>D). Furthermore, commonly adducted species (Na+, K+, and Zn2+) observed in experimental spectra broaden these distributions, making it increasingly difficult to distinguish and deconvolute the proteoforms (see <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.analchem.5c05771/suppl_file/ac5c05771_si_001.pdf">Figure S2</a>). Here, we employ Orbitrap-based CDMS/DMT to overcome this m/z overlap to resolve and quantify hybrid tetramer formation and T4 binding.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Anal_Chem_2025_97_49_27057_27063_Figure_1_A_Theoretical_mass_spectra_of_WT_TTR_and_CT_TTR_homotetramers_47232849d9.jpg" alt="Anal. Chem. 2025, 97, 49, 27057–27063: Figure 1. (A) Theoretical mass spectra of WT-TTR and CT-TTR homotetramers. (B) Zoomed-in view of the 14+ WT-TTR and 15+ CT-TTR distributions to show the large overlap of the proteoforms. (C) Theoretical mass spectra of WT-TTR and CT-TTR homotetramers with thyroxine (T4) binding. (D) Zoomed-in view of the 14+ WT-TTR and 15+ CT-TTR distributions where the 0 and 1 T4 have overlapping distributions. These theoretical distributions do not contain any commonly adducted species (Na+, K+, Zn2+), which broaden the distributions and create more overlap even for the proteoforms in the range 3300–3900 m/z (see Figure S2)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Anal_Chem_2025_97_49_27057_27063_Figure_1_A_Theoretical_mass_spectra_of_WT_TTR_and_CT_TTR_homotetramers_47232849d9.jpg 187w," sizes="100vw" width="1417" height="1182"><h5>Thyroxine Binding to TTR Homotetramers</h5>Thyroxine is the less active form of a thyroid hormone important for metabolism that binds to TTR for transport throughout plasma and cerebral spinal fluid. <a href="javascript:void(0);">(31)</a> Turnover of T4 into T3 (active) occurs after dissociation, where it can then modulate gene expression via binding to nuclear receptors. <a href="javascript:void(0);">(32)</a> The TTR tetramer gains substantial stability as it binds up to 2 T4 at the tetramer interface, decreasing tetramer dissociation and SUE. <a href="javascript:void(0);">(33)</a> This has been shown previously to decrease the amyloidogenic rate of TTR, which led to tafamidis (similar in structure to T4) to be designed and is currently the only drug that is approved for clinical treatment of TTR amyloidosis. <a href="javascript:void(0);">(34)</a> Measuring the difference in binding affinity between TTR proteoforms may provide insight into regions of TTR or TTR intermolecular/intramolecular interactions that are important for binding of small molecules such as tafamidis and T4.DMT was further applied to quantify T4 binding to WT- and CT-TTR homotetramers, highlighting the technique’s utility in probing protein–ligand interactions. <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.5c05771#fig3">Figure 3</a>A shows a representative mass spectrum of TTR homotetramers in the presence of T4. While ligand-bound species can be observed, homotetramers cannot be confidently resolved across the entire 3000–5000 m/z range. By leveraging the charge-resolving capabilities of DMT, we achieve separation of the homotetramers (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.analchem.5c05771#fig3">Figure 3</a>B,C) and the 0, 1, and 2 ligand-bound states. The 2D plot clearly shows the gain in resolution achieved when charge is simultaneously measured with m/z by separating the 14+ and 15+ charge states for WT and CT, respectively, in the range 3900–4200 m/z. Additionally, using ion mobility does not provide the same kind of 2D resolution due to the similarity in arrival times for the homotetramer proteoforms, as evidenced by <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.analchem.5c05771/suppl_file/ac5c05771_si_001.pdf">Figures S4 and S5</a> (see the Supporting Information). These data provide evidence that when ion mobility is able to separate out species that overlap in m/z yet differ in mobility, CDMS could be an alternative or used complementarily since the species likely also differ in charge.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Anal_Chem_2025_97_49_27057_27063_Figure_3_A_Mass_spectra_of_WT_TTR_and_CT_TTR_homotetramers_with_thyroxine_binding_61b1e98a94.jpg" alt="Anal. Chem. 2025, 97, 49, 27057–27063: Figure 3. (A) Mass spectra of WT-TTR and CT-TTR homotetramers with thyroxine binding. Ligand binding can be resolved, but homotetramers cannot be confidently identified. Charge states labeled are for WT; CT is always one charge state higher at the same m/z range. (B) DMT heatmap resolving WT-TTR and CT-TTR homotetramers with the additional measurement of charge. (C) Deconvoluted mass spectrum from DMT analysis. Both homotetramers and their ligand binding are resolved, and fit for analysis is shown in red. (D) Mole fraction of each homotetramer and their thyroxine binding species. Error bars represent the standard deviation of replicates (n = 3). The significance for each ligand state is in respect to the left adjacent state. *p &lt; 0.05." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Anal_Chem_2025_97_49_27057_27063_Figure_3_A_Mass_spectra_of_WT_TTR_and_CT_TTR_homotetramers_with_thyroxine_binding_61b1e98a94.jpg 181w," sizes="100vw" width="1266" height="1094"><h4>Conclusion</h4>This work demonstrates that quantitatively, Orbitrap-based CDMS/DMT can determine differences in protein-complex stability and ligand binding. By directly comparing WT- with CT-TTR, we show that the CT tag accelerates tetramer dissociation while enhancing T4 binding affinity. These results indicate that the C-terminus, located at the dimer–dimer interface, plays a more significant role in TTR stability and ligand recognition than previously appreciated. <a href="javascript:void(0);">(12)</a> Even the addition of a short peptide sequence perturbs noncovalent interactions at the interface, likely altering solvent accessibility, and the dynamics of the thyroxine binding channel. <a href="javascript:void(0);">(37,54)</a> Such findings underscore the importance of evaluating how seemingly minor sequence modifications, such as affinity tags, can influence fundamental biophysical properties.Beyond TTR, this work positions quantitative CDMS as a broadly applicable strategy for analyzing heterogeneous protein systems, where charge domain separation overcomes the resolution limits of m/z domain measurements in traditional nMS experiments. While much of the field has emphasized extending CDMS to ultrahigh masses and maximal resolution, <a href="javascript:void(0);">(16,18)</a> our results highlight the underexplored potential of applying the method in the 1000–10,000 m/z range common to nMS experiments. This capability is directly relevant to heterogeneous membrane protein complexes, which often display broad distributions of oligomeric states, PTMs, lipid, and ligand adducts that complicate spectra. <a href="javascript:void(0);">(48,55)</a> Similarly, mAbs can oligomerize and exhibit mass heterogeneity from variable glycosylation, affecting pharmacokinetics, effector function, and stability. <a href="javascript:void(0);">(19)</a> CDMS may resolve and quantify such proteoforms in a single experiment without the need for charge reduction or chemical derivatization. <a href="javascript:void(0);">(48,56,57)</a>

Dissecting Heterogeneous Populations of Protein-Complex Samples Using Direct Mass Technology

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Starting with defining the basics terms used to describe chromatographic separations and finishing with a strategy for running and interpreting a scouting gradient, in this talk we will look at the basic essentials needed to efficiently develop an HPLC method.
<blockquote>
Presenter: Paul Altiero (Application Engineer, Agilent Technologies, Inc.)
</blockquote>
<blockquote>
Paul Altiero is an Applications Engineer located at the Agilent Little Falls site in Wilmington, DE. Prior to coming to Agilent in 2009, Paul worked in the pharmaceutical industry performing LCMS bioanalysis supporting Drug Discovery DMPK. While at Agilent, Paul has been a field service engineer for LCMS as well as the integrating program manager for the RapidFire high throughput LCMS system. In his current role Paul’s responsibilities include technical support and application assistance for LCMS consumables.
</blockquote>

Agilent Technologies: Essentials of Good HPLC Method Development

Essentials of Good HPLC Method Development

In this talk we will look at the basic essentials needed to efficiently develop an HPLC method.

In this webinar we will discuss some important considerations to help maximize resolution when developing an HPLC method.
<ul>
<li>What should I consider in selecting a column for my separation goals?</li>
<li>What method parameters can I adjust to reduce the chance of method variability?</li>
<li>What role does my HPLC instrument play in optimizing a method?</li>
</ul>
Maximizing resolution with a robust and rugged method requires a strategy that addresses both chemistry and instrumentation.
<blockquote>
Presenter: Mark Powell (Application Engineer, Agilent Technologies, Inc.)
</blockquote>
<blockquote>
Mark Powell is an Applications Engineer located at Agilent’s Little Falls site in Wilmington, DE. Before joining Agilent in 2011, Mark worked in the pharmaceutical industry synthesizing, purifying and analyzing drug candidates. He provides applications assistance and technical support for Agilent’s HPLC columns and consumables.
</blockquote>

Agilent Technologies: Tips to Help Maximize Resolution

Tips to Help Maximize Resolution

In this webinar we will discuss some important considerations to help maximize resolution when developing an HPLC method.

Polymer analysis is becoming ever more complex and important in product design and upholding safety standards across a wide range of industries. Analysis of polymeric materials has never been more key to meeting both your supply chain and customer requirements. The meet the experts series in designed to give you an insight into how polymer separation techniques can help you meet some of these analytical challenges, learn how to get the separation methods/techniques and truly interrogate your GPC/SEC data. There is also an added bonus to discuss all of your polymer separation related questions to our panel of polymer analysis experts.
<h3>Session 1 - An Introduction to Polymer Analysis - A Lesson in GPC Fundamentals – Don Trinite</h3>
<ul>
<li>Introduction to Size-Exclusion Chromatography
</li>
<li>Understand how the technique aids polymer analysis
</li>
<li>Determination of Molecular Weight, Distribution, and Polydispersity
</li>
</ul>
<blockquote>
Presenter: Don Trinite (Senior Principal Technical Support Specialist, Waters Corporation)
</blockquote>
<blockquote>
Don Trinite has been working at Waters Corporation for thirty-five years. Don is a Senior Principal Technical Support Specialist for Waters Technologies Corporation for the last twenty-nine years. He is involved in developing seminars for training users in methods development and methods transfer and troubleshooting for HPLC, UPLC, IC, GPC, Prep LC and UPC² ™ He is a key person in amino acid applications development and support. Recently, he has been involved with the Andrew+™ Robotic system, testing protocols for various amino acid sample protocols to automate different aspects of sample handling for these types of samples. He is also involved in troubleshooting for Data Handling and Networking Systems. Don has also developed methods for users and has had several application notes published. Prior to this, Don was in the field service organization for Waters as Senior Service Specialist and Service Manager for five years. He was previously employed in the chemical industry by UOP, Des Plaines, Illinois, in a chromatography service group supporting chemical synthesis, production and natural product research as a Senior Research Technician. Don has a total of 43 years of experience in the LC field.
</blockquote>

Waters Corporation: An Introduction to Polymer Analysis - A Lesson in GPC Fundamentals

An Introduction to Polymer Analysis - A Lesson in GPC Fundamentals

Introduction to Size-Exclusion Chromatography. Understand how the technique aids polymer analysis. Molecular Weight, Distribution, and Polydispersity.

An Introduction to Polymer Analysis - GPC Fundamentals

A calibration curve or regression is always plotted with the instrument responses on the dependent vertical (y -) axis and the standard concentrations on the horizontal (x -) axis. As a linearity of calibration regression minimizes random errors of the instrument, we aim to find the best straight line to pass through these calibration points.
<ul>
<li>What are the estimated errors and confidence limits we can estimate for the slope (or gradient) and the y-intercept of the line?
</li>
<li>What is the uncertainty of the analyte concentration found on the calibration curve?
</li>
<li>What are the least concentration of the measurand can be detected with, say 95% confidence?
</li>
<li>In other words, what is the limit of detection (LOD) of this method?
</li>
</ul>
This one-day training program is designed to answer all the above questions and others related to the subjects of linear regression of instrument calibration and detection limits including IDL, MDL and LOQ.
<h3>Training Program (8:00 - 10:30 CEST)</h3>
<ul>
<li>Introduction</li>
<li>Different meanings for correlation and regression</li>
<li>Assumptions of linear regression and correlation</li>
<li>Residual analysis – what and how</li>
<li>Uncertainty estimation of linear calibration regression</li>
<li>Must your linear regression always pass through its origin (0,0)?</li>
<li>The most controversial subject in analytical chemistry – Limit of Detection LOD</li>
<li>Different definitions for LOD and its basic concepts</li>
<li>Differences between IDL, MDL, LOQ</li>
<li>Determining LOD by linear calibration model</li>
</ul>
<h5>Who should attend:</h5>
This course is designed for those who want to know more about the sampling procedures and how it affects the validity of their test results, including:
<ul>
<li>Laboratory analysts</li>
<li>Laboratory QA/QC personnel</li>
<li>Lab directors / managers</li>
<li>Personnel who carry out tasks such as method validation and uncertainty estimation</li>
</ul>
<h5>Pre-requisites</h5>
The participants are assumed to have had some basic statistical knowledge such as the calculation of mean, standard deviation, coefficient of variation, relative standard deviation, etc.
Participants are to have a laptop with Microsoft Excel installed and have some very basic knowledge of how to use the program.
<blockquote>
Presenter: Yeoh Guan Huah
</blockquote>
<blockquote>
Mr Yeoh Guan Huah has B.Sc. (Hons) and M.Sc. degrees in Chemistry and was former Managing Director of ALS Technichem Singapore (1978-2006), former East Asia Regional Manager of ALS Group of Laboratories in Hong Kong and China (2006-2016). He is now the senior partner of GLP Consulting, Singapore.
</blockquote>
<blockquote>
Currently Mr Yeoh is an appointed External Lead Assessor and Technical Assessor of SAC-SINGLAS till 31 March 2025 and had represented DSM/SIRIM Malaysia as a active member of ISO Technical Committee TC 69 (Application of Statistical Methods) and Convenor of AHG1 from 2005-2016.
</blockquote>

Agilent Technologies: Uncertainty of Linear Calibration Curve & Detection Limit

Uncertainty of Linear Calibration Curve & Detection Limit (Part 1)

We will answer all questions related to the subjects of linear regression of instrument calibration and detection limits including IDL, MDL and LOQ.

Uncertainty of Linear Calibration Curve & Detection Limit

In this webinar, join our low flow LC expert who will review micro and nano LC fundamentals, and present a selective approach to trap and column strategies to increase method performance. They will provide chromatographic examples on forward and backward trap elute strategies, trap selectivity comparisons, and micro and nano LC column selectivities, and will explain how to find the best selectivity strategy for your application.
<h5>Key learning objectives</h5>
<ul>
<li>Understand the current state of micro and nano LC and hardware advances</li>
<li>Understand trap and elute – forward and backward comparison</li>
<li>Discover trap and column selectivity strategies</li>
<li>Learn how to find the best selectivity for your application</li>
</ul>
<h5>Who should attend?</h5>
<ul>
<li>Any scientist who is interested in improving and expanding their micro and Nano LC performance and would like to increase their knowledge of current trap and column strategies and hardware advancements</li>
</ul>
<blockquote>
Presenter: Roxana Eggleston-Rangel (Senior Applications Scientist, Phenomenex)
</blockquote>
<blockquote>
Presenter: Dora Wells (SelectScience)
</blockquote>

SelectScience: What You Need to Know: Micro and Nano Low Flow LC Essentials

What You Need to Know: Micro and Nano Low Flow LC Essentials

In this webinar we will review micro and nano LC fundamentals, and present a selective approach to trap and column strategies to increase method performance.

<h3>Two-dimensional liquid chromatography (2D-LC): A tutorial focusing on best practice</h3>
In recent years, 2D-LC has evolved from a niche technology to one that is in demand, especially when it comes to the analysis of complex samples, or hidden or co-eluting compounds. Whether you are starting out with 2D-LC or want to refresh your knowledge, this two-part webinar series will give you a comprehensive introduction to the technique, including best practices.
<h4>Session 1: Key concepts of 2D-LC</h4>
Learn about the four modes of 2D-LC: heart-cutting, multiple heart-cutting, selective and full comprehensive, and find out how they relate conceptually. Plus, get advice on how to choose complementary separations for the first and second dimension (RP, IEX, HILIC, etc.), how to optimize cycle time, and how to maximize second dimension sensitivity.
<h5>Who should attend?</h5>
New and experienced 2D-LC users, scientists, and lab managers or technical decision makers in pharma, biopharma, food, energy &amp; chemicals, environmental, material, forensics, and life science research
<blockquote>
Presenter: Dr. Dwight Stoll (Professor of Chemistry, Gustavus Adolphus College. St. Peter, MN, USA)
</blockquote>
<blockquote>
Dr. Dwight Stoll completed his undergraduate work at Minnesota State University, Mankato, in plant biology and biochemistry. Upon graduation in 1999, he took a job in industry as a research technician with ZirChrom Separations, Inc. For his graduate work, he studied with Professor Peter Carr in the Chemistry Department at the University of Minnesota from 2001–2007, where he worked on the development of fast, comprehensive two-dimensional liquid chromatography.
</blockquote>
<blockquote>
In the fall of 2008, Stoll was hired as an Assistant Professor in the Chemistry Department at Gustavus Adolphus College, where he mainly teaches quantitative and instrumental analysis courses. In 2014, he was promoted to Associate Professor, and in 2018, he was promoted to Professor. Currently, he is co-chair of the Chemistry Department. Active research projects in his laboratory touch upon most aspects of multi-dimensional separation methodologies, including optimization strategies, characterization of selectivity in reversed-phase HPLC, instrument development, and applications in biopharmaceutical analysis.
</blockquote>
<blockquote>
Stoll is the author or co-author of more than 62 peer-reviewed publications and three book chapters in the area of separation science, is a named co-inventor on four granted patents, and has instructed numerous short courses in two-dimensional liquid chromatography. In 2011, he was the recipient of LCGC’s Emerging Leader in Chromatography Award. In 2014, he added to The Analytical Scientist’s list of ‘Top 40 Under 40’ analytical scientists. In 2017, he received the Georges Guiochon Faculty Fellowship and an Agilent Technologies Thought Leader Award, which will support research in his laboratory on the development of 2D-LC methodologies for biopharmaceutical analysis.
</blockquote>

Agilent Technologies: Two-dimensional liquid chromatography (2D-LC): A tutorial focusing on best practice

Key concepts of 2D-LC

Learn about the four modes of 2D-LC: heart-cutting, multiple heart-cutting, selective and full comprehensive, and find out how they relate conceptually.

High Performance Liquid Chromatography (HPLC) is an important technique used in Analytical Laboratories for the identification and quantitation of different analytes/compounds in samples. It is the most versatile and dependable chromatographic technique for quality control.
In this seminar, we will cover the complete LC workflow, starting from mobile phase to detection, the different parts involved in this flow path and the different consumables associated with each module of LC system and their importance in acquiring chromatographic results.
<h5>Please join us in this webinar to learn about:</h5>
<ul>
<li>Introduction to LC workflow</li>
<li>Sample flow path and the respective modules involved from Mobile phase to Detection.</li>
<li>Importance of small consumable parts of each module for smooth running of LC system.</li>
<li>How to enhance productivity with new innovative technology products in day-to-day chromatography analysis.</li>
</ul>
<h5>Who Should Attend:</h5>
<ul>
<li>Users of Liquid chromatography in Pharma, Academia &amp; Applied Markets</li>
<li>Analytical scientists, chemists, and lab managers trying to enhance their lab productivity.</li>
</ul>
<blockquote>
Presenter: Dr. Ramaprasad Ganni (Technical Support Specialist, Agilent Technologies)
</blockquote>
<blockquote>
Ramaprasad Ganni has 21 years of experience in the field of Chromatography and Column chemistries. Working with Agilent technologies India, since 2008 in Chemistries and Supplies division. Previously Worked for Elsevier GmbH, and Millipore India Pvt, Ltd. Responsible for Workflow solutions development, providing technical support, and trainings. Experienced in Sample prep, HPLC, GC, Mass spec and GPC Chromatography technologies .
</blockquote>

Agilent Technologies: Mobile Phase to Detector - Complete Workflow

Mobile Phase to Detector - Complete Workflow

In this seminar, we will cover the complete LC workflow, starting from mobile phase to detection

A UV-VIS spectrometer is a powerful and versatile tool for material analysis. It is capable of providing very quick information of sample composition and concentration for quality control in addition to indicating how samples change versus time or temperature.
In this webinar, we will go over what is happening when we take a UV-VIS measurement, discuss how measurement parameters can improve our data collection, and look at applications like concentration and kinetics.
<blockquote>
Presenter: Scott Melis, PhD (Application Scientist, Agilent Technologies, Inc.)
</blockquote>
<blockquote>
Scott Melis has a PHD in Physics from Georgetown University in Washington, DC and started with Agilent in 2021 as a Molecular Spectroscopy Application Scientist. In his graduate work, Scott studied nanoparticle formation and growth for a variety of applications. Projects that he worked with include developing processes to deposit and characterize semiconducting molecular nano-cocrystals for use in optoelectronic devices as well as controlling polymer nanoparticle size through rapid mixing conditions.
</blockquote>

Agilent Technologies: Understanding Your UV-Vis Measurements

Understanding Your UV-Vis Measurements

What is happening when we take a UV-VIS measurement, how measurement parameters can improve our data collection, and look at applications.

This presentation is the first in a series that will introduce you to the most widely used analytical technique in the world – high performance liquid chromatography (HPLC). HPLC is a separation technique that allows you to separate, identify and quantitate the components in a sample. The objective of this session is to provide you with an entry level understanding of this technique. Topics to be discussed include chromatographic theory, the different modes of HPLC, a generic look at HPLC instrumentation, and where HPLC is used.
In later sessions in this series some of the items discussed under these topics (i.e., columns, detectors and troubleshooting) will be discussed in greater detail.
<blockquote>
Presenter: Pamela A. Perrone (Senior Principal LC Technical Education Specialist, Perkinelmer)
</blockquote>
<blockquote>
Pam has been with PerkinElmer for almost 39 years. She had the opportunity to work as a summer intern and was offered full-time employment after completing her Ph.D. in Analytical Chemistry at the University of Rhode Island. Currently Pam is in the role she finds most fulfilling and challenging which is as the global support and trainer for both service and customers for LC and chromatography data handling. To help someone learn and be successful in their job is very rewarding.
</blockquote>
<blockquote>
Pam has a passion for three things (besides LC, of course!): her family, the UConn women’s basketball team and Broadway. She believes there is a Broadway song that fits almost every occasion.
</blockquote>

PerkinElmer: Liquid Chromatography 2022 Learning Series

Introduction to HPLC

This presentation is the first in a series that will introduce you to the most widely used analytical technique in the world – HPLC.

One of the key features in developing a HPLC chromatographic method is the selection HPLC column. It is the focus of this presentation to review the key factors that go into HPLC column selection. These factors include column length, column diameter, particle size of the column packing media, pore size of the column packing media, column type and stationary phase of the packing media.
We explain how column selection can be a match in developing an optimized separation of analyte classes and ultimately how this selection can help ensure a robust separation method. We show how practical compromises can be made in selecting the best column for required separation.
<blockquote>
Presenter: Matthew Przybyciel, Ph.D. (Research and Development Leader, PerkinElmer)
</blockquote>
<blockquote>
Matthew has a PhD in Analytical Chemistry from the University of South Carolina. The focus of his research was the development of new stationary phases for capillary gas chromatography. In the late 1990’s he worked in ES Industries focusing its efforts exclusively on chromatography column products for HPLC and SFC where Matt was Vice President and Technical Director for chromatography products. He has successfully developed and commercialized a number of chromatographic stationary phases and columns for high performance chromatographic separations for HPLC and SFC.
</blockquote>
<blockquote>
In 2021, ES Industries became part of PerkinElmer, Inc. and Matthew now is product development leader at PerkinElmer. As part of his work, he has also published a number of articles on the development and application of new stationary phases for GC, HPLC and SFC.
</blockquote>

Introduction to LC Column Selection

One of the key features in developing a HPLC chromatographic method is the selection HPLC column.

LCMS webinars focusing on Academy/Principles - page 25