News from LabRulezLCMS Library - Week 17, 2026

<p><strong>This study investigates the optimization and functional properties of kombucha enriched with </strong><i><strong>Prunus spinosa</strong></i><strong> juice and bee products (propolis, pollen, and bee bread). Advanced analytical techniques (HPLC–DAD, LC–MS/MS) revealed enhanced phenolic, amino acid, and antioxidant profiles, with bee bread enrichment showing the highest overall bioactive content and sensory acceptance.</strong></p><p>Propolis-enriched kombucha exhibited the strongest in vitro antidiabetic activity, while pollen enrichment provided the highest amino acid levels. Despite partial losses during digestion, significant retention of bioactives suggests potential postdigestive benefits. These findings highlight the potential of enriched kombucha as a functional beverage with nutraceutical value.</p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00008"><strong>From Chemistry to Functionality: HPLC–DAD/LC–MS/MS Characterization of Bee Product-Enriched Prunus spinosa L. Kombucha with In Vitro Antidiabetic Activity and Bioaccessibility</strong></a></h4><p>ACS Omega 2026, 11, 8, 14048–14063</p><p><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsomega.6c00008">https://doi.org/10.1021/acsomega.6c00008</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>Kombucha tea, a traditionally fermented beverage, is believed to have originated as early as 200–300 BC. In recent decades, it has attracted growing global interest due to its recognized health-promoting properties and its deep cultural roots. <a href="javascript:void(0);">(1,2)</a> Sweetened tea derived from <i>Camellia sinensis</i> is transformed into kombucha through fermentation carried out by a SCOBY (Symbiotic Culture of Bacteria and Yeast), a mixed culture of yeasts and bacteria that forms a cellulose-rich biofilm throughout the process. <a href="javascript:void(0);">(3)</a> SCOBY represents a symbiotic consortium of microorganisms that comprises various yeast genera, including <i>Saccharomyces</i>, <i>Schizosaccharomyces</i>, <i>Zygosaccharomyces</i>, <i>Brettanomyces/Dekkera</i>, and <i>Pichia</i>, as well as both acetic and lactic acid bacteria. <a href="javascript:void(0);">(2)</a> During fermentation, the sugars formed contribute a mild sweetness to the tea, whereas the organic acids synthesized by acetic acid bacteria are responsible for its distinctive sour flavor. <a href="javascript:void(0);">(4)</a> Kombucha tea is traditionally brewed using black tea; however, alternative versions can also be produced with other types of tea. <a href="javascript:void(0);">(5)</a> It has been reported that herbal infusions and fruit juices can also be used for fermentation with the kombucha culture, and due to their chemical composition, these substrates have been shown to serve as promising alternatives. <a href="javascript:void(0);">(6)</a> The antioxidant potential of kombucha tea is primarily linked to its abundant phenolics, with flavonoids playing a significant contributory role. <a href="javascript:void(0);">(7)</a> A high anthocyanin content tends to enhance the antioxidant capacity. <a href="javascript:void(0);">(8)</a> The antioxidant activity of kombucha tea can be improved by incorporating anthocyanin-rich fruits. Accordingly, the use of wild fruits with high anthocyanin content─such as <i>Prunus spinosa</i> L. (blackthorn), <i>Prunus laurocerasus</i> (cherry laurel), and <i>Rubus idaeus</i> (red raspberry)─has been considered a practical way to improve the beverage’s nutritional, functional and sensorial properties. <a href="javascript:void(0);">(9)</a></p><p>Propolis is a naturally occurring material that honeybees gather from the resins of different trees and the buds of various plants. <a href="javascript:void(0);">(28,29)</a> Propolis is a chemically complex matrix comprising more than 300 identified components, including flavonoids, phenolic acids, and their ester derivatives, and is associated with a wide range of biological activities such as antiviral, antioxidant, antimicrobial, anti-inflammatory, antitumor, hepatoprotective, and immunomodulatory effects. <a href="javascript:void(0);">(30−34)</a> Minerals and vitamins are also present in high concentrations in propolis. <a href="javascript:void(0);">(28)</a> Bee pollen is formed when bees agglutinate pollen grains using their saliva, nectar, or honey and store them in granular form in their honeycomb cells. The key role of bee pollen is to ensure the colony’s survival by providing the necessary nutrients, thereby supporting development and maintenance processes. <a href="javascript:void(0);">(35)</a> Bee pollen is a natural product rich in bioactive compounds. More than 250 bioactive compounds have been identified in bee pollen derived from different plant species. These basic chemical components include lipids, amino acids, proteins, carbohydrates, fatty acids, phenolic compounds, carotenoids, flavonoids, vitamins, and bioelements. <a href="javascript:void(0);">(36)</a> Exhibits antihyperglycemic, anti-inflammatory, hepatoprotective, nephroprotective, and anticancer properties thanks to its rich and versatile component mixture. <a href="javascript:void(0);">(37,38)</a> Many of these have demonstrated antioxidant, antimicrobial, antidiabetic. They have shown promising results in the treatment of diabetes and obesity, and their potential as natural therapeutic agents has been evaluated. They have been found to increase insulin sensitivity, reduce oxidative stress, regulate appetite, adjust obesity-related hormone levels, and strengthen antioxidant defense systems. <a href="javascript:void(0);">(39,40)</a> The antimicrobial effect of bee bread has been extensively studied. Bakour et al. found that different bacterial species and fungi are sensitive to the hydromethanolic bee bread extract. <a href="javascript:void(0);">(41)</a> A vital bee byproduct, bee bread is formed after the pollen gathered by bees is enriched with nectar and bee saliva, leading to an ensuing lactic acid fermentation process inside the hives. <a href="javascript:void(0);">(42)</a> Bee bread is high in nutrition, with significant carbohydrates, proteins and lipids. It is a rich source of vitamins, phenolic compounds, minerals and essential amino acids, and exhibits multiple chemical activities. <a href="javascript:void(0);">(43,44)</a></p><p>The Response Surface Method (RSM) is one of the statistical optimization approaches. This method uses empirical modeling techniques to identify relationships between independent variables and the system response using experimental data. It also allows for determining optimal conditions for achieving the desired outcome by evaluating the effects of process variables on the system response. <a href="javascript:void(0);">(45−47)</a></p><p>Previous studies have not extensively investigated the sensory parameters (color, taste, smell, and general acceptance) of kombucha tea enriched with <i>P. spinosa</i> L. and bee products using RSM. The primary aim of this study was to optimize the sensory properties of kombucha formulations enriched with <i>P. spinosa</i> L. and bee-derived ingredients (bee bread, propolis, and pollen) using RSM to determine the most acceptable formulation. While many studies have examined kombucha enriched with various fruit and plant infusions, none have specifically investigated the impact of <i>P. spinosa</i> L. and bee products on kombucha’s sensory and bioactive properties. The study also evaluated the total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity (DPPH•), total anthocyanin content (TAC), phenolic profile (HPLC), microbiological quality, and levels of organic acids amino acids and in vitro bioaccessibility in the developed samples, as well as their antidiabetic activity. RSM-based optimization offers a new methodological approach. It is expected to yield valuable data. This data will improve the functional and disease-fighting properties of kombucha.</p><h4><strong>2. Materials and Methods</strong></h4><h5><strong>2.9. Analysis of Phenolic Compounds (HPLC)</strong></h5><p>Chromatographic separation was carried out on an ACE Genix C18 column (250 × 4.6 mm, 5 μm; Agilent), following the procedure reported by Portu et al. (2017). <a href="javascript:void(0);">(56)</a> Phenolics were characterized using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1260+LC"><strong>Agilent 1260 HPLC</strong></a>–DAD system operated under gradient conditions at 30 °C with a flow rate of 0.80 mL/min. The mobile phase comprised solvent A (water supplemented with 0.1% phosphoric acid) and solvent B, applied according to a predefined gradient program. Samples were injected at a volume of 10 μL, and chromatograms were recorded at 280, 320, and 360 nm. Quantitative results were reported as μg/mL.</p><h5><strong>2.10. Analysis of Organic Acids (HPLC)</strong></h5><p>HPLC was used to determine organic acid profiles, following a method adapted with minor modifications from Coelho et al. <a href="javascript:void(0);">(57)</a> Organic acids were analyzed using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1260+LC"><strong>Agilent 1260 Infinity LC HPLC system</strong></a> fitted with refractive index (RID) and diode array (DAD) detectors. Samples were filtered through a 0.45 μm syringe filter, and a 20 μL aliquot was injected into an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1260+LC"><strong>Agilent Hi-Plex H ion-exchange column</strong></a> (300 × 7.7 mm). Chromatographic separation was carried out using 10.0 mM H<sub>2</sub>SO<sub>4</sub> as the mobile phase at a flow rate of 0.6 mL/min, with the column temperature set to 65 °C and the RID cell maintained at 35 °C. The total run time was 20 min. DAD additionally monitored tartaric, pyruvic, and acetic acids at 210 nm. Quantitative determination was carried out using external calibration standards, and the results were reported in g/L.</p><h5><strong>2.11. Determination of Free Amino Acids (LC–MS)</strong></h5><p>Amino acid composition was determined using a method adapted with minor modifications from the procedure described by Bilgin et al. (2019). <a href="javascript:void(0);">(58)</a> A gradient elution scheme involving mobile phases A and B was applied to obtain chromatographic separation at a flow rate of 0.7 mL/min, yielding a total run duration of 7.5 min. Tandem mass spectrometric analyses were performed on an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10"><strong>Agilent 6460 triple quadrupole LC–MS instrument</strong></a> (Agilent Technologies, Waldbronn, Germany) fitted with an electrospray ionization (ESI) interface. The mass spectrometer was operated with a gas temperature of 150 °C, a gas flow rate of 10 L/min, a nebulizer pressure of 40 psi, and a capillary voltage of +2000 V. All analyses were carried out in triplicate, and the results were reported as mg/100 mL.</p><h4><strong>3. Results and Discussion</strong></h4><h5><strong>3.4. Phenolic Compounds (HPLC)</strong></h5><p>As presented in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00008#tbl1">Table 1</a>, the total quantified phenolic content differed significantly among the treatments (<i>p</i> &lt; 0.05). The control sample (BKT) exhibited the lowest phenolic concentration (106.35 ± 2.25 μg/mL), whereas pollen supplementation (PL-BKT) resulted in a substantial increase, reaching 159.23 ± 4.82 μg/mL. Further enhancement of the phenolic pool was achieved through propolis (PB-BKT: 178.66 ± 5.04 μg/mL) and bee bread (BB-BKT: 186.15 ± 0.97 μg/mL) supplementation. No statistically significant difference was observed between PB-BKT and BB-BKT in total quantified phenolics; however, both treatments exhibited significantly higher values than PL-BKT and the control, indicating a more pronounced phenolic enrichment.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_8_14048_14063_Table_1_Changes_in_Total_Phenolic_Compounds_of_Kombucha_Samples_Subjected_to_BKT_PB_BKT_PL_BKT_and_BB_BKT_5a50c7851f.jpg" alt="ACS Omega 2026, 11, 8, 14048–14063: Table 1. Changes in Total Phenolic Compounds of Kombucha Samples Subjected to BKT, PB-BKT, PL-BKT and BB-BKT. Data are presented as mean ± standard deviation (n = 3). Values denoted by different letters within the same row indicate statistically significant differences (p &lt; 0.05). BKT: Blackthorn kombucha tea; PB-BKT: Propolis blackthorn kombucha tea; PL-BKT: Pollen blackthorn kombucha tea; BB-BKT: Bee bread blackthorn kombucha tea. n.d.: could not be detected." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_8_14048_14063_Table_1_Changes_in_Total_Phenolic_Compounds_of_Kombucha_Samples_Subjected_to_BKT_PB_BKT_PL_BKT_and_BB_BKT_5a50c7851f.jpg 159w," sizes="100vw" width="1054" height="1034"></p><p>The compound-level distribution suggests that the observed elevation in total phenolics is driven by selective, matrix-dependent enrichment. Bee bread is a functional food thanks to its bioactive compounds and chemical diversity. <a href="javascript:void(0);">(72)</a> The marked increase in chlorogenic acid concentration in BB-BKT, to 79.04 ± 4.66 μg/mL, was a key contributor to the elevated total phenolic content in this group. In contrast, chlorogenic acid remained undetectable or present at negligible levels in the other treatments. Likewise, the enrichment of thermosonicated kiwi juice with bee bread resulted in a statistically significant elevation in chlorogenic acid levels. <a href="javascript:void(0);">(68)</a> The highest catechin hydrate concentration was observed in PL-BKT (121.2 ± 3.36 μg/mL), indicating that pollen supplementation preferentially enhances the flavan-3-ol fraction. In contrast, the markedly elevated levels of quercetin (47.32 ± 2.07 μg/mL) and naringenin (17.59 ± 0.38 μg/mL) detected in PB-BKT support the flavonoid-enriching effect of propolis (<i>p</i> &lt; 0.05). In another study, propolis supplementation of thermosonicated yellow cherry juice was found to increase quercetin content significantly. Additionally, the exclusive emergence of chrysin in the enriched samples, particularly its pronounced accumulation in BB-BKT (66.42 ± 1.84 μg/mL), suggests that bee bread supplementation can substantially reshape the specific phenolic signature of the matrix. This interpretation is further supported by the highest rosmarinic acid level observed in BB-BKT (3.05 ± 0.24 μg/mL). Conversely, the absence of caffeic acid and the flavones fraction across all treatments (n.d.) indicates that the applied interventions exerted limited effects on specific phenolic subclasses.</p><h5><strong>3.5. Free Amino Acids (LC–MS)</strong></h5><p>The PL-BKT sample, which was supplemented with pollen, exhibited the highest free amino acid content (see <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00008#tbl2">Table 2</a>). This outcome differed significantly from all other groups (<i>p</i> &lt; 0.05). Significant increases were observed in proline (47.37 mg/100 mL), leucine (4.10 mg/100 mL), and lysine (4.54 mg/100 mL), indicating that pollen, due to its high protein content, increases amino acid release during fermentation. In contrast, amino acid levels were lower in the sample supplemented with propolis alone (PB-BKT), and some amino acids (e.g., glutamic acid, histidine, threonine) were not statistically different from the BKT sample (<i>p</i> &gt; 0.05).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_8_14048_14063_Table_2_Effects_of_BKT_PB_BKT_PL_BKT_and_BB_BKT_on_Free_Amino_Acids_mg_100_m_L_f377c7c7e0.jpg" alt="ACS Omega 2026, 11, 8, 14048–14063: Table 2. Effects of BKT, PB-BKT, PL-BKT and BB-BKT on Free Amino Acids (mg/100 mL). The mean value, along with the standard deviation, is given for the data (n = 3). Values indicated by different letters within the same row differ significantly (p &lt; 0.05). BKT: Blackthorn kombucha tea; PB-BKT: Propolis blackthorn kombucha tea; PL-BKT: Pollen blackthorn kombucha tea; BB-BKT: Bee bread blackthorn kombucha tea." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_8_14048_14063_Table_2_Effects_of_BKT_PB_BKT_PL_BKT_and_BB_BKT_on_Free_Amino_Acids_mg_100_m_L_f377c7c7e0.jpg 159w," sizes="100vw" width="1055" height="1033"></p><h4><strong>4. Conclusions</strong></h4><p>This study demonstrated that kombucha enriched with <i>P. spinosa</i> L. juice and bee products (propolis, pollen, and bee bread) provides a robust model for functional beverage design through RSM-based optimization and advanced analytical characterization (HPLC–DAD, LC–MS/MS). From a sensory perspective, the highest overall acceptability was observed in the bee bread-enriched sample (8.32), which also exhibited the strongest antioxidant response (TPC 257.10 mg GAE/100 mL; TFC 36.16 mg CE/100 mL; DPPH• 57.05 μmol TE/mL). HPLC–DAD data revealed that total phenolics increased to 159.23–186.15 μg/mL following enrichment, and that marker compounds, such as chlorogenic acid and catechin derivatives, varied depending on the formulation. LC–MS/MS supported an increase in free amino acids (particularly proline) with pollen supplementation. Propolis addition highlighted the antidiabetic potential, yielding the highest enzyme inhibition (α-glucosidase: 40.7%; α-amylase: 42.02%). Although a partial reduction in bioactives was observed during in vitro digestion, the preservation of phenolic/flavonoid recovery within approximately the 21–30% range indicates that postdigestive activity may be sustained. Future studies are recommended to expand biomarkers using targeted/untargeted LC–HRMS-based metabolomic approaches, to correlate SCOBY microbiota (16S/ITS) with chemical profiles, and to evaluate shelf life and packaging stability (phenolic degradation kinetics, volatile profile), as well as in vivo/preclinical validation (glycemic response, bioaccessibility). A limitation of this work is that batch-specific botanical origin (palynological) identification of the commercial bee products was not performed; therefore, compositional variability due to botanical source cannot be fully excluded.</p></div>

From Chemistry to Functionality - HPLC–DAD/LC–MS/MS Characterization of Bee Product-Enriched Prunus spinosa L. Kombucha with In Vitro Antidiabetic Activity and Bioaccessibility

<p><strong>In the week of April 27, 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. Separation Science: Analytical Excellence Week 2026: Part 1- Understanding Pharmacopoeia and ACS Compliance</strong></h5><ul><li>Tu, 28.4.2026 09:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6130">Registration</a></li></ul><p>Discover the critical role of compliant reagents in your lab and learn how to ensure that your analyses meet regulatory standards.</p><h5><strong>2. Agilent Technologies: PFAS &amp; Our Planet: Environmental Monitoring</strong></h5><ul><li>Tu, 28.4.2026 11:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6140">Registration</a></li></ul><p>Explore evolving PFAS regulations and practical LC-MS/MS workflows for monitoring PFAS in water, soil, and air, including contamination control, sample prep, and reporting strategies.</p><h5><strong>3. Restek Corporation: Air Sampling Media Selection and Analysis</strong></h5><ul><li>Tu, 28.4.2026 14:30 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6160">Registration</a></li></ul><p>This presentation will give an overview of the selection of air sampling media, covering both whole air and sorbent sampling techniques.</p><h5><strong>4. Tosoh Bioscience: Tosoh Tuesday - What is MALS &amp; When is it Useful?</strong></h5><ul><li>Tu, 28.4.2026 15:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6145">Registration</a></li></ul><p>Join Tosoh Tuesday to explore MALS fundamentals, practical implementation, and real-world SEC-MALS applications for polymer and biologics characterization.</p><h5><strong>5. Technology Networks: Mass Spectrometry in Drug Discovery: Covalent and Non-Covalent Screening Applications</strong></h5><ul><li>Tu, 28.4.2026 17:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6114">Registration</a></li></ul><p>Identifying small-molecule binders for complex biological targets remains a major challenge in early drug discovery.</p><h5><strong>6. Thermo Fisher Scientific: From Sample to Structure: Crosslinking MS Made Practical - Part 1</strong></h5><ul><li>Tu, 28.4.2026 17:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6115">Registration</a></li></ul><p>Crosslinking mass spectrometry (XL-MS) is transforming how researchers map protein–protein and protein–nucleic acid interactions.</p><h5><strong>7. SelectScience: Why researchers turn to reversed phase chromatography when purifying GLP-1</strong></h5><ul><li>Tu, 28.4.2026 17:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6117">Registration</a></li></ul><p>Purifying synthetic peptides is a critical step in developing advanced biotherapeutics like GLP-1 receptor agonists.</p><h5><strong>8. Phenomenex: Fundamentals First Bio Series – SEC for Biologics</strong></h5><ul><li>We, 29.4.2026 10:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6149">Registration</a></li></ul><p>Master SEC fundamentals for biopharma—covering principles, column selection, and troubleshooting with real-world applications in mAbs, AAVs, and nucleic acids.</p><h5><strong>9. SCIEX: Extending the power of LC-MS/MS: advanced workflows with QTRAP®</strong></h5><ul><li>We, 29.4.2026 10:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6147">Registration</a></li></ul><p>Discover how QTRAP® workflows enhance LC-MS/MS performance with MS³ selectivity, improving sensitivity, reducing interference, and enabling confident analysis in complex matrices.</p><h5><strong>10. Metrohm: The Basics of Titration explained</strong></h5><ul><li>We, 29.4.2026 15:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6129">Registration</a></li></ul><p>In this webinar, Robert Tobler, Senior Application Specialist Titration at Metrohm International Headquarters, will give you an introduction to the basics of titration.</p><h5><strong>11. Separation Science: Analytical Excellence Week 2026: Part 2 - Benefits of PFAS-Tested Consumables</strong></h5><ul><li>Th, 30.4.2026 09:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6131">Registration</a></li></ul><p>Gain insights into the importance of PFAS-tested additives and solvents for reliable analysis and their impact on the quality of your results.</p><h5><strong>12. Evosep Biosystems: Next-Gen HCP Analysis in Biopharma</strong></h5><ul><li>Th, 30.4.2026 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6151">Registration</a></li></ul><p>Discover how LC-MS is advancing biopharma workflows through scalable, high-throughput proteomics.</p><h5><strong>13. Bruker: Bridging Molecules and Genes: Integrating MALDI Imaging and Spatial Transcriptomics on a Single Tissue Section</strong></h5><ul><li>Th, 30.4.2026 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6132">Registration</a></li></ul><p>Explore how integrating MALDI Imaging with spatial transcriptomics reveals tissue heterogeneity, molecular gradients, and disease-relevant patterns through advanced multimodal workflows.</p><h5><strong>14. Thermo Fisher Scientific: From Sample to Structure: Crosslinking MS Made Practical - Part 2</strong></h5><ul><li>Th, 30.4.2026 17:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6116">Registration</a></li></ul><p>Crosslinking mass spectrometry (XL-MS) is transforming how researchers map protein–protein and protein–nucleic acid interactions.</p>

Webinars LabRulezLCMS Week 18/2026

News from LabRulezLCMS Library - Week 18, 2026

<p><strong>This study investigates the impact of emitter positioning on signal intensity in nanoflow electrospray ionization mass spectrometry. By systematically varying emitter position in three dimensions, the authors demonstrate that signal robustness improves at greater distances along the z-axis, with consistent performance maintained within 1–2 mm of the optimal position.</strong></p><p>Signal behavior was consistent across the m/z range, indicating that precise repositioning for different analytes is unnecessary. These findings provide practical guidance for improving robustness and reproducibility in nanoflow LC–MS/MS proteomics workflows.</p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/jasms.5c00441"><strong>Regarding Emitter Positioning for Nanoflow Electrospray Ionization with a High-Capacity Inlet Capillary</strong></a></h4><p>Noah M. Lancaster, Scott T. Quarmby, Katherine A. Overmyer, and Joshua J. Coon*</p><p>J. Am. Soc. Mass Spectrom. 2026, 37, 3, 754–759</p><p><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/jasms.5c00441">https://doi.org/10.1021/jasms.5c00441</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>Owing to its high sensitivity, nanoflow electrospray liquid chromatography coupled with tandem mass spectrometry (nESI-LC-MS/MS) is widely used for protein sequence analysis. <a href="javascript:void(0);">(1,2)</a> Whereas high-flow electrospray sources typically have relatively fixed emitter alignments and are more robust to changes in emitter positioning, nESI sources frequently require manual alignment in three dimensions. <a href="javascript:void(0);">(3,4)</a> Since the number of ions reaching the MS system can vary depending on the location of the nESI emitter, positioning is a critical aspect of achieving robust and reproducible results.</p><p>Multiple reports illustrate the relationship between emitter positioning and electrospray signal intensity; <a href="javascript:void(0);">(2,4−10)</a> however, these studies typically examine a single dimension or evaluate emitters and flow regimes not typical of shotgun proteomic workflows. To understand these relationships, we investigate here the effect of emitter positioning on signal intensity when using fused-silica capillaries with integrated emitters at a flow rate of 300 nL/min, a common choice across the field. <a href="javascript:void(0);">(11−17)</a> Experiments were conducted using an Orbitrap Ascend mass spectrometer, a notable example of instruments commonly used in proteome analysis, <a href="javascript:void(0);">(18)</a> which features a nanoflow source and asymmetric inlet capillary (high-capacity transfer tube, HCTT). This work represents, to our knowledge, the first report in the literature characterizing how emitter positioning impacts signal intensity for nanoflow electrospray ionization into an inlet capillary without radial symmetry.</p><p>Since most nESI emitters are manually positioned, understanding the precision in positioning that is required could assist in reducing batch-to-batch variation that is common in larger proteomic experiments. Further, recent efforts to improve the throughput of proteome analysis feature the use of multiple columns with multiple emitters aligned with the source at the same time. <a href="javascript:void(0);">(19−23)</a> By performing measurements in all three dimensions, this report provides insight for implementing such a multiemitter setup.</p><h4><strong>Experimental Section</strong></h4><p>Emitter positioning measurements were performed by infusing BSA peptides at 300 nL/min into an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/748"><strong>Orbitrap Ascend mass spectrometer</strong></a> (Thermo Scientific) using a fused silica capillary (360 μm O.D, 75 μm I.D.) with integrated emitter (∼10 μm opening). <a href="javascript:void(0);">(24)</a> The emitter was aligned to the inlet using the source camera and the <i>x</i>/<i>y</i>/<i>z</i> micrometer on a NanoSpray Flex source (Thermo Scientific). The origin of the coordinate system used here was defined as the center of the inlet capillary opening. In particular, the <i>z</i> = 0 plane is orthogonal to the opening of the inlet capillary, and the <i>x</i> = 0 and <i>y</i> = 0 planes are parallel to the inlet capillary axis. Top- and side-view images of the starting position are shown in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/jasms.5c00441/suppl_file/js5c00441_si_001.pdf">Figure S2</a>. Code used for data analysis is available at <a target="_blank" rel="noopener noreferrer" href="https://github.com/coongroup/EmitterPositioning">https://github.com/coongroup/EmitterPositioning</a>. Raw data is available at MassIVE accession number MSV000100165. Additional experimental and data analysis details are described in the <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/jasms.5c00441/suppl_file/js5c00441_si_001.pdf">Supporting Information</a>.</p><h4><strong>Results and Discussion</strong></h4><p>We characterized signal intensity dependence on emitter position for nanoflow electrospray by infusing a mixture of peptides generated via trypsin digestion of purified BSA in an Orbitrap-quadrupole linear ion trap hybrid mass spectrometer (Orbitrap Ascend Tribrid). To ensure LC–MS applicability, we used an emitter size (∼10 μm orifice) and flow rate (300 nL/min) identical to those typical of capillary LC–MS. <a href="javascript:void(0);">(11−17)</a> The resulting MS1 analysis produced numerous ions across the <i>m</i>/<i>z</i> range (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/jasms.5c00441/suppl_file/js5c00441_si_001.pdf">Figure S3</a>). We selected two of these ions, a doubly protonated species (LGEYGFQNALIVR at <i>m</i>/<i>z</i> 740.4) and a triply protonated species (RHPEYAVSVLLR at <i>m</i>/<i>z</i> 480.6), for their strong signal intensity and stability over the time range of the experiments here (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/jasms.5c00441/suppl_file/js5c00441_si_001.pdf">Figures S4 and S5</a>). With the above setup, we tracked those <i>m</i>/<i>z</i> peaks as a function of emitter position in three dimensions and recorded intensity distributions (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/jasms.5c00441#fig1">Figure 1</a>). <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/jasms.5c00441#fig1">Figure 1</a>C presents the ion signal across x-positions, where the <i>x</i> = 0 plane is aligned to the central axis of the inlet capillary and the dashed lines depict the MS inlet opening width in this dimension (0.6 mm, <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/jasms.5c00441/suppl_file/js5c00441_si_001.pdf">Figure S6</a>). <a href="javascript:void(0);">(25,26)</a> Strikingly, we observe that the signal is relatively stable and high across two millimeters of the <i>x</i>-dimension. Further, greater than 50% of the ion signal is retained at distances up to ∼five times the width of the inlet (i.e., full width half max (fwhm)). <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/jasms.5c00441#fig1">Figure 1</a>D presents the same concept but in the y-dimension, where good signal stability is also observed across a similar length of two millimeters; however, the larger inlet opening in the y-dimension (1.6 mm) does not appear to proportionally impact the width of the intensity distribution. The ion plume is roughly symmetrical in both <i>x</i>- and <i>y</i>-dimensions; <a href="javascript:void(0);">(8)</a> therefore, it is expected that the smaller inlet width in the <i>x</i>-dimension would have a higher probability of being in the high density region of the plume than in the <i>y</i>-dimension. Proportional to inlet opening, the <i>x</i> position of the emitter would consequently have less of an effect on the intensity than the <i>y</i> position. We note the intensity distributions appear to be slightly off-center; however, we find the centroids of the distributions (−0.1 and 0.3 mm, <i>x</i>- and <i>y</i>-respectively, <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/jasms.5c00441/suppl_file/js5c00441_si_001.pdf">Figure S7</a>) were within the tolerance of the micrometer. <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/jasms.5c00441#fig1">Figure 1</a>E presents the ion intensities as a function of emitter position in the <i>z</i>-dimension. Impressively, signal continues to be observed up to ∼7 mm removed from the inlet at levels close to 50% of the highest. Not surprisingly, the highest signal is observed at the closest position, but, as noted above, that signal gradually declines with increasing distance. The relative intensities of these two peptide ions remain fairly constant at all observed z positions (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/jasms.5c00441/suppl_file/js5c00441_si_001.pdf">Figure S8</a>). Overall, identical trends were observed for both peptides over all dimensions. As these peptides are fairly high signal intensity, we wanted to confirm that the trends reported here were not biased by signal saturation, so we extracted signal intensities for low intensity features and compared the <i>x</i>, <i>y</i>, and <i>z</i> intensity distributions (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/jasms.5c00441/suppl_file/js5c00441_si_001.pdf">Figure S9</a>). This analysis confirms that these two peptides are representative of global trends for our tryptic peptide sample.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Am_Soc_Mass_Spectrom_2026_37_3_754_759_Figure_1_1_D_Positioning_Experiments_A_The_xyz_coordinate_system_used_for_this_study_c971dc3d2f.jpg" alt="J. Am. Soc. Mass Spectrom. 2026, 37, 3, 754–759: Figure 1. 1D Positioning Experiments. (A) The x/y/z coordinate system used for this study. (B) An x/y perspective of the inlet with coordinate definitions. (C) Intensity dependence on x-position for two selected peptides (at y = 0 and z = −0.9). Dotted lines indicate the estimated position of the inlet capillary edges. (D) Intensity dependence on y-position for two selected peptides (at x = 0 and z = −0.9). Dotted lines indicate the estimated position of the inlet capillary edges. (E) Intensity dependence on z-position for two selected peptides (at x = 0 and y = 0)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Am_Soc_Mass_Spectrom_2026_37_3_754_759_Figure_1_1_D_Positioning_Experiments_A_The_xyz_coordinate_system_used_for_this_study_c971dc3d2f.jpg 94w," sizes="100vw" width="996" height="1652"></p><h4><strong>Conclusions</strong></h4><p>We report the dependence of peptide signal intensity on emitter positioning for nanoflow electrospray. Specifically, within 1–2 mm in any dimension, one can achieve reasonably consistent and robust signal. Distinct intensity profiles we observed here for the <i>x</i>- and <i>y</i>-dimensions likely arise from the asymmetric shape of the ion capillary. We demonstrate improved robustness of signal intensity to <i>x</i>/<i>y</i> variation at increasing <i>z</i> distances, an observation that will be helpful for positioning multiple emitters, for example. <a href="javascript:void(0);">(19−23)</a></p><p>We provide evidence that the effect of the emitter position on signal intensity does not strongly depend on the analyte for bottom-up proteomics analysis. This report provides insight into the role of emitter positioning on signal intensity for bottom-up proteomics and represents the first characterization of the effect of emitter position on electrospray signal intensity on an instrument with an inlet capillary lacking radial symmetry. Future areas of interest would be an examination of how trends vary across different flow rates or emitter types, as well as assessing how the exact intensity profiles vary across instruments with differences in their atmospheric interface.</p></div>

Regarding Emitter Positioning for Nanoflow Electrospray Ionization with a High-Capacity Inlet Capillary

<p><strong>In the week of May 4, 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. Merck: Recent Advances in Chromatographic Approaches in Characterizing Antibody-Drug Conjugates</strong></h5><ul><li>Tu, 5.5.2026 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6146">Registration</a></li></ul><p>Explore advanced LC-MS and HIC strategies for comprehensive ADC characterization, including impurity profiling, DAR analysis, and MS-compatible workflows.</p><h5><strong>2. Waters Corporation: Empower Tips: Ask Me Anything About Updates in Empower Software to Advance Your Compliance</strong></h5><ul><li>Tu, 5.5.2026 16:00 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5975">Registration</a></li></ul><p>Discover the latest Empower CDS updates and learn how new features enhance compliance, security, USP calculations, and risk reduction in modern chromatography labs.</p><h5><strong>3. Metrohm: Electrochemiluminescence: The technique that should be on your radar for sensor development</strong></h5><ul><li>We, 6.5.2026 09:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6158">Registration</a></li></ul><p>In this webinar, we will explore what makes ECL a unique technique for sensor development providing key hints and examples.</p><h5><strong>4. Phenomenex: How To Start Up and Shut Down LC and LC-MS Instruments</strong></h5><ul><li>We, 6.5.2026 10:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6180">Registration</a></li></ul><p>Learn practical LC and LC-MS startup and shutdown procedures, column care, and system checks in this webinar. Gain system-agnostic best practices to protect instruments and maximize column lifetime.</p><h5><strong>5. Thermo Fisher Scientific: Integrated LC-MS CDS workflow for automated ADC and DAR analysis</strong></h5><ul><li>We, 6.5.2026 11:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6135">Registration</a></li></ul><p>In this webinar, we’ll present an automated, high-throughput intact ADC workflow using LC-MS under native conditions on the Thermo Scientific Orbitrap Exploris 240 mass spectrometer.</p><h5><strong>6. Waters Corporation: Improving Sensitivity and Throughput in Early Drug Development with Inert LC Technologies</strong></h5><ul><li>We, 6.5.2026 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6152">Registration</a></li></ul><p>Discover how Waters MaxPeak Premier Columns improve LC method development with better sensitivity, peak shape, and robustness for acidic analytes in early drug research.</p><h5><strong>7. Thermo Fisher Scientific: Keeping Up with Evolving Regulatory Requirements: Addressing Analytical Challenges for Known and Unknown Nitrosamines</strong></h5><ul><li>Th, 7.5.2026 05:30 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6166">Registration</a></li></ul><p>Join our webinar to see how advanced LC-MS tools can help you confidently quantify known nitrosamines and uncover unknown impurities.</p><h5><strong>8. Waters Corporation: Transform Your Daily Lab Operations with Automation - Proteomics Workflows</strong></h5><ul><li>Th, 7.5.2026 11:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6165">Registration</a></li></ul><p>Explore how automated liquid handling improves reproducibility, scalability, and efficiency in proteomics workflows across digestion, desalting, and peptide analysis.</p><h5><strong>9. SCIEX: Assessing biotherapeutic stability through forced degradation using intact and subunit icIEF-UV/MS workflows</strong></h5><ul><li>Th, 7.5.2026 11:30 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6181">Registration</a></li></ul><p>Explore icIEF-UV/MS workflows for biotherapeutic stability studies. Learn how to identify charge variants and degradation pathways in monoclonal antibodies with enhanced analytical confidence.</p><h5><strong>10. Metrohm: Environmental waters: A new generation of high-capacity IC columns makes analysis more efficient</strong></h5><ul><li>Th, 7.5.2026 15:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6155">Registration</a></li></ul><p>Learn how next-generation high-capacity IC columns enable compliant, robust analysis of environmental waters with improved detection limits and streamlined workflows.</p><h5><strong>11. Waters Corporation: Add a New Dimension - MS Imaging for Environmental Analysis</strong></h5><ul><li>Th, 7.5.2026 15:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5927">Registration</a></li></ul><p>Learn about MS imaging and discover key benefits of Desorption Electro Spray Ionisation (DESI) for environmental analysis.</p><h5><strong>12. US HUPO: Raising the Bar: Quality Assurance and Data Integrity in MALDI MSI</strong></h5><ul><li>Th, 7.5.2026 19:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6133">Registration</a></li></ul><p>Join US HUPO experts to explore quality assurance and data integrity in MALDI MSI, covering best practices for reliable imaging workflows and reproducible spatial omics data.</p><h5><strong>13. Agilent Technologies: Impurity Characterization of GLP-1 Receptor Agonists via RPLC and HILIC</strong></h5><ul><li>Th, 7.5.2026 19:00 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5998">Registration</a></li></ul><p>Explore RPLC and HILIC strategies for GLP-1 impurity profiling. Learn method development tips to optimize separation, resolution, and regulatory-ready characterization.</p><h5><strong>14. Agilent Technologies: Accelerating LC/MS Impurity Screening Followed by ECD-Based Sequence Analysis for GLP-1 Analogs</strong></h5><ul><li>Fr, 8.5.2026 11:00 CET</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5945">Registration</a></li></ul><p>Episode 4 of Peptide 360 presents a staged LC/MS workflow for rapid GLP-1 impurity screening followed by ECD-based sequence analysis for precise structural confirmation and faster decisions.</p>

Webinars LabRulezLCMS Week 19/2026

News from LabRulezLCMS Library - Week 19, 2026

<p><strong>This study investigates the pathogenesis of neurosyphilis through cerebrospinal fluid proteomics using LC–MS/MS and ELISA validation. Comparative analysis identified 234 differentially expressed proteins, with major alterations linked to lysosomal dysfunction, axon guidance, and neurodegeneration pathways.</strong></p><p>Key lysosomal proteins, including PSAP and DNASE2, together with the axonal protein EPHA4, were significantly downregulated in neurosyphilis patients. These proteins showed potential as biomarkers for neurodegeneration and may provide new insights into disease mechanisms, diagnostics, and therapeutic targets.</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.jproteome.5c00689"><strong>Cerebrospinal Fluid Proteome Reveals Dysregulation of Lysosomal and Axonal Proteins in Neurosyphilis</strong></a></h4><p>Huayang Tang*, Junjie Hu, Yifan Wu, Jinping Gao, Wenjun Wang, Xiaodong Zheng, Ran Zhang, Bo Liang, Fusheng Zhou, Ze Guo*</p><p>J. Proteome Res. 2026, 25, 2, 713–722</p><p><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acs.jproteome.5c00689">https://doi.org/10.1021/acs.jproteome.5c00689</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>Neurosyphilis (NS) is a group of clinical syndromes characterized by neuroinflammation caused by <i>Treponema pallidum</i> (<i>T. pallidum</i>), which occurs in all stages of syphilis. <a href="javascript:void(0);">(1,2)</a> NS is classified based on clinical presentation as either asymptomatic (ANS) or symptomatic (SNS), and based on disease stage as either early (occurring 1–2 years after primary infection) or late. <a href="javascript:void(0);">(1)</a> <i>T. pallidum</i> can cause local nonspecific and central nervous system (CNS) inflammation, leading to possible degenerative changes. <a href="javascript:void(0);">(3)</a> The immune response of CNS in neurosyphilis is comparable to that observed in other neuroinflammatory diseases. Consequently, the presence of neurological symptoms may not reliably indicate active CNS inflammation in syphilis patients. <a href="javascript:void(0);">(3)</a> A cerebrospinal fluid (CSF) proteomic study identified three biomarkers (SEMA7A, SERPINA3, and ITIH4) that contribute to NS and have potential implications for diagnosis and therapeutic strategies. <a href="javascript:void(0);">(4)</a> Through integrated proteomics and single-cell transcriptomics analyses, Toll-like/NF-κB signaling was identified as the key pathway involved in CNS damage caused by <i>T. pallidum</i>. <a href="javascript:void(0);">(5)</a> Despite an increasing understanding of the biomarkers and possible neuroinflammatory damage mechanisms of neurosyphilis, the mechanisms of neurodegeneration caused by <i>T. pallidum</i> infection remain elusive. <a href="javascript:void(0);">(6)</a></p><p>Patients with neurosyphilis exhibit stage-specific profiles of soluble factors in their CSF, which are linked to both immune status and the extent of neuronal damage. <a href="javascript:void(0);">(7)</a> In this study, we analyzed differentially expressed proteins (DEPs) in the CSF proteomes from 28 individuals, including neurosyphilis (NS), suspected neurosyphilis (spNS) and non-neurosyphilis (nNS) individuals, by LC–MS/MS and analyzed a public data set of the neurosyphilis CSF proteome. Validation was then performed by ELISA in an independent group comprising 110 samples. The results revealed that neuroinflammation by <i>T. pallidum</i> infection results in dysregulation of the key proteins in lysosomal and axonal pathway, which suggesting a potential contribution to neurodegeneration in neurosyphilis.</p><h4><strong>Materials and Methods</strong></h4><h5><strong>LC–MS/MS</strong></h5><p>The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and loaded onto a custom-made reversed-phase column (15 cm × 75 μm). Using an <strong>EASY-nLC 1000 UPLC system</strong>, a gradient was run at 400 nL/min as follows: 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23% to 35% in 8 min, up to 80% in 3 min, and held at 80% for 3 min. Peptides were ionized via an NSI source at 2.0 kV and analyzed online by a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/559"><strong>Q Exactive Plus mass spectrometer</strong></a><strong>.</strong> Full scans (<i>m</i>/<i>z</i> 350–1800) were acquired in the Orbitrap at 70,000 resolution. The top precursors were selected for MS/MS with an NCE of 28, and fragments were detected at 17,500 resolution. A data-dependent method cycled between one MS scan and 20 MS/MS scans with a 15.0 s dynamic exclusion. AGC was set at 5E4. MS/MS data were processed with MaxQuant (v.1.5.2.8) using Trypsin/P as the enzyme (up to 2 missed cleavages). Precursor mass tolerance was 20 ppm (first search) and 5 ppm (main search); fragment mass tolerance was 0.02 Da. Fixed modification: carbamidomethyl (C); variable modification: oxidation (M). The FDR threshold was &lt;1%, and the minimum peptide score was &gt;40.</p><h5><strong>ELISA</strong></h5><p>CSF levels of host proteins were quantified using commercial ELISA kits according to the manufacturers’ protocols. Proteins analyzed included PSAP (OKEH07124, Aviva, USA), CTSL (abx150963, Abbexa, UK), NPC2 (A-QEK07857, Biogradetech, USA), and DNASE2 (abx386953, Abbexa, UK). Briefly, 100 μL of CSF was added to precoated plates and incubated at 37 °C for 2 h. After washing, 100 μL of detection antibody was added to each well and incubated at 37 °C for another 2 h. Following a wash, 100 μL of streptavidin-HRP working solution was added and incubated at room temperature for 20 min in the dark. The reaction was stopped with 50 μL of stop solution, and the optical density was measured at 450 nm.</p><h4><strong>Results</strong></h4><h5><strong>Downregulation of Key Lysosomal Proteins in CSF from Patients With Neurosyphilis</strong></h5><p>By analyzing the DEPs in NS vs nNS, we observed a significant enrichment of downregulated proteins in the lysosome pathway (<i>P</i> = 0.007) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig2">Figure 2</a>A), with 9 lysosomal proteins showing significant downregulation (<i>P</i><sub>adj</sub> &lt; 0.05) in the NS group (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jproteome.5c00689/suppl_file/pr5c00689_si_001.zip">Table S4</a>). To validate this finding, we examined the public CSF proteomics data set of neurosyphilis (PXD033034) and identified 18 proteins in the lysosomal pathway that were significantly downregulated in patients with NS (<i>P</i><sub>adj</sub> &lt; 0.05) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jproteome.5c00689/suppl_file/pr5c00689_si_001.zip">Table S4</a>). PPI network analysis determined the top 10 hub genes within the lysosome pathway (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig2">Figure 2</a>B). A Venn diagram revealed 3 proteins (PSAP, CTSL, and NPC2) shared across our current data, the public data set (PXD033034) and the top 10 hub genes (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig2">Figure 2</a>C). The mass spectrometry results indicated that the levels of PSAP (<i>P</i><sub>adj</sub> = 0.043), CTSL (<i>P</i><sub>adj</sub> = 0.028) and NPC2 (<i>P</i><sub>adj</sub> = 0.013) were significantly lower in the NS group compared to the nNS group (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig2">Figure 2</a>D). The PXD033034 data set revealed consistent changes in the PSAP, CTSL, and NPC2 levels in the NS group (<i>P</i><sub>adj</sub> &lt; 0.05) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jproteome.5c00689/suppl_file/pr5c00689_si_001.zip">Figure S1 and Table S4</a>). In addition, associations between the lysosomal protein DNASE2 and NS were observed in both the present study (<i>P</i><sub>adj</sub> = 0.003) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig2">Figure 2</a>D) and the PXD033034 data set (<i>P</i><sub>adj</sub> &lt; 0.05) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jproteome.5c00689/suppl_file/pr5c00689_si_001.zip">Figure S1 and Table S4</a>), with subgroup analysis revealing a significantly lower expression level of DNASE2 in the SNS group compared to the ANS group (<i>P</i> = 0.035) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig2">Figure 2</a>D and <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jproteome.5c00689/suppl_file/pr5c00689_si_001.zip">Table S3</a>). Further validation by ELISA (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jproteome.5c00689/suppl_file/pr5c00689_si_001.zip">Table S5</a>) in an independent group (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#tbl1">Table 1</a>) confirmed significantly downregulated CSF levels of PSAP (<i>P</i> &lt; 0.0001), CTSL (<i>P</i> &lt; 0.0001), NPC2 (<i>P</i> = 0.0024) and DNASE2 (<i>P</i> &lt; 0.0001) in patients with NS (n = 21) than in nNS individuals (n = 56) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig2">Figure 2</a>E) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jproteome.5c00689/suppl_file/pr5c00689_si_001.zip">Table S6</a>), as well as decreased in the SNS subgroup (<i>P</i> &lt; 0.05) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jproteome.5c00689/suppl_file/pr5c00689_si_001.zip">Figure S2 and Table S6</a>). Receiver operating characteristic (ROC) curve analysis supported PSAP (AUC = 0.91, 95% CI: 0.83–0.99) and DNASE2 (AUC = 0.95, 95% CI: 0.91–0.99) for distinguishing NS from nNS and as potential biomarkers for assessing neurodegeneration in NS (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig2">Figure 2</a>F). These results indicate that <i>T. pallidum</i> infection may lead to lysosomal dysfunction in CNS.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Proteome_Res_2026_25_2_713_722_Figure_2_Downregulation_of_key_lysosomal_proteins_in_the_cerebrospinal_fluid_of_neurosyphilis_patients_bbccd95f44.jpg" alt="J. Proteome Res. 2026, 25, 2, 713–722: Figure 2. Downregulation of key lysosomal proteins in the cerebrospinal fluid of neurosyphilis patients. (a) KEGG pathway analysis demonstrated that downregulated proteins in patients with NS were significantly enriched in the lysosome pathway, as well as in axon guidance and neurodegeneration. (b) The top 10 hub genes involved in the lysosome pathway were identified using the STRING network data set and PPI analysis. (c) A Venn diagram revealed 3 proteins (PSAP, CTSL, and NPC2) shared across our current data, the public data set (PXD033034) and the top 10 hub genes. (d) The CSF levels of PSAP (Padj = 0.043), CTSL (Padj = 0.028), DNASE2 (Padj = 0.003) and NPC2 (Padj = 0.013) were significantly decreased in the NS group than in the nNS group. Subgroup analysis found a significantly lower expression level of DNASE2 in the SNS group than in the ANS group (P = 0.035). (e) PSAP, DNASE2, CTSL and NPC2 levels in CSF were tested by ELISA in the nNS (n = 56) and NS (n = 21) groups. Compared with those in the nNS group, significantly lower PSAP (P &lt; 0.0001), CTSL (P &lt; 0.0001), DNASE2 (P &lt; 0.0001) and NPC2 (P = 0.0024) levels were detected in the NS group. (f) ROC curve analysis for NS revealed that PSAP had an AUC = 0.91 (95% CI: 0.83–0.99), DNASE2 had an AUC = 0.95 (95% CI: 0.91–0.99), CTSL had an AUC = 0.85 (95% CI: 0.76–0.95), and NPC2 had an AUC = 0.72 (95% CI: 0.59–0.85). NS: neurosyphilis; nNS: non-neurosyphilis; ANS: asymptomatic neurosyphilis; SNS: symptomatic neurosyphilis. ROC: receiver operating characteristic; AUC: area under the curve. * P &lt; 0.05, ** P &lt; 0.01, ****P &lt; 0.0001." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Proteome_Res_2026_25_2_713_722_Figure_2_Downregulation_of_key_lysosomal_proteins_in_the_cerebrospinal_fluid_of_neurosyphilis_patients_bbccd95f44.jpg 140w," sizes="100vw" width="2100" height="2339"></p><h5><strong>Downregulation of Key Axonal Proteins Correlated with Lysosomal Protein Reduction in Neurosyphilis CSF</strong></h5><p>Lysosomes play crucial roles in neuronal health by maintaining axonostasis. <a href="javascript:void(0);">(10)</a> The enrichment analysis indicated that the downregulated proteins in NS were implicated in the biological processes of axonostasis (such as axon development and axonogenesis) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig1">Figure 1</a>F) and the pathway of axon guidance (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig2">Figure 2</a>A). Our proteomic data revealed a total of 24 downregulated (<i>P</i><sub>adj</sub> &lt; 0.05) axonal proteins and 1 upregulated (<i>P</i><sub>adj</sub> &lt; 0.05) axonal protein in the CSF of patients with NS (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jproteome.5c00689/suppl_file/pr5c00689_si_001.zip">Table S9</a>). Furthermore, the PXD033034 data set revealed 55 downregulated (<i>P</i><sub>adj</sub> &lt; 0.05) and 4 upregulated (<i>P</i><sub>adj</sub> &lt; 0.05) axonal proteins in NS (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jproteome.5c00689/suppl_file/pr5c00689_si_001.zip">Table S9</a>). The top 10 hub genes involved in the biological processes of axon development and axonogenesis were identified by PPI network analysis (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig4">Figure 4</a>A). Considering the overlap of our current data, the PXD033034 data set and the top 10 hub genes (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig4">Figure 4</a>B), one key protein, EPHA4, was identified across 3 data sets, with significant downregulation in our current data (<i>P<sub>adj</sub></i> = 0.011) and in the PXD033034 data set (<i>P<sub>adj</sub></i> = 0.028) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig4">Figure 4</a>C). Correlation analysis further revealed a positive correlation between the levels of PSAP and EPHA4 (<i>r</i> = i 0.83, <i>P</i> &lt; 0.05) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jproteome.5c00689#fig4">Figure 4</a>D). Reduced levels of PSAP and EPHA4 in CSF have been implicated in neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). <a href="javascript:void(0);">(11,12)</a> Our findings suggest that coordinated dysregulation of lysosomal and axonal proteins may contribute to neurodegeneration in NS.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Proteome_Res_2026_25_2_713_722_Figure_4_Key_axonal_protein_downregulated_in_CSF_from_patients_with_neurosyphilis_66f5533e94.jpg" alt="J. Proteome Res. 2026, 25, 2, 713–722: Figure 4. Key axonal protein downregulated in CSF from patients with neurosyphilis. (a) The top 10 hub genes involved in axonostasis were identified by the STRING network data set and PPI analysis. (b) A Venn diagram revealed 1 protein (EPHA4) shared across DEPs related to axonostasis in our current data and in the public data set (PXD033034) and the top 10 hub genes. (c) EPHA4 was significantly downregulated both in our present study (Padj = 0.011) and in the PXD033034 data set (Padj = 0.028). (d) Correlation analysis demonstrated that the EPHA4 level was significantly positively correlated (P &lt; 0.05) with the levels of PSAP (r = 0.83), CTSL (r = 0.6), NPC2 (r = 0.69), DNASE2 (r = 0.61) and SEMA7A (r = 0.83). NS: neurosyphilis; nNS: non-neurosyphilis. * P &lt; 0.05." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Proteome_Res_2026_25_2_713_722_Figure_4_Key_axonal_protein_downregulated_in_CSF_from_patients_with_neurosyphilis_66f5533e94.jpg 178w," sizes="100vw" width="1250" height="1096"></p><h4><strong>Conclusions</strong></h4><p>The present study reveals the associations of dysregulated proteins in lysosomal and axonal pathways with neurosyphilis, and potential evidence of shared pathophysiological mechanisms between neuroinflammation caused by <i>T. pallidum</i> infection and neurodegenerative diseases.</p><p>However, this study has certain limitations. First, the use of ELISA to validate a limited number of proteins has certain limitations, such as the inherent cross-reactivity of ELISA. In the future, we can optimize the validation experiments by adding techniques such as Western blotting for verification. Second, this study uses a cross-sectional sampling method for research, which limits our ability to infer disease progression and treatment responses. In the future, we will conduct follow-up on the subjects, continue collecting cerebrospinal fluid samples after treatment, and observe whether the protein dysregulation is reversible after treatment. For spNS patients, follow-up should also be conducted to determine if they have transformed into NS, which will be very valuable for validating the predictive utility of our biomarkers. Finally, although multiple discovery and validation cohort studies have been conducted, we currently lack direct mechanistic evidence. Future work will investigate whether exposing neural cell coculture models to outer-membrane antigens of <i>T. pallidum</i> reproduces the lysosome and axon alterations observed in CSF, clarifying their role in neural damage of neurosyphilis and potential biomarkers for diagnosis and treatment of neurodegeneration in neurosyphilis.</p></div>

Cerebrospinal Fluid Proteome Reveals Dysregulation of Lysosomal and Axonal Proteins in Neurosyphilis

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Cerebrospinal Fluid Proteome Reveals Dysregulation of Lysosomal and Axonal Proteins in Neurosyphilis

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