A newly developed portable capillary liquid chromatograph was investigated for the separation of various pharmaceutical and illicit drug compounds. The system consists of two high‐pressure syringe pumps capable of delivering capillary‐scale flow rates at pressures up to 10 000 psi. Capillary liquid chromatography columns packed with sub‐2 μm particles are housed in cartridges that can be inserted into the system and easily connected through high‐pressure fluidic contact points by simply applying a specific, predetermined torque rather than using standard fittings and less precise sealing protocols. Several over‐the‐counter analgesic drug separations are demonstrated, along with a simple online measurement of tablet dissolution. Twenty illicit drug compounds were also separated across six targeted drug panels. The results described in this study demonstrate the capability of this compact liquid chromatography instrument to address several important drug‐related applications while simplifying system operation, and greatly reducing solvent usage and waste generation essential for onsite analysis.
Human tear protein biomarkers are useful for detecting both ocular and systemic diseases. Unfortunately, existing tear film sampling methods (Schirmer strips (SS) and microcapillary tubes (MCT)) have significant drawbacks. Here we present an alternative tear protein sampling method using soft contact lenses (SCLs). First, we optimized SCL protein sampling in vitro, then performed in vivo studies in 6 human subjects. Using Etafilcon A SCLs and 4M guanidine-HCl for protein removal, we sampled an average of 60 +/- 31 ug of protein per eye. We also performed objective and subjective assessments of all sampling methods. Proteomic analysis by mass spectrometry (MS) revealed unique subsets of proteins for SS and SS/MCT methods. Additionally, SCLs had reduced levels of reflex tear protein zymogen granule protein 16 homolog B (ZG16B) versus SS and MCT. These experiments demonstrate SCLs as an accessible tear sampling method which may minimize reflex tearing and potentially aid biomarker discovery.
Soft contact lenses (SCLs) have recently been introduced as an alternative method for human tear protein sampling. However, SCLs are available in a variety of chemical compositions which affect protein binding specificity. Here we analyzed 8 different SCL materials to identify an optimal lens for tear protein sampling. Polymer contamination, mass spectrometry (MS) sample preparation method, total protein capture, individual protein specificity, and SCL cost were all assessed. Using a filter-aided sample prep (FASP) method with 4M guanidine for protein removal, only etafilcon A and verofilcon A did not have significant polymer contamination. Polymer was successfully removed using phosphate buffered saline (PBS) with S-Trap columns for all SCL materials, though yielding a slightly lower number of protein identifications per sample. Minor quantitative differences were observed between SCL materials. However, we also saw significant intersubject variation in protein abundance. Of all the assessed SCL materials, verofilcon A lenses yielded the most total protein while comfilcon A and senofilcon A had the least protein variability. As a newly released daily disposable modality (Precision 1, Alcon), verofilcon A has one of the longest predictable production schedules and one of the lowest costs per lens, making it beneficial for large-scale experiments and diagnostics. Furthermore, we demonstrate how protein binding bias with SCL tear sampling is useful for intra-experiment normalization. Overall, these experiments have led us to optimize our previous protocol for SCL tear protein sampling, highlighting important differences between SCL materials and identifying etafilcon A and verofilcon A as optimal materials for tear protein sampling.
Kinetoplastid pathogens (such as Trypanosoma brucei, Trypanosoma cruzi, and Leishmania species) infect millions of people yearly causing a significant health burden worldwide. To better treat these diseases, improved therapeutics are needed. However, many candidates in drug discovery pipelines have an unknown mode‐of‐action (MOA), limiting their development. Hence, there is a great need for methods to identify the MOA of these drug leads. We have adapted a Proteome Integral Solubility Alteration assay, a variation of thermal proteome profiling, to identify drug targets in Trypanosoma brucei. This assay identifies likely drug targets by detecting proteins with altered stability due to drug treatment (see figure). To validate this approach for identifying a drug's MOA, we used eflornithine that is a known ornithine decarboxylase (ODC) suicide inhibitor. We initially identified approximately 16,300 peptides and 1,400 protein groups per sample, indicating we obtained about 17% proteome coverage. Off‐line fractionation was optimized to increase peptide and proteome coverage. Criteria used to classify proteins as potential drug targets include 3 or more peptides per protein with p‐values < 0.05 (2‐sided Student's T‐test) in the same direction when comparing drug to vehicle‐control treatments. Along with ODC being identified as an eflornithine target, other potential drug targets identified include aspartate aminotransferase and tryparedoxin peroxidase. These may be additional direct or indirect drug targets as eflornithine is an amino acid analog and trypanothione synthesis is downstream of ODC. We are applying this methodology to identify the MOA for hit compounds identified in phenotypic screening assays. We expect this assay will improve drug development pipelines for Trypanosoma brucei and related kinetoplastid diseases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.