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Analytes, from sample preparation, until entering an analytical instrument, are prone to adsorb to surfaces, driven by the chemical properties of the surface and the liquids they are dissolved in. This problem can be addressed with internal standards when a single or few known analytes are quantified that are usually not available in omics. However, minimal to no loss of analytes is the aim. Here, we present a novel assay for qualifying and quantifying interactions responsible for adsorption of molecules to surfaces (APS) by using LC-MS/MS-based differential quantitative analysis. To reflect a broad range of chemical interactions with surfaces, a reference mixture of thousands of tryptic peptides, with known compositions was selected, representing a variety of different chemical characteristics. The assay was tested by investigating the adsorption properties of several different vials with different surface chemistries. A significant number of hydrophobic peptides adsorbed to conventional polypropylene vials. In contrast, only few peptides adsorbed to polypropylene vials, assigned as low-protein-binding. The highest number of peptides adsorbed to glass vials driven by electrostatic interactions. In summary, the new assay is suitable to characterize adsorption properties of different surfaces and to approximate the loss of analytes during sample preparation., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

In a fragment-based approach using NMR spectroscopy, benzyloxyacetohydroxamic acid-derived inhibitors of the bacterial deacetylase LpxC bearing a substituent to target the uridine diphosphate-binding site of the enzyme were developed. By appending privileged fragments via a suitable linker, potent LpxC inhibitors with promising antibacterial activities could be obtained, like the one-digit nanomolar LpxC inhibitor ( S )- 13j [ K i ( Ec LpxC C63A) = 9.5 nM; K i ( Pa LpxC): 5.6 nM]. To rationalize the observed structure-activity relationships, molecular docking and molecular dynamics studies were performed. Initial in vitro absorption-distribution-metabolism-excretion-toxicity (ADMET) studies of the most potent compounds have paved the way for multiparameter optimization of our newly developed isoserine-based amides.

Fractionation of proteoforms is currently the most challenging topic in the field of proteoform analysis. The need for considering the existence of proteoforms in experimental approaches is not only important in Life Science research in general but especially in the manufacturing of therapeutic proteins (TPs) like recombinant therapeutic antibodies (mAbs). Some of the proteoforms of TPs have significantly decreased actions or even cause side effects. The identification and removal of proteoforms differing from the main species, having the desired action, is challenging because the difference in the composition of atoms is often very small and their concentration in comparison to the main proteoform can be low. In this study, we demonstrate that sample displacement batch chromatography (SDBC) is an easy-to-handle, economical, and efficient method for fractionating proteoforms. As a model sample a commercial ovalbumin fraction was used, containing many ovalbumin proteoforms. The most promising parameters for the SDBC were determined by a screening approach and applied for a 10-segment fractionation of ovalbumin with cation exchange chromatography resins. Mass spectrometry of intact proteoforms was used for characterizing the SDBC fractionation process. By SDBC, a significant separation of different proteoforms was obtained., (© 2023 The Authors. PROTEOMICS published by Wiley-VCH GmbH.)

The FDA announced a boxed warning for tigecycline due to progression of infections caused by Gram-negative bacteria and increased risk of mortality during treatment. Plasma exposure of tigecycline might not prevent bacteraemia in these cases from the focuses. Hence, we evaluated intensified dosing regimens and breakpoints that might suppress bloodstream infections, caused by progression of infection by e.g., Gram-negatives. A pharmacometric model was built from tigecycline concentrations (100-600 mg daily doses) against clinical Klebsiella pneumoniae isolates (MIC 0.125-0.5 mg/L). Regrowth occurred at clinically used doses and stasis was only achieved with 100 mg q8h for the strain with the lowest studied MIC of 0.125 mg/L. Stasis at 24 h was related to fAUC/MIC of 38.5. Our study indicates that even intensified dosing regimens might prevent bloodstream infections only for MIC values ≤0.125 mg/L for tigecycline. This indicates an overly optimistic breakpoint of 1 mg/L for Enterobacterales, which are deemed to respond to the tigecycline high dose regimen (EUCAST Guidance Document on Tigecycline Dosing 2022)., Competing Interests: Declaration of Competing Interest All authors, nothing to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Lassa fever is endemic in large parts of West Africa. The recommended antiviral treatment is ribavirin. Two treatment regimens are currently endorsed in Nigeria: the "McCormick regimen" based on a study published in 1986 and the "Irrua regimen" constituting a simplified schedule developed at the Irrua Specialist Teaching Hospital, Nigeria. Evidence for the safety and efficacy of ribavirin in Lassa fever patients is poor and pharmacokinetic data for both regimens are lacking., Methods: Polymerase chain reaction-confirmed Lassa fever patients with mild to moderate disease severity were invited to participate in this prospective, observational pharmacokinetic study. Pharmacokinetics of ribavirin, clinical, virologic, and clinical laboratory parameters were assessed., Results: Using a population pharmacokinetic approach, plasma concentrations of ribavirin were best described by a 3-compartment model. Drug exposure was remarkably consistent between participants. Overall, drug clearance was 28.5% lower in female compared with male participants. Median (5th-95th percentile) time above half maximal inhibitory concentration (IC50) was 37.3% (16.9%-73.1%), 16.7% (8.2%-58.5%), and 9.6% (4.9%-38.4%) on days 1, 7, and 8, respectively. Clinical laboratory parameters indicated reduction of cell damage and development of hemolytic anemia in the course of the treatment period., Conclusions: This observational study characterizes the pharmacokinetics of ribavirin in the treatment of Lassa fever indicating consistent exposure across patients. Whereas only a short time interval of concentrations above the IC50 implies rather low antiviral efficacy in vivo, the prominent reduction of cell damage markers might point to indirect-potentially anti-inflammatory-effects of ribavirin. The role of ribavirin in the treatment of Lassa fever requires further scrutiny., Competing Interests: Potential conflicts of interest. S. G. reports support for attending meetings and/or travel from Keystone Symposia. E. O. E. and S. O. report other financial or nonfinancial interests in CEPI—Lassa Epidemiological Programme as a study team member at ISTH, Nigeria. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Hemodialysis fails to remove protein-bound uremic toxins that are attributed with high cardiovascular risk. Application of adsorption materials is a viable strategy, but suitable biocompatible adsorbents are still not available. Here, we demonstrate that adsorbents based on the bottom-up assembly of the intrinsically biocompatible protein cage ferritin are applicable for toxin adsorption. Due to the size-exclusion effect of its pores, only small molecules such as uremic toxins can enter the protein cage. Protein redesign techniques that target selectively the inner surface were used to introduce anchor sites for chemical modification. Porous crystalline adsorbents were fabricated by bottom-up assembly of the protein cage. Linkage of up to 96 phenylic or aliphatic molecules per container was verified by ESI-MS. Materials based on unmodified ferritin cages can already adsorb the uremic toxins. The adsorption capacity could be increased by about 50% through functionalization with hydrophobic molecules reaching 458 μg g -1 for indoxyl sulfate. The biohybrid materials show no contamination with endotoxins and do not activate blood platelets. These findings demonstrate the great potential of protein-based adsorbents for the clearance of uremic toxins: modifications enhance toxin adsorption without diminishing the biocompatibility of the final protein-based material.

For patients with myelofibrosis, allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative treatment to date. Busulfan-based conditioning regimens are commonly used, although high inter-individual variability (IIV) in busulfan drug exposure makes individual dose selection challenging. Since data regarding the IIV in patients with myelofibrosis are sparse, this study aimed to develop a population pharmacokinetic (PopPK) model of busulfan and its metabolite sulfolane in patients with myelofibrosis. The influence of patient-specific covariates on the pharmacokinetics of drug and metabolite was assessed using non-linear mixed effects modeling in NONMEM ® . We obtained 523 plasma concentrations of busulfan and its metabolite sulfolane from 37 patients with myelofibrosis. The final model showed a population clearance (CL) and volume of distribution (V d ) of 0.217 L/h/kg and 0.82 L/kg for busulfan and 0.021 L/h/kg and 0.65 L/kg for its metabolite. Total body weight (TBW) and a single-nucleotide polymorphism of glutathione-S-transferase A1 (GSTA1 SNP) displayed a significant impact on volume of distribution and metabolite clearance, respectively. This is the first PopPK-model developed to describe busulfan's pharmacokinetics in patients with myelofibrosis. Incorporating its metabolite sulfolane into the model not only allowed the characterization of the covariate relationship between GSTA1 and the clearance of the metabolite but also improved the understanding of busulfan's metabolic pathway.

Urotensin II (UII) and UII-related peptide (URP) are vasoactive peptide hormones causing strong vasoconstriction or vasodilation, depending on the type of blood vessel. In humans, the active forms are resulting from proteolytic cleavage of their inactive precursor protein. In blood plasma, a defined protease converting the inactive UII and URP precursors into their active forms has not been identified yet. Using mass spectrometry-based enzyme screening for detecting UII- and URP-converting enzymes, the human plasma fraction Cohn IV-4 was chromatographed, and the resulting fractions were screened for UII- or URP-generating activity. Plasma kallikrein (PK) as a UII- and URP-generating protease was identified. URP generation was also found for the serine protease factor XIa, plasmin, thrombin, and, to a smaller extent, factor XIIa. It was demonstrated that in the Cohn IV-4 fraction, PK accounts for a significant amount of UII- and URP-generating activity.

Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the TΨC-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 Å resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon.

Purpose: This study aimed to examine the degradation of tigecycline in Mueller Hinton broth (ca-MHB), as knowledge about bacterial susceptibility is key for therapeutic decisions., Methods: Antioxidative stabilizers were evaluated on tigecycline stability in a quantitative chromatography assay and tigecycline induced kill against Staphylococcus aureus (ATCC29213) was determined in time kill studies., Results: Ascorbic acid caused rapid degradation of tigecycline and resulted in loss of antibacterial activity. Tigecycline was stabilized in aged broth by 2% pyruvate and bacterial growth, and tigecycline killing was similar to fresh broth without supplementation, but independent of age., Conclusion: Our results underline the importance of using freshly prepared ca-MHB or the need for stabilizers for tigecycline susceptibility testing while using aged ca-MHB.

Colloidal approaches allow for the synthesis of Au nanoclusters (NCs) with atomic precision and sizes ranging from a few to hundreds of atoms. In most of the cases, these processes involve a common strategy of thiol etching of initially polydisperse Au nanoparticles into atomically precise NCs, resulting in the release of Au-thiolate complexes as byproducts. To the best of our knowledge, neither the removal of these byproducts nor the mass spectra in the relevant mass region were shown in previous studies. A thorough analysis of inorganic byproducts in the synthesis of [Au25(PPh3)10(PET)5X2]2+ NC, abbreviated as Au25 NC, reveals that published protocols lead to Au25 NCs in vanishingly small quantities compared to their byproducts. Three purification methods are presented to separate byproducts from the desired Au25 NCs which are proposed to be applicable to other promising Au NC systems. Additionally, critical factors for a successful synthesis of Au25 NCs are identified and discussed including the role of residual water. An important finding is that the etching duration is very critical and must be monitored by UV-Vis spectroscopy resulting in synthetic yields as high as 40%.

Herein, we demonstrate that meticulous and in-depth analysis of the reaction mechanisms of nanoparticle formation is rewarded by full control of the size, shape, and crystal structure of superparamagnetic iron oxide nanocrystals during synthesis. Starting from two iron sources, iron(II) and iron(III) carbonate, a strict separation of oleate formation from the generation of reactive pyrolysis products and concomitant nucleation of iron oxide nanoparticles was achieved. This protocol enabled us to analyze each step of nanoparticle formation independently in depth. The progress of the entire reaction was monitored via matrix-assisted laser desorption ionization time-of-flight mass spectrometry and gas chromatography, thus providing insight into the formation of various iron oleate species prior to nucleation. Interestingly, due to the intrinsic strongly reductive pyrolysis conditions of the oleate intermediates and redox process in early stages of the synthesis, pristine iron oxide nuclei were composed exclusively from wüstite irrespective of the oxidation state of the iron source. Controlling the reaction conditions provided a very broad range of size- and shape-defined monodispersed iron oxide nanoparticles. Curiously, after nucleation, star-shaped nanocrystals were obtained that underwent metamorphism toward cubic-shaped particles. Electron energy loss spectroscopy tomography revealed ex post oxidation of the primary wustite nanocrystal, providing a full 3D image of Fe 2+ and Fe 3+ distribution within. Overall, we developed a highly flexible synthesis, yielding multi-gram amounts of well-defined iron oxide nanocrystals of different sizes and morphologies.

This study aimed at developing a rapid chromatographic assay to monitor phosphorylation sites in peptides. For the analysis of nociceptive signal transduction pathways, the detection of phosphorylated proteins/peptides plays a fundamental role. To get further insights in the phosphorylation mechanism of protein kinase C-ε (PKC-ε) and protein kinase A (PKA), potential targets were divided into subsections resulting in peptides that contain only one possible phospho-binding site. The use of high-performance thin-layer chromatography (HPTLC) offers the possibility of a high throughput of samples and the advantage of a quick sample clean-up. A combined strategy of an effect-directed overlay procedure on the TLC plate using specific antibodies (immunostaining, HPTLC-IS) as well as a parallel, direct mass spectrometric methodology by HPTLC-MALDI-TOF-MS was developed. With regard to HPTLC-IS, validation of the data exhibited a lower limit of detection than the traditionally used protein derivatization reagent fluorescamine. Besides the identification of the phosphorylated peptides, a semi-quantitative estimation can be performed with HPTLC-IS., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The investigation of venoms has many clinical, pharmacological, ecological and evolutionary outcomes. The Crotalus spp. venom can cause hemorrhage, neurotoxicity, myotoxicity, coagulopathy and hypotension. Although neurotoxicity and hemorrhage usually does not occur for the same species, the rare Venezuelan species Crotalus vegrandis presents both characteristic. Different from the other species it has a restricted ecological niche and geographical distribution. Nevertheless, it has a raising medical importance as this rattlesnake population is increasing. Few works describe its neurotoxic and hemorrhagic features, but other toxins might play an important role in envenomation. We combined proteomic methods to identify for the first time the main components of it venom: 2D SDS-PAGE and gel-filtration chromatography for protein mixture decomplexation; LC-MS(2) of low molecular mass fractions and tryptic peptides; bioinformatic identification of toxin families and specific protein species based on unique peptide analysis and sequence database enriched with species-specific venom gland transcripts; and finally polyclonal anti-crotamine Western-blotting. Our results point to a broad arsenal of toxins in C. vegrandis venom: PIII and PII metalloproteases, crotoxin subunits, other phospholipases, isoforms of serine proteases and lectins, l-amino-acid oxidase, nerve growth factor, as well as other less abundant toxins., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In recent years, protein chemistry tends inexorably toward the analysis of more complex proteins, proteoforms, and posttranslational protein modifications. Although mass spectrometry developed quite fast correspondingly, sample preparation and separation of these analytes is still a major issue and quite challenging. For many years, electrophoresis seemed to be the method of choice; nonetheless its variance is limited to parameters such as size and charge. When taking a look at traditional (thin-layer) chromatography, further parameters such as polarity and different mobile and stationary phases can be utilized. Further, possibilities of detection are manifold compared to electrophoresis. Similarly, two-dimensional separation can be also performed with thin-layer chromatography (TLC). As the revival of TLC developed enormously in the last decade, it seems to be also an alternative to use high performance thin-layer chromatography (HPTLC) for the separation of proteins. The aim of this study was to establish an HPTLC separation system that allows a separation of protein mixtures over a broad polarity range, or if necessary allowing to modify the separation with only few steps to improve the separation for a specific scope. Several layers and solvent systems have been evaluated to reach a fully utilized and optimized separation system., (Copyright © 2015 Elsevier B.V. All rights reserved.)