Protocol for constructing tumor-targeting ANM-PROTACs for degradation of transcription factors.

AS1411-NCL-MDM2-based proteolysis-targeting chimeras (ANM-PROTACs) are capable of inducing selective degradation of transcription factors (TFs) in tumor cells. Here, we present a protocol for constructing ANM-PROTACs. We describe steps for molecular design of the ANM-PROTACs, assembly and characterization of the ANM-PROTACs, and initial assessment of in vitro TF degradation potency. We then detail procedures for validation of selective degradation of TFs via proteomic analysis. This protocol has been successfully applied to degrade various TFs across multiple tumor cell lines. For complete details on the use and execution of this protocol, please refer to Fu et al.1.

Development of a simultaneous analytical method for the dietary exposure determination of ß-Carboline alkaloids in foods.

As ß-carboline (ßC) alkaloids, posing potential health risks, are present in a wide variety of foods, determining the exposure degrees of food to these alkaloids from dietary activity is key to ensuring food safety. Here, we developed a rapid and sensitive simultaneous analytical method for six ßC alkaloids in food. We optimized the buffered QuEChERS method, which includes a clean-up process through dispersive solid phase extraction, to extract the target compounds from food matrices; then, these compounds were detected via liquid chromatography-tandem mass spectrometry. We established calibration ranges for each target compound and matrix within the range of 0.05-250 µg/kg, and verified linearity (R2 ≥ 0.99) and limit of quantitation (≤1.63 µg/kg). Furthermore, we validated trueness (85.8%-118.8%) and precision (≤18.7%) at three levels within the calibration range, including the lowest and highest concentrations. Finally, we employed the developed method to determine the ßC alkaloid contents in 304 samples of 41 food items and dietary exposure of six ßC alkaloids resulting from daily intake. Although ßC alkaloids were detected in 86.2% of the samples, exposure level to the 41 food items was insufficient to cause toxicity.

Aerodynamic thermal breakup droplet ionization combined with a ß-irradiation source for mass-spectrometric analysis of samples in a nonpolar solvent.

A method is proposed for increasing the number of ions during mass-spectrometric analysis of samples in a nonpolar solvent (benzene). For this purpose, aerodynamic thermal breakup droplet ionization (ATBDI) with the impact of ß-radiation on the aerosol droplets used in ATBDI was evaluated. This modification of the method, which we named ß-ATBDI, allows to shift a nonvolatile analyte (trinitrotoluene in the negative ionization region and cocaine in the positive ionization region, as an example) into a gas phase as an aerosol at room temperature (in contrast to atmospheric pressure chemical ionization). In addition, ß-ATBDI enables a researcher to distinguish mass spectrometric peaks of the compounds located in an aerosol droplet from compounds located outside the droplet, i.e., to identify background peaks. Also briefly discussed the ionization of two antibiotics-azithromycin in methylene chloride and sulfadiazine in salt water with ß-ATBDI, ATBDI and electrospray ionization source.

Development and application of UPLC-Q-Orbitrap HRMS methods for the determination of eight metabolites of p-chloronitrobenzene in human urine.

Accurate, reliable, and sensitive methods for the determination of eight metabolites of p-chloronitrobenzene (p-CNB) were developed based on ultra-performance liquid chromatography - quadrupole - orbitrap high resolution mass spectrometry (UPLC-Q-Orbitrap HRMS). The free and conjugated forms of metabolites were determined before and after urine samples were hydrolyzed with acid. Subsequently, three solid phase extraction steps were used for concentration and purification. The calibration curves of the eight metabolites exhibited good linearity with an R2 of >0.999, and the precision was good as well, with the coefficient of variations of intra-day and inter-day being lower than 7.0 % and 8.5 %, respectively. Analytical accuracy for all metabolites varied within ranges of 76.0-102.9 %, and the limit of detection and limit of quantification of all the metabolites varied within ranges of 0.2-7.7 µg/L and 0.6-25.6 µg/L in human urine, respectively. In addition, the application potential of the proposed methods were evaluated by applying them to the determination of metabolites in the urine of workers exposed to p-CNB, and these results showed that these methods were accurate, reliable, and sensitive, which makes them an excellent choice for detecting the metabolites of p-CNB in the urine of exposed workers.

Myocardial Infarction Suppresses Protein Synthesis and Causes Decoupling of Transcription and Translation.

Gene expression involves transcription, translation, and mRNA and protein degradation. Advanced RNA sequencing measures mRNA levels for cell state assessment, but mRNA level does not fully reflect protein level. Identifying heart cell proteomes and their stress response is crucial. Using a cardiomyocyte-specific mouse model, we tracked protein synthesis after myocardial infarction. Our results showed that myocardial infarction suppresses protein synthesis and unveils a decoupling of translation and transcription regulation in cardiomyocytes.

To stay or not to stay intact as an allergen: the endolysosomal degradation assay used as tool to analyze protein immunogenicity and T cell epitopes.

Antigen uptake and processing of exogenous proteins is critical for adaptive immunity, particularly for T helper cell activation. Proteins undergo distinct proteolytic processing in endolysosomal compartments of antigen-presenting cells. The resulting peptides are presented on MHC class II molecules and specifically recognized by T cells. The in vitro endolysosomal degradation assay mimics antigen processing by incubating a protein of interest with a protease cocktail derived from the endolysosomal compartments of antigen presenting cells. The kinetics of protein degradation is monitored by gel electrophoresis and allows calculation of a protein's half-life and thus endolysosomal stability. Processed peptides are analyzed by mass spectrometry and abundant peptide clusters are shown to harbor T cell epitopes. The endolysosomal degradation assay has been widely used to study allergens, which are IgE-binding proteins involved in type I hypersensitivity. In this review article, we provide the first comprehensive overview of the endolysosomal degradation of 29 isoallergens and variants originating from the PR-10, Ole e 1-like, pectate lyase, defensin polyproline-linked, non-specific lipid transfer, mite group 1, 2, and 5, and tropomyosin protein families. The assay method is described in detail and suggestions for improved standardization and reproducibility are provided. The current hypothesis implies that proteins with high endolysosomal stability can induce an efficient immune response, whereas highly unstable proteins are degraded early during antigen processing and therefore not efficient for MHC II peptide presentation. To validate this concept, systematic analyses of high and low allergenic representatives of protein families should be investigated. In addition to purified molecules, allergen extracts should be degraded to analyze potential matrix effects and gastrointestinal proteolysis of food allergens. In conclusion, individual protein susceptibility and peptides obtained from the endolysosomal degradation assay are powerful tools for understanding protein immunogenicity and T cell reactivity. Systematic studies and linkage with in vivo sensitization data will allow the establishment of (machine-learning) tools to aid prediction of immunogenicity and allergenicity. The orthogonal method could in the future be used for risk assessment of novel foods and in the generation of protein-based immunotherapeutics.

The Open SprayBot: A high-throughput paper spray mass spectrometry platform for disease screening.

Newborn disease screening increases survival, improves quality of life and reduces treatment costs for healthcare systems. Mass spectrometry (MS) is an effective method for metabolic screening. However, conventional analytical methods require biofluid handling and cooling conditions during transport, making the logistics difficult and expensive, especially for remote regions. 'Paper-spray' (PS) ionization generates a charged solvent spray from samples deposited on paper strips. Therefore, samples can be applied on a suitable matrix and shipped as dried spots to diagnostic laboratories with standard postal or messenger services. We built a robotic platform, the 'Open SprayBot', to automatically analyze paper-deposited samples via PS-MS and increase the sample throughput. The system is operated via RUMBA32 and Arduino Mega boards. A commercial syringe pump and power supply provide solvent application and electrical current required for PS-MS. The usability of the Open SprayBot was demonstrated by quantifying palmitoyl-l-carnitine, a common biomarker in newborn screening.

Chinese traditional medicine DZGP beneficially affects gut microbiome, serum metabolites and recovery from rheumatoid arthritis through mediating NF-κB signaling pathway.

Rheumatoid arthritis (RA) is globally treated with several commercially available anti-inflammatory and analgesic drugs, which pose adverse side effects in many cases. Due to increasing population affected by autoimmune disorder of joints inflammation, it is crucial to use natural therapies, which are less toxic at metabolic level and promote gut health. In this study, we investigated the potential role of a locally developed traditional Chinese medicine (TCM), namely Duzheng tablet (DZGP) in controlling the RA. For this purpose, we introduced RA in male mice and divided them into 5 different groups. High throughput transcriptome analysis of synovial cells after DZGP treatment in arthritic mice revealed a significant alteration of gene expression. The correlation analysis of transcriptome with metabolites revealed that DZGP specifically targeted the B cells mediated immunity pathways. Treatment with DZGP inhibited the cytokines production, while reducing the production of inflammatory TNF-α, which led to the alleviation of inflammatory response in arthritic mice. Additionally, we applied integrated approach using 16S rDNA sequencing to understand the microbial population in relation to metabolites accumulation. The results showed that DZGP promoted the healthy gut microbiota by maintaining the ratio of Firmicutes and Bacteroidota and introduction of two additional phyla namely, Verrucomicrobiota and Cyanobacteria. Therefore, it is concluded that DZGP offers an advantage over commercial drug by changing the metabolic profile, gut microbiota while exhibiting lower cellular toxicity.

Development and validation of stability-indicating method of etrasimod by HPLC/DAD/MS/MS technique with greenness profiling.

Etrasimod, a novel selective sphingosine-1-phosphate receptor modulator, was recently approved by the U.S. Food and Drug Administration and the European Medicinal Agency for the treatment of moderately to severely active ulcerative colitis in adults. In this research, the forced degradation study as an integral part of drug product and packaging development, which generates data on degradation mechanisms, is published. The development and validation of the stability-indicating method using a superior high-performance liquid chromatography technique coupled with a diode array detector and tandem mass spectrometer was performed to support the forced degradation study and monitor the formation of degradation products. Etrasimod demonstrated good stability under elevated temperature and basic stress conditions, while acidic hydrolysis, oxidative, and photolytic degradation produced eight degradation products. The knowledge of degradation products will be useful in the long-term stability study for establishing the acceptance criteria of etrasimod as a drug substance and dosage form during quality control and stability assessment. The eco-friendliness of the developed forced degradation procedure was evaluated using various greenness appraisal tools. The green metric tools showed that the forced degradation procedure obeys eco-friendly conditions.

Applying Spatial Metabolomics To Investigate Age- and Drug-Induced Neurochemical Changes.

In an era when population aging is increasing the burden of neurodegenerative conditions, deciphering the mechanisms underlying brain senescence is more important than ever. Here, we present a spatial metabolomics analysis of age-induced neurochemical alterations in the mouse brain using negative ionization mode mass spectrometry imaging. The age-dependent effects of the acetylcholinesterase inhibitor tacrine were simultaneously examined. For ultrahigh mass resolution analysis, we utilized a Fourier-transform ion cyclotron resonance spectrometer. To complement this, a trapped ion mobility spectrometry time-of-flight analyzer provided high speed and lateral resolution. The chosen approach facilitated the detection and identification of a wide range of metabolites, from amino acids to sphingolipids. We reported significant, age-dependent alterations in brain lipids which were most evident for sulfatides and lysophosphatidic acids. Sulfatide species, which are mainly localized to white matter, either increased or decreased with age, depending on the carbon chain length and hydroxylation stage. Lysophosphatidic acids were found to decrease with age in the detailed cortical and hippocampal subregions. An age-dependent increase in the glutamine/glutamate ratio, an indicator of glia-neuron interconnection and neurotoxicity, was detected after tacrine administration. The presented metabolic mapping approach was able to provide visualizations of the lipid signaling and neurotransmission alterations induced by early aging and can thus be beneficial to further elucidating age-related neurochemical pathways.

Accurate non-ceruloplasmin bound copper: a new biomarker for the assessment and monitoring of Wilson disease patients using HPLC coupled to ICP-MS/MS.

OBJECTIVES: Assessment of Wilson disease is complicated, with neither ceruloplasmin, nor serum or urine copper, being reliable. Two new indices, accurate non-ceruloplasmin copper (ANCC) and relative ANCC were developed and applied to a cohort of 71 patients, as part of a Wilson Disease Registry Study. METHODS: Elemental copper-protein speciation was developed for holo-ceruloplasmin quantitation using strong anion exchange chromatography coupled to triple quadrupole inductively coupled plasma mass spectrometry. The serum proteins were separated using gradient elution and measured at m/z 63 (63Cu+) and 48 (32S16O+) using oxygen reaction mode and Cu-EDTA as calibration standard. The ANCC was calculated by subtraction of the ceruloplasmin bound copper from the total serum copper and the RelANCC was the percentage of total copper present as the ANCC. RESULTS: The accuracy of the holo-ceruloplasmin measurement was established using two certified reference materials, giving a mean recovery of 94.2 %. Regression analysis between the sum of the copper containing species and total copper concentration in the patient samples was acceptable (slope=0.964, intercept=0, r=0.987) and a difference plot, gave a mean difference for copper of 0.38 µmol/L. Intra-day precision for holo-ceruloplasmin at serum copper concentrations of 0.48 and 3.20 µmol/L were 5.2 and 5.6 % CV and the intermediate precision at concentrations of 0.80 and 5.99 µmol/L were 6.4 and 6.4 % CV, respectively. The limit of detection (LOD) and lower limit of quantification (LLOQ) for holo-ceruloplasmin were 0.08 and 0.27 µmol/L as copper, respectively. CONCLUSIONS: ANCC and Relative ANCC are important new diagnostic and monitoring biomarker indices for Wilson disease (WD).

Mass Spectrometry Identifies Taenia solium Proteins in Sera of Patients With and Without Parenchymal Neurocysticercosis.

Neurocysticercosis (NCC), a major cause of global acquired epilepsy, results from Taenia solium larval brain infection. T. solium adult worms release large numbers of infective eggs into the environment contributing to high levels of exposure in endemic areas. This study identifies T. solium proteins in the sera of individuals with and without NCC using mass spectrometry to examine exposure in endemic regions. Forty-seven patients (18-51 years), 24 parenchymal NCC (pNCC), 8 epilepsy of unknown aetiology, 7 glioma, 8 brain tuberculoma, and 7 healthy volunteers were studied. Trypsin digested sera were subject to liquid chromatography-tandem mass spectrometry and spectra of 375-1700 m/z matched against T. solium WormBase ParaSite database with MaxQuant software to identify T. solium proteins. Three hundred and nineteen T. solium proteins were identified in 87.5% of pNCC and 56.6% of non-NCC subjects. Three hundred and four proteins were exclusive to pNCC sera, seven to non-NCC sera and eight in both. Ten percent, exhibiting immune-modulatory properties, originated from the oncosphere and cyst vesicular fluid. In conclusion, in endemic regions, T. solium proteins are detected in sera of individuals with and without pNCC. The immunomodulatory nature of these proteins may influence susceptibility and course of infection.

Starch phosphorylation-A needle in a haystack.

Phosphoesterification is the only naturally occurring covalent starch modification identified to date, and it has a major impact on overall starch metabolism. The incorporation of phosphate groups mediated by dikinases [α-glucan, water dikinase (GWD), EC 2.7.9.4; phosphoglucan, water dikinase (PWD), EC 2.7.9.5] massively alters the starch granule properties; however, previous studies did not determine whether the starch-related dikinases bind the phosphate to the glucosyl units within the amylopectin molecules in a specific pattern or randomly. In order to answer this challenging question, a number of approaches were initially pursued until a protocol could be established that enabled a massive step forward in the in vitro analysis of phosphorylated glucan chains obtained from starch. For this purpose, phosphorylation by GWD was investigated, including the final state of phosphorylation i.e., the state of substrate saturation when GWD lacks further free hydroxyl groups at OH-C6 for the catalysis of monophosphate esters. Since the separated phosphorylated glucan chains were required for the analysis, isoamylase digestion was performed to cleave the α-1,6-glycosidic bonds and to allow for the removal of the huge number of existing neutral chains by means of anion exchange chromatography. Via Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) MS and MALDI-MS/MS, the phosphorylated α-glucan chains were analysed, and the position of the phosphate group within the chain in relation to the reducing end was determined. Here, we demonstrate a protocol that enables the analysis of phosphorylated oligosaccharides, even in small quantities.

Mapping the Periostin splice isoforms in atopic dermatitis and an in vitro asthma model - A multi-platform analysis using mass spectrometry and RT-qPCR.

Periostin is a matricellular protein known to be alternatively spliced to produce ten isoforms with a molecular weight of 78-91 kDa. Within the extracellular matrix, periostin attaches to cell surfaces to induce signaling via integrin-binding and actively participates in fibrillogenesis, orchestrating the arrangement of collagen in the extracellular environment. In atopic diseases such as atopic dermatitis (AD) and asthma, periostin is known to participate in driving the disease-causing type 2 inflammation. The periostin isoforms expressed in these diseases and the implication of the alternative splicing events are unknown. Here, we present two universal assays to map the expression of periostin isoforms at the mRNA (RT-qPCR) and protein (PRM-based mass spectrometry) levels. We use these assays to study the splicing profile of periostin in AD lesions as well as in in vitro models of AD and asthma. In these conditions, periostin displayed overexpression with isoforms 3 and 5 standing out as highly overexpressed. Notably, isoforms 9 and 10 exhibited a divergent pattern relative to the remaining isoforms. Isoforms 9 and 10 are often overlooked in periostin research and this paper presents the first evidence of their expression at the protein level. This underlines the necessity to include isoforms 9 and 10 in future research addressing periostin splice isoforms. The assays presented in this paper hold the potential to improve our insight into the splicing profile of periostin in tissues and diseases of interest. The application of these assays to AD lesions and in vitro models demonstrated their potential for identifying isoforms of particular significance, warranting a further in-depth investigation.

Identification of Predictive Biomarkers of Lameness in Transition Dairy Cows.

The aim of this study was to identify with a high level of confidence metabolites previously identified as predictors of lameness and understand their biological relevance by carrying out pathway analyses. For the dairy cattle sector, lameness is a major challenge with a large impact on animal welfare and farm economics. Understanding metabolic alterations during the transition period associated with lameness before the appearance of clinical signs may allow its early detection and risk prevention. The annotation with high confidence of metabolite predictors of lameness and the understanding of interactions between metabolism and immunity are crucial for a better understanding of this condition. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with authentic standards to increase confidence in the putative annotations of metabolites previously determined as predictive for lameness in transition dairy cows, it was possible to identify cresol, valproic acid, and gluconolactone as L1, L2, and L1, respectively which are the highest levels of confidence in identification. The metabolite set enrichment analysis of biological pathways in which predictors of lameness are involved identified six significant pathways (p < 0.05). In comparison, over-representation analysis and topology analysis identified two significant pathways (p < 0.05). Overall, our LC-MS/MS analysis proved to be adequate to confidently identify metabolites in urine samples previously found to be predictive of lameness, and understand their potential biological relevance, despite the challenges of metabolite identification and pathway analysis when performing untargeted metabolomics. This approach shows potential as a reliable method to identify biomarkers that can be used in the future to predict the risk of lameness before calving. Validation with a larger cohort is required to assess the generalization of these findings.

Skin Surface Sebum Analysis by ESI-MS.

The skin surface is an important sample source that the metabolomics community has only just begun to explore. Alterations in sebum, the lipid-rich mixture coating the skin surface, correlate with age, sex, ethnicity, diet, exercise, and disease state, making the skin surface an ideal sample source for future noninvasive biomarker exploration, disease diagnosis, and forensic investigation. The potential of sebum sampling has been realized primarily via electrospray ionization mass spectrometry (ESI-MS), an ideal approach to assess the skin surface lipidome. However, a better understanding of sebum collection and subsequent ESI-MS analysis is required before skin surface sampling can be implemented in routine analyses. Challenges include ambiguity in definitive lipid identification, inherent biological variability in sebum production, and methodological, technical variability in analyses. To overcome these obstacles, avoid common pitfalls, and achieve reproducible, robust outcomes, every portion of the workflow-from sample collection to data analysis-should be carefully considered with the specific application in mind. This review details current practices in sebum sampling, sample preparation, ESI-MS data acquisition, and data analysis, and it provides important considerations in acquiring meaningful lipidomic datasets from the skin surface. Forensic researchers investigating sebum as a means for suspect elimination in lieu of adequate fingerprint ridge detail or database matches, as well as clinical researchers interested in noninvasive biomarker exploration, disease diagnosis, and treatment monitoring, can use this review as a guide for developing methods of best-practice.

Effect on serum metabolomics of rats with premature ovarian insufficiency by Zhibian (BL54) through Shuidao (ST28) acupuncture.

OBJECTIVE: To analyze the serum metabolic targets of the "Zhibian (BL54) through Shuidao (ST28)" acupuncture technique in cyclophosphamide (CTX)-induced premature ovarian insufficiency (POI) model rats and to elucidate the potential molecular mechanism of acupuncture in improving POI. METHODS: We used an intraperitoneal injection of CTX to establish the POI rat model (POI group) and compared serum hormone levels and ovarian histopathological changes to evaluate the effect of the Zhibian (BL54) through Shuidao (ST28) technique (ZS + POI group) on ovarian function. Then, nontargeted metabolomics was performed using rat serum by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). RESULTS: After acupuncture intervention, the serum hormone levels and ovarian pathological morphology of POI rats were effectively improved. Moreover, UPLC-Q-TOF/MS results showed that the ZS + POI group showed a significant reversal of the levels of 6 differential metabolites. Among them, the levels of four serum metabolic markers, divanillyltetrahydrofuran ferulate, trans-ferulic acid, tryptamine, and neuraminic acid, increased significantly. Further analysis of biological effects showed that all metabolites were involved in the regulation of reproductive hormone levels and antioxidant and antiapoptotic effects. CONCLUSIONS: The "Zhibian (BL54) through Shuidao (ST28)" acupuncture method may improve the ovarian function of POI rats by regulating serum metabolite markers to exert antioxidant and antiapoptotic effects, which provides a theoretical basis for the clinical application of acupuncture in the treatment of POI.

Native Mass Spectrometry-Centric Approaches Revealed That Neuropeptides Frequently Interact with Amyloid-ß.

Amyloid-ß (Aß) aggregates are recognized as initiators of Alzheimer's disease, and their interaction with the nervous system contributes to the progression of neurodegeneration. Herein, we investigated the frequency at which neuropeptides interact with Aß and affect the aggregation kinetics and cytotoxicity of Aß. To this end, we established a native mass spectrometry (MS)-centric workflow for screening Aß-interacting neuropeptides, and six out of 12 neuropeptides formed noncovalent complexes with Aß species in the MS gas phase. Thioflavin-T fluorescence assays and gel separation indicated that leptin and cerebellin decreased Aß aggregation, whereas kisspeptin increased this process. In addition, leptin and cerebellin attenuated Aß-induced cytotoxicity, which was independent of the influence of metal ions. Leptin can chelate copper from copper-bound Aß species, reducing the cytotoxicity caused by the aggregation of Aß and metal ion complexes. Overall, our study demonstrated that neuropeptides frequently interact with Aß and revealed that leptin and cerebellin are potential inhibitors of Aß aggregation, providing great insight into understanding the molecular mechanism of Aß interacting with the nervous system and facilitating drug development.

Assessing Per- and Polyfluoroalkyl Substances in Fish Fillet Using Non-Targeted Analyses.

Per- and polyfluoroalkyl substances (PFAS) are a class of thousands of man-made chemicals that are persistent and highly stable in the environment. Fish consumption has been identified as a key route of PFAS exposure for humans. However, routine fish monitoring targets only a handful of PFAS, and non-targeted analyses have largely only evaluated fish from heavily PFAS-impacted waters. Here, we evaluated PFAS in fish fillets from recreational and drinking water sources in central North Carolina to assess whether PFAS are present in these fillets that would not be detected by conventional targeted methods. We used liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) to collect full scan feature data, performed suspect screening using an in-house library of 100 PFAS for high confidence feature identification, searched for additional PFAS features using non-targeted data analyses, and quantified perfluorooctanesulfonic acid (PFOS) in the fillet samples. A total of 36 PFAS were detected in the fish fillets, including 19 that would not be detected using common targeted methods, with a minimum of 6 and a maximum of 22 in individual fish. Median fillet PFOS levels were concerningly high at 11.6 to 42.3 ppb, and no significant correlation between PFOS levels and number of PFAS per fish was observed. Future PFAS monitoring in this region should target more of these 36 PFAS, and other regions not considered heavily PFAS contaminated should consider incorporating non-targeted analyses into ongoing fish monitoring studies.

Aqueous Ionic Liquid Mixtures as Minimal Models of Lipid Bilayer Membranes.

We introduce aqueous ionic liquid (IL) mixtures, specifically mixtures of 1-butyl-3-imidazoliumtetrafluoroborate (BMImBF4), with water as a minimal model of lipid bilayer membranes. Imidazolium-based ILs are known to form clustered nanoscale structures in which local inhomogeneities, micellar or lamellar structures, are formed to shield hydrophobic parts of the cation from the polar cosolvent (water). To investigate these nanostructures, dynamic light scattering (DLS) on samples with different mixing ratios of water and BMImBF4 was performed. At mixing ratios of 50% and 45% (v/v), small and homogeneous nanostructures can indeed be detected. To test whether, in particular, these stable nanostructures in aqueous mixtures may mimic the effects of phospholipid bilayer membranes, we further investigated their interaction with myelin basic protein (MBP), a peripheral, intrinsically disordered membrane protein of the myelin sheath. Using dynamic light scattering (DLS), continuous wave (CW) and pulse electron paramagnetic resonance (EPR), and small-angle X-ray scattering (SAXS) on recombinantly produced, "healthy" charge variants rmC1WT and double cysteine variant C1S17CH85C, we find that the size and the shape of the determined nanostructures in an optimum mixture offer model membranes in which the protein exhibits native behavior. SAXS measurements illuminate the size and shape of the nanostructures and indicate IL-rich "beads" clipped together by functional MBP, one of the in vivo roles of the protein in the myelin sheath. All the gathered data combined indicate that the 50% and 45% aqueous IL mixtures can be described as offering minimal models of a lipid mono- or bilayer that allow native processing and potential study of at least peripheral membrane proteins like MBP.