Drug targets are biological macromolecules that bind drug molecules in vivo. Therefore, the system-wide identification of drug targets plays a vital role in fully understanding the mechanism of drug action, efficacy, and side effects. The unbiased screening of drug targets may accelerate the process of drug discovery and candidate screening. Mass spectrometry is a key tool for large-scale protein identification and accurate quantification owing to its high acquisition speed, resolution, and sensitivity. Mass spectrometry-based proteomics has been widely used for drug-target screening. It can systematically identify the protein-target landscape of a drug and elucidate drug-protein interactions. Commonly used drug-target characterization methods, such as labeling-based affinity enrichment, require the chemical derivatization of drug molecules, which is not only time-consuming but may also affect the affinity of the drug towards its targets. Furthermore, the spatial effects of the derivatization groups may block interactions between the drug and its targets. Considering the disadvantages of affinity-enrichment methods, strategies that do not require chemical derivatization have received widespread attention. Proteins may undergo denaturation, unfolding, and precipitation under different conditions such as high temperatures, extreme pH, denaturants, and mechanical stress. Binding to small-molecule drugs may alter the folding balance of target proteins. The conformational stability of target proteins can be stabilized by binding with drugs, and protein-drug complexes are more resistant than free proteins to the precipitation induced by different conditions. Based on this mechanism, various large-scale drug-target identification methods using protein precipitation have been developed by combining proteomics and mass spectrometry analysis, including thermal proteome profiling and solvent-, mechanical stress-, and pH-induced protein precipitation. These methods have been successfully applied to the characterization of small-molecule drug targets. In this review, we describe the protein precipitation-based methods used for the high-throughput discovery of drug targets and elucidation of the interactions between drugs and proteins in the past decade. We also summarize the characteristics of each method and discuss their application potential in drug-efficacy evaluation and drug discovery.

Racecadotril, an anti-secretory medication, has been used as an adjuvant in an oral rehydration therapy for children experiencing severe diarrhea. Racecadotril is quickly converted to thiorphan, an active metabolite, after oral treatment, which mediates all subsequent activities. An efficient and rapid liquid chromatography-tandem mass spectrometry method was developed and fully validated to measure thiorphan in human plasma, using thiorphan-d7 as an internal standard. The extraction method used was protein precipitation while chromatographic separation was achieved using InertSil CN-3 (50 × 2.1 mm, 5 µm column). The assay was linear over the concentration range of 1-200 ng/ml with correlation coefficients of ≥0.9991. The intra- and inter-day precisions were less than 10.0 % for all concentrations investigated. 0.02 % aqueous formic acid and methanol (30:70 v: v) were used as mobile phases, with an analysis time of less than 1 min. This method proved stable under several conditions. The developed method worked well in a three-period pharmacokinetic bioequivalence study after a single oral administration of 100 mg racecadotril to 15 healthy Jordanian volunteers under fasting conditions.

Two protocols of protein extraction from Arthrospira platensis (spirulina) microalgae to study their proteome by mass spectrometry (MS) are here presented. The first is based on an aqueous buffer solution of Tris-HCl and the second on cold acetone. The identification of proteins was carried out by a bottom-up approach, which involves enzymatic digestion of extracted proteins followed by either matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) MS or liquid chromatography (LC) coupled with electrospray ionization (ESI) and Fourier-transform tandem MS. While MALDI-TOF MS allowed for a fast peptide mass fingerprinting (PMF) check yet identifying less than 20 proteins in the extracted samples, the data-dependent acquisitions (DDA) mode of reversed-phase (RP) LC-ESI tandem FTMS/MS separations allowed us to recognize more than one hundred proteins by searching into dedicated spectral libraries. The application of MALDI-TOF MS analysis was found, however, of great support for preliminary investigations of cyanobacteria samples before proceeding with the RPLC-ESI-MS/MS DDA investigation, which definitively allows for a qualitative proteome analysis also of minor spirulina proteins in processed foodstuffs. Although the protein content in spirulina can be influenced by cultivation and environmental conditions, e.g., light intensity, climate, and water/air quality, here the qualitative chemical profiles of the examined samples were characterized by similar composition in high-quality proteins as phycocyanins and phycoerythrins.

Virus-like particles (VLPs) are a promising class of biopharmaceuticals for vaccines and targeted delivery. Starting from clarified lysate, VLPs are typically captured by selective precipitation. While VLP precipitation is induced by step-wise or continuous precipitant addition, current monitoring approaches do not support the direct product quantification, and analytical methods usually require various, time-consuming processing and sample preparation steps. Here, the application of Raman spectroscopy combined with chemometric methods may allow the simultaneous quantification of the precipitated VLPs and precipitant owing to its demonstrated advantages in analyzing crude, complex mixtures. In this study, we present a Raman spectroscopy-based Process Analytical Technology (PAT) tool developed on batch and fed-batch precipitation experiments of Hepatitis B core Antigen VLPs. We conducted small-scale precipitation experiments providing a diversified data set with varying precipitation dynamics and backgrounds induced by initial dilution or spiking of clarified Escherichia coli-derived lysates. For the Raman spectroscopy data, various preprocessing operations were systematically combined allowing the identification of a preprocessing pipeline, which proved to effectively eliminate initial lysate composition variations as well as most interferences attributed to precipitates and the precipitant present in solution. The calibrated partial least squares models seamlessly predicted the precipitant concentration with R 2 of 0.98 and 0.97 in batch and fed-batch experiments, respectively, and captured the observed precipitation trends with R 2 of 0.74 and 0.64. Although the resolution of fine differences between experiments was limited due to the observed non-linear relationship between spectral data and the VLP concentration, this study provides a foundation for employing Raman spectroscopy as a PAT sensor for monitoring VLP precipitation processes with the potential to extend its applicability to other phase-behavior dependent processes or molecules.

BACKGROUND: Therapeutic drug monitoring of treatment with therapeutic antibodies is hampered by the application of a wide range of different methods in the quantification of serum levels. LC-MS based methods could significantly improve comparability of results from different laboratories, but such methods are often considered complicated and costly. We developed a method for LC-MS/MS based quantification of 11 therapeutic antibodies concomitantly measured in a single run, with emphasis on simplicity in sample preparation and low cost.
RESULTS: After a single-step sample purification using caprylic acid precipitation to remove interfering proteins, the sample underwent proteolysis followed by LC-MS/MS analysis. Infliximab is used as internal standard for sample preparation while isotope-labeled signature peptides identified for each analyte are internal standards for the LC-MS/MS normalization. The method was validated according to recognized guidelines, and pipetting steps can be performed by automated liquid handling systems. The total precision of the method ranged between 2.7 and 7.3 % (5.1 % average) across the quantification range of 4-256 μg/ml for all 11 drugs, with an average accuracy of 96.3 %. Matrix effects were xamined in 55 individual patient samples instead of the recommended 6, and 147 individual patient samples were screened for interfering compounds.
UNASSIGNED: Our method for simultaneous quantification of 11 t-mAb in human serum allows an unprecedented integration of robustness, speed and reduced complexity, which could pave the way for uniform use in research projects and clinical settings alike. In addition, the first LC-MS protocol for signature peptide-based quantification of durvalumab is described. This high throughput method can be readily adapted to a drug panel of choice.

Pesticides play an important role in forensic toxicology and are usually classified as a single class of chemicals. Despite their commonly perceived unity, pesticides encompass a spectrum of compounds, including organophosphates, carbamates, pyrethroids or organochlorines, among others, each with varying degrees of toxicity. Pesticide analysis in post-mortem samples can be difficult due to the complexity of the samples and to the high toxicity of these compounds. The aim of this study was to develop and validate an easy to use, sensitive, and robust method, using ultra-performance liquid chromatography-tandem mass spectrometry to be incorporated in the routine flow for pesticide analysis in post-mortem blood samples. Described herein is a streamlined, expeditious, yet highly efficient method facilitating the screening, qualitative assessment, and quantitative confirmation of 15 pesticides, including acetamiprid, azinphos-ethyl, bendiocarb, carbofuran, chlorfenvinphos, dimethoate, imidaclopride, malathion, methiocarb, methomyl, parathion, pirimicarb, strychnine, tetrachlorvinphos, and thiacloprid in post-mortem blood, recognizing the pivotal role blood plays in forensic investigations. The developed method was linear from 10 to 200 ng/mL; limits of detection were between 1 and 10 ng/mL, depending on the compound; it was successfully evaluated a dilution ratio of 1-2, 5 and 10; and 8 substances showed maximum stability for the time interval studied. This UHPLC-MS/MS method is useful and a powerful tool in a toxicology lab because it is fast, simple, effective, and trustworthy. The results of this validation highlight the robustness of the analytical method, providing a valuable tool for the accurate and sensitive detection of pesticides in post-mortem blood. Poised for routine implementation, this method has already found success in suspected intoxication cases, promising to elevate the standards of forensic pesticide analysis.

The protein extraction method based on the phenol solution and combined with protein precipitation with ammonium acetate in methanol and purification in the same solution, and additionally in acetone and ethanol, is recommended for proteomic studies of plant tissues. The obtained protein samples do not require additional nucleic acid digestion and removal of interfering contaminations. The presented protocol was used to analyze the proteome of common buckwheat flowers and leaves.

Nicotinamide adenine dinucleotide (NAD) is a cofactor in redox reactions and an essential mediator of energy metabolism. The redox balance between NAD+ and NADH affects various diseases, cell differentiation, and aging, and in recent years there has been a growing need for measurement techniques with improved accuracy. However, NAD(H) measurements, representing both NAD+ and NADH, have been limited by the compound\'s properties. We achieved highly sensitive simultaneous measurement of NAD+ and NADH under non-ion pairing, mobile phase conditions of water, or methanol containing 5 mM ammonium acetate. These were achieved using a simple pre-treatment and 7-min analysis time. Use of the stable isotope 13C5-NAD+ as an internal standard enabled validation close to BMV criteria and demonstrated the robustness of NAD(H) determination. Measurements using this method showed that brain NAD(H) levels correlate strongly with plasma NAD(H) levels in the same mouse, indicating that NAD(H) concentrations in brain tissue are reflected in plasma. As NAD(H) is involved in various neurodegenerative diseases and cerebral ischemia, as well as brain diseases such as mitochondrial myopathies, monitoring changes in NADH levels in plasma after drug administration will be useful for development of future diagnostics and therapeutics.

Aims: Cyanoenone triterpenoids penetrate the CNS, exhibiting biological activity via the nuclear factor E2-related factor (Nrf2) pathway. This is the first report on methods for the quantification of cyanonenone triterpenoids\' distribution in various CNS tissues by LC-MS/MS. Materials & methods: The analyte was extracted from brain tissue homogenate using protein precipitation and supported liquid extraction. Results & conclusion: The assay validated a quantification range of 3.00-3000 ng/g in brain tissue samples as low as 5 mg. All parameters, including interference (≤20% at LLOQ) and accuracy/precision (15%, with 20% at LLOQ), met acceptance criteria. This assay supported a CNS distribution study, analyzing more than 10 mouse brain regions successfully.

Soy protein concentrates (SPCs) are common food ingredients. They typically contain 65% (w/w) protein and ∼30% (w/w) carbohydrate. SPCs can be obtained with various protein precipitation conditions. A systematic study of the impact of these different protein precipitation protocols on the SPC protein composition and physical properties is still lacking. Here, SPCs were prepared via three different protocols, that is, isoelectric (pH 3.5-5.5), aqueous ethanol (50%-70% [v/v]), and Ca2+ ion (5-50 mM) based precipitations, and analyzed for (protein) composition, protein thermal properties, dispersibility, and water-holding capacity. SPCs precipitated at pH 5.5 or by adding 15 mM Ca2+ ions had a lower 7S/11S globulin ratio (∼0.40) than that (∼0.50) of all other SPC samples. Protein in SPCs obtained by isoelectric precipitation denatured at a significantly higher temperature than those in ethanol- or Ca2+ -precipitated SPCs. Precipitation with 50%-60% (v/v) ethanol resulted in pronounced denaturation of 2S albumin and 7S globulin fractions in SPCs. Additionally, increasing the precipitation pH from 3.5 to 5.5 and increasing the Ca2+ ion concentration from 15 to 50 mM caused a strong decrease of both the dispersibility of the protein in SPC and its water-holding capacity at pH 7.0. In conclusion, this study demonstrates that the SPC production process can be directed to obtain ingredients with versatile protein physicochemical properties toward potential food applications. PRACTICAL APPLICATION: This study demonstrates that applying different protein precipitation protocols allows obtaining SPCs that vary widely in (protein) composition and physical properties (such as protein dispersibility and water-holding capacity). These varying traits can greatly influence the suitability of SPCs as functional ingredients for specific applications, such as the production of food foams, emulsions, gels, and plant-based meat alternatives. The generated knowledge may allow targeted production of SPCs for specific applications.