Polar metabolomics using trichloroacetic acid extraction and porous graphitic carbon stationary phase.

INTRODUCTION: Accurately identifying and quantifying polar metabolites using untargeted metabolomics has proven challenging in comparison to mid to non-polar metabolites. Hydrophilic interaction chromatography and gas chromatography-mass spectrometry are predominantly used to target polar metabolites. OBJECTIVES: This study aims to demonstrate a simple one-step extraction combined with liquid chromatography-mass spectrometry (LC-MS) that reliably retains polar metabolites. METHODS: The method involves a MilliQ + 10% trichloroacetic acid extraction from 6 healthy individuals serum, combined with porous graphitic carbon liquid chromatography-mass spectrometry (LC-MS). The coefficient of variation (CV) assessed retention reliability of polar metabolites with logP as low as - 9. QreSS (Quantification, Retention, and System Suitability) internal standards determined the method's consistency and recovery efficiency. RESULTS: The method demonstrated reliable retention (CV < 0.30) of polar metabolites within a logP range of - 9.1 to 5.6. QreSS internal standards confirmed consistent performance (CV < 0.16) and effective recovery (70-130%) of polar to mid-polar metabolites. Quality control dilution series demonstrated that ~ 80% of annotated metabolites could be accurately quantified (Pearson's correlation coefficient > 0.80) within their concentration range. Repeatability was demonstrated through clustering of repeated extractions from a single sample. CONCLUSION: This LC-MS method is better suited to covering the polar segment of the metabolome than current methods, offering a reliable and efficient approach for accurate quantification of polar metabolites in untargeted metabolomics.

Extraction of Proteins from Green Tissues of Plants and Phyllosphere.

The proteomic approach plays a key role to characterize a biological system at any given time. In recent years, advances in proteomics have led to an increasing application in all biological fields, including plant matrices and associated microbiome studies. However, extracting adequate protein samples remains the most critical step for any plant proteomics study. The protein extraction protocols proposed for the phyllosphere involve an initial leaf washing step; however, this is an approach only applicable if interest is restricted to epiphytes. A metaproteomic approach is required to obtain an overall picture and consequently an extraction that considers proteins derived from the plant, epiphytic and endophytic microorganisms. The most commonly used extractions for plant tissue involve the use of phenol or TCA-acetone. However, for efficient protein recovery is essential to remove interfering components abundant in plant tissues, such as polysaccharides, lipids, and phenolic compounds. A well-proven protocol on the basis of a combination of TCA-acetone and phenol extraction is presented here, obtaining some cleaned protein pellets, suitable for electrophoresis and subsequent proteomics studies. Important points for the success of this protocol are (i) a proper sampling and sample preparation, (ii) maintaining samples at a low temperature during extraction and using protease inhibitors, (iii) an initial step in TCA-acetone to remove part of the interfering substances, and (iv) careful recovery of the phenolic phase. Furthermore, the protocol is timesaving and can be completed in one working day.

Computational Binding Analysis of Ethyl 3,3,5,5-Tetracyano-2-Hydroxy-2-Methyl-4,6-Diphenylcyclohexane-1-Carboxylate in Calf Thymus DNA.

In the present paper, several computational binding analyses were performed on ethyl 3,3,5,5-tetracyano-2-hydroxy-2-methyl-4,6-diphenylcyclohexane-1-carboxylate which was newly synthesized by three-component condensation of benzaldehyde with ethyl acetoacetate and malononitrile in the presence of trichloroacetic acid, and the structure was finally proved by X-ray analysis. The visualization of molecular interaction was carried out through Hirshfeld surface analysis and ESP. The atomic charges, HOMO, LUMO, and electrostatic potential were also studied to explore the insight of the molecule deeper, and then, natural bonding orbitals (NBO) and non-linear optical properties (NLO) were calculated to reveal the interactions that happen to be between the filled and vacant orbitals. Afterwards, molecular docking studies predicted the compound binding mode fits in the minor groove of DNA and remained interacts via stable bonding as validated by molecular dynamics simulations. The binding energy estimation also affirmed domination van der Waals and electrostatic energies. Lastly, the compound was found as good drug-like molecule and had good pharmacokinetic profile with exception of toxic moieties.

Amyloid-Templated Palladium Nanoparticles for Water Purification by Electroreduction.

Electrocatalysis offers great promise for water purification but is limited by low active area and high uncontrollability of electrocatalysts. To overcome these constraints, we propose hybrid bulk electrodes by synthesizing and binding a Pd nanocatalyst (nano-Pd) to the electrodes via amyloid fibrils (AFs). The AFs template is effective for controlling the nucleation, growth, and assembly of nano-Pd on the electrode. In addition, the three-dimensional hierarchically porous nanostructure of AFs is beneficial for loading high-density nano-Pd with a large active area. The novel hybrid cathodes exhibit superior electroreduction performance for the detoxification of hexavalent chromium (Cr6+ ), 4-chlorophenol, and trichloroacetic acid in wastewater and drinking water. This study provides a proof-of-concept design of an AFs-templated nano-Pd-based hybrid electrode, which constitutes a paradigm shift in electrocatalytic water purification, and broadens the horizon of its potential engineered applications.

High-Performance Trichloroacetic Acid Sensor Based on the Intramolecular Hydrogen Bond Formation and Disruption of a Specially Designed Fluorescent o-Carborane Derivative in the Film State.

Discriminative and sensitive detection of environmentally important and health-related trichloroacetic acid (TCA) suffers from various problems such as bulky instruments and time-consuming operation as well as complex sample processing. Herein, we present a rapid, sensitive, and specific method for the detection of gaseous TCA using a fluorescent single-molecule array. An o-carborane-based benzothiazole derivative (CB-BT-OCH3) with specific fluorescence properties was specifically designed and utilized to fabricate a film-based single-molecule array. It was revealed that the fluorescent film is photochemically stable and extremely sensitive to TCA vapor, depicting an observable fluorescence color change from green to blue. The experimental detection limit is 0.2 ppm, which is lower than the safety limit (1 ppm) required by the threshold limit values and biological exposure indices. In addition, the film could show detectable intensity change within 0.2 s. On the basis of multiple signal responses, a conceptual two-channel-based fluorescent TCA sensor was developed. Importantly, the proposed conceptual sensor paves a new route to the development of specific fluorescent film-based sensor arrays with a single fluorophore as sensing units.

Novel Core-Sheath Cu/Cu2O-ZnO-Fe3O4 Nanocomposites with High-Efficiency Chlorine-Resistant Bacteria Sterilization and Trichloroacetic Acid Degradation Performance.

In order to solve two issues of chlorine-resistant bacteria (CRB) and disinfection byproducts (DBPs) in tap water after the chlorine-containing treatment process, an innovative core-sheath nanostructured Cu/Cu2O-ZnO-Fe3O4 was designed and synthesized. The fabrication mechanism of the materials was then systematically analyzed to determine the component and valence state. The properties of CRB inactivation together with trichloroacetic acid (TCAA) photodegradation by Cu/Cu2O-ZnO-Fe3O4 were investigated in detail. It was found that Cu/Cu2O-ZnO-Fe3O4 displayed excellent antibacterial activity with a relatively low cytotoxicity concentration due to its synergism of nanowire structure, ion release, and reactive oxygen species generation. Furthermore, the Cu/Cu2O-ZnO-Fe3O4 nanocomposite also exhibited outstanding photocatalytic degradation activity on TCAA under simulated sunlight irradiation, which was verified to be dominated by the surface reaction through kinetic analysis. More interestingly, the cell growth rate of Cu/Cu2O-ZnO-Fe3O4 was determined to be 50% and 10% higher than those of Cu/Cu2O and Cu/Cu2O-ZnO after 10 h incubation, respectively, manifesting a weaker cytotoxicity. Therefore, the designed Cu/Cu2O-ZnO-Fe3O4 could be a promising agent for tap water treatment.

The impact of food-grade carrageenans and consumer age on the in vitro proteolysis of whey proteins.

The food additive carrageenan (E407) (CGN) is a family of sulphated galactans widely used in numerous processed foods, including dairy. There are various indications that CGN may hinder digestive proteolysis. This study sought to link CGN macromolecular characteristics to its implications on digestive proteolysis of whey protein isolate (WPI) in toddlers, adults and seniors. Size exclusion chromatography and dynamic laser scattering reveal commercial CGN samples differ in molecular weight distributions, zeta-potentials and flow behavior of WPI-CGN mixtures. Moreover, κ-CGN, ι-CGN and λ-CGN were found to contain low MW (<200 kDa) fractions at levels of 6.36 ± 2.11% (w/w), 3.64 ± 1.06% (w/w) and 2.08 ± 1.41% (w/w), respectively. In vitro human digestion of WPI-CGN mixtures and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of digesta indicate that CGN alters the breakdown of alpha-lactalbumin, beta-lactoglobulin and lactoferrin differentially in toddlers, adults and seniors digestion conditions. Interestingly, proteomic analyses indicate there is a possible correlation between CGN degree of sulphation and the release of bioactive peptide homologues in the gut lumen. Moreover, these analyses indicate CGN compromises the bioaccessibility of essential amino acids. Altogether, this study shows CGN may attenuate whey digestive proteolysis. This effect should be taken in account by food manufacturers and regulatory agencies in view of the rising levels of exposure to CGN in the human diet.

Highly sensitive UPLC-MS/MS method for the quantification of paromomycin in human plasma.

A highly sensitive method was developed to quantitate the antileishmanial agent paromomycin in human plasma, with a lower limit of quantification of 5 ng/mL. Separation was achieved using an isocratic ion-pair ultra-high performance liquid chromatographic (UPLC) method with a minimal concentration of heptafluorobutyric acid, which was coupled through an electrospray ionization interface to a triple quadrupole - linear ion trap mass spectrometer for detection. The method was validated over a linear calibration range of 5 to 1000 ng/mL (r2≥0.997) with inter-assay accuracies and precisions within the internationally accepted criteria. Volumes of 50 µL of human K2EDTA plasma were processed by using a simple protein precipitation method with 40 µL 20 % trichloroacetic acid. A good performance was shown in terms of recovery (100 %), matrix effect (C.V. ≤ 12.0 %) and carry-over (≤17.5 % of the lower limit of quantitation). Paromomycin spiked to human plasma samples was stable for at least 24 h at room temperature, 6 h at 35 °C, and 104 days at -20 °C. Paromomycin adsorbs to glass containers at low concentrations, and therefore acidic conditions were used throughout the assay, in combination with polypropylene tubes and autosampler vials. The assay was successfully applied in a pharmacokinetic study in visceral leishmaniasis patients from Eastern Africa.

Challenges in the bioanalysis of tetracyclines: Epimerisation and chelation with metals.

Tetracyclines (TCs) are important broad spectrum antibiotics which are active against gram-positive and gram-negative bacteria. TCs readily form epimers, especially under weakly acidic conditions. The epimers are reported to have different antibacterial and toxicological properties and pose a significant challenge for selective bioanalysis, being isobaric with the parent drug and possessing very similar physicochemical properties. During the development, validation and use of bioanalytical methods for minocycline in plasma, urine and renal dialysate there were two unexpected findings. The first was that the analyte and the internal standard, tetracycline, were found to be unexpectedly stable and resistant towards epimerisation in the presence of the deproteinising agent, trichloroacetic acid (TCA). The second was that keeping minocycline spiked dialysate in a freezer led to significant losses which were worse for low concentrations at lower storage temperatures. Investigations into the stability of tetracycline, minocycline, omadacycline and tigecycline in aqueous acidic solutions, under typical analytical conditions, revealed that TCA acts as a stabiliser with respect to both epimerisation and other degradation pathways for these TCs. This gives the rarely used TCA a significant advantage over the commonly used deproteinising agents such as acetonitrile when analysing TCs. Studies of the recoveries of tetracycline and tigecycline from frozen renal dialysis buffer demonstrated similar losses to those for minocycline. These were assigned to deposition of insoluble Mg2+ or Ca2+ complexes on freezing, as pre-storage treatment of the samples by incubation in EDTA coated tubes at room temperature prevented the losses. Minocycline was stable in renal dialysis buffer samples when frozen, for up to ca. 3 months, when this treatment was employed. The TCs were analysed using LC-MS/MS based methods developed in-house using the assay that was originally developed for minocycline in plasma, urine and dialysate as a template.

Malondialdehyde determination in raw and processed meat products by UPLC-DAD and UPLC-FLD.

The objective of this study was to develop an accurate and fast method to determine malondialdehyde (MDA) levels in raw and processed meat. This method is based on extraction with trichloroacetic acid (TCA), reaction with 2-thiobarbituric acid (TBA) and quantification with ultraperformance liquid chromatography with a fluorescence detector (UPLC-FLD) with ʎexcitation = 530 nm and ʎemission = 550 nm and with a diode array detector (UPLC-DAD) with ʎabsorbance = 532 nm. The method tested was compared with the TBARS spectrophotometric method with ʎabsorbance = 532 nm. The concentration of MDA was similar for most of the matrices in both UPLC methods, except for cooked ham and frankfurter sausage. The TBARS spectrophotometric method overestimated the MDA concentration in all the matrices. Therefore, the use of both chromatographic methods, especially UPLC-FLD, to determine MDA would be more advisable than the classic TBARS method to avoid overestimation in meat and processed meat products.

Electrochemically Sensing of Trichloroacetic Acid with Iron(II) Phthalocyanine and Zn-Based Metal Organic Framework Nanocomposites.

Rapid and accurate determination of disinfection byproducts (DBPs) has become an emerging need for environmental monitoring and has yet to be realized in electrochemical sensors with metal organic framework (MOF)-based materials. In this study, a highly sensitive electrochemical sensor for trichloroacetic acid (TCAA) detection based on iron(II) phthalocyanine (PcFe) and a Zn-based metal organic framework (ZIF-8) composite is fabricated. As an electrode material, ZIF-8 possesses a large surface area and porous structure, which exhibits high absorbability; meanwhile, PcFe (II), as the sensing element, undergoes a reduction process from PcFe (II) to PcFe (I) during the sensing process. In the presence of TCAA, PcFe (I) is reoxidized by TCAA, which shifts the reaction equilibrium and accelerates the electron transfer on the electrode interface. By analyzing the reduction current of PcFe (II), the quantitative detection of TCAA is realized. The sensor shows a limit of detection (LOD) of 1.89 nM, which is superior to other reported TCAA sensors, as well as a high sensitivity (826 µΑ/µM). Moreover, the good selectivity and stability of this sensing platform demonstrate its capability and promise in determination of trace DBPs. The reported sensor provides a new strategy for electrochemical detection of DBPs and could expand the applications of MOFs in emerging technologies for monitoring contaminants.

Quantitation of Super Basic Peptides in Biological Matrices by a Generic Perfluoropentanoic Acid-Based Liquid Chromatography-Mass Spectrometry Method.

Peptides represent a promising modality for the design of novel therapeutics that can potentially modulate traditionally non-druggable targets. Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are two large families that are being explored extensively as drug delivery vehicles, imaging reagents, or therapeutic treatments for various diseases. Many CPPs and AMPs are cationic among which a significant portion is extremely basic and hydrophilic (e.g., nona-arginine). Despite their attractive therapeutic potential, it remains challenging to directly analyze and quantify these super cationic peptides from biological matrices due to their poor chromatographic behavior and MS response. Herein, we describe a generic method that combines solid phase extraction and LC-MS/MS for analysis of these peptides. As demonstrated, using a dozen strongly basic peptides, low µM concentration of perfluoropentanoic acid (PFPeA) in the mobile phase enabled excellent compound chromatographic retention, thus avoiding co-elution with solvent front ion suppressants. PFPeA also had a charge reduction effect that allowed the selection of parent/ion fragment pairs in the higher m/z region to further reduce potential low molecular weight interferences. When the method was coupled to the optimized sample extraction process, we routinely achieved low digit ng/ml sensitivity for peptides in plasma/tissue. The method allowed an efficient evaluation of plasma stability of CPPs/AMPs without fluorescence derivatization or other tagging methods. Importantly, using the widely studied HIV-TAT CPP as an example, the method enabled us to directly assess its pharmacokinetics and tissue distribution in preclinical animal models.

Identification of N-terminal protein processing sites by chemical labeling mass spectrometry.

RATIONALE: Proteins undergo post-translational modifications and proteolytic processing that can affect their biological function. Processing often involves the loss of single residues. Cleavage of signal peptides from the N-terminus is commonly associated with translocation. Recent reports have suggested that other processing sites also exist. METHODS: The secreted proteins from S. aureus N315 were precipitated with trichloroacetic acid (TCA) and amidinated with S-methyl thioacetimidate (SMTA). Amidinated proteins were digested with trypsin and analyzed with a high-resolution orbitrap mass spectrometer. RESULTS: Sixteen examples of Staphylococcus aureus secretory proteins that lose an N-terminal signal peptide during their export were identified using this amidination approach. The N-termini of proteins with and without methionine were identified. Unanticipated protein cleavages due to sortase and an unknown protease were also uncovered. CONCLUSIONS: A simple N-terminal amidination based mass spectrometry approach is described that facilitates identification of the N-terminus of a mature protein and the discovery of unexpected processing sites.

Measurement of Radioactivity in Nucleic Acids.

Radioactive isotopes are used as tracers to monitor the progress of many reactions used to synthesize DNA and RNA. To calculate the efficiency of such reactions, it is necessary to measure accurately the proportion of the radioactive precursor incorporated into the desired product. An effective method to achieve this goal is differential precipitation of the nucleic acid products with trichloroacetic acid (TCA).

Secretome analysis of nerve repair mediating Schwann cells reveals Smad-dependent trophism.

Schwann cells promote nerve regeneration by adaptation of a regenerative phenotype referred to as repair mediating Schwann cell. Down-regulation of myelin proteins, myelin clearance, formation of Bungner's bands, and secretion of trophic factors characterize this cell type. We have previously shown that the sphingosine-1-phosphate receptor agonist Fingolimod/FTY720P promotes the generation of this particular Schwann cell phenotype by activation of dedifferentiation markers and concomitant release of trophic factors resulting in enhanced neurite growth of dorsal root ganglion neurons. Despite its biomedical relevance, a detailed characterization of the corresponding Schwann cell secretome is lacking, and the impact of FTY720P on enhancing neurite growth is not defined. Here, we applied a label-free quantitative mass spectrometry approach to characterize the secretomes derived from primary neonatal and adult rat Schwann cells in response to FTY720P. We identified a large proportion of secreted proteins with a high overlap between the neonatal and adult Schwann cells, which can be associated with biologic processes such as development, axon growth, and regeneration. Moreover, FTY720P-treated Schwann cells release proteins downstream of Smad signaling known to support neurite growth. Our results therefore uncover a network of trophic factors involved in glial-mediated repair of the peripheral nervous system.-Schira, J., Heinen, A., Poschmann, G., Ziegler, B., Hartung, H.-P., Stühler, K., Küry, P. Secretome analysis of nerve repair mediating Schwann cells reveals Smad-dependent trophism.

Formation kinetics of disinfection byproducts in algal-laden water during chlorination: A new insight into evaluating disinfection formation risk.

It is necessary and important to investigate the formation of disinfection byproducts (DBPs) in water treatment systems for the management of disinfection formation risk. In the present study, the formation potential of trichloromethane (TCM) and haloacetic acids in different algal metabolites were compared, and the formation kinetics of these DBPs were investigated. The results indicated that DBP formation potential, the traditional index widely used to evaluate the disinfection formation risk, can represent neither the total precursors of DBPs nor the possible generated amounts of DBPs in drinking water systems. Kinetic analyses showed that the formation of DBPs could be well described by a classical second-order reaction kinetic model and that the actual concentrations of DBPs during chlorination were predictable with the model. The formation of DBPs in drinking water treatment systems was highly dependent on the total precursors of DBPs in water and the formation rate of DBPs with chlorine; the latter is usually underestimated in previous studies. Because of their high reactivity, TCM in hydrophilic extracellular organic matter and trichloroacetic acid in all algal metabolites should be serious considerations in the management of disinfection formation risk.

Modified TCA/acetone precipitation of plant proteins for proteomic analysis.

Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.

Formation of disinfection by-products during chlorination of organic matter from phoenix tree leaves and Chlorella vulgaris.

To better understand the precursor of disinfection by-products (DBPs) and provide useful information for water utilities to manage the drinking water, a study of DBP formation was conducted through chlorination of leaf organic matter (OM) from phoenix tree and algal OM from Chlorella vulgaris. DBPs investigated include trichloromethane (TCM), trichloroacetic acid (TCAA), dichloroacetic acid (DCAA), chloroacetic acid (CAA), dichloroacetonitrile (DCAN) and trichloroacetonitrile (TCNM). Results show that the specific yields (µg/mg C) of C-DBPs (TCM, CAA, DCAA and TCAA) from leaf OM were higher but the specific yields of N-DBPs (DCAN and TCNM) were lower than those from algal OM. Correlation analysis revealed that C-DBPs yields (µg/L) were significantly (p < 0.01) interrelated with each other (r = 0.937-0.996), and for each C-DBP, the hydrophobic OM contributed more to their formation (61-90% of total yields) as compared with hydrophilic OM. In spite of these characteristics, an in-depth examination was conducted revealing that the hydrophobicity and aromaticity of C-DBPs precursors were in the order of TCAA > DCAA & TCM > CAA. DCAN precursors were highly variable: they were dominated by hydrophobic OM (leaf OM: 86%) or hydrophilic OM (algal OM: 61%). Hydrophilic OM was the most important precursor for TCNM (76-79% of total yields), followed by hydrophobic neutral and base substances (29-45% of total yields), but the hydrophobic acids exhibited an inhibition role in TCNM formation.

Utilizing two detectors in the measurement of trichloroacetic acid in human urine by reaction headspace gas chromatography.

A reaction headspace gas chromatography (HS-GC) technique was investigated for quantitatively analyzing trichloroacetic acid in human urine. This method is based on the decomposition reaction of trichloroacetic acid under high-temperature conditions. The carbon dioxide and chloroform formed from the decomposition reaction can be respectively detected by the thermal conductivity detection HS-GC and flame ionization detection HS-GC. The reaction can be completed in 60 min at 90°C. This method was used to quantify 25 different human urine samples, which had a range of trichloroacetic acid from 0.52 to 3.47 mg/L. It also utilized two different detectors, the thermal conductivity detector and the flame ionization detector. The present reaction HS-GC method is accurate, reliable and well suitable for batch detection of trichloroacetic acid in human urine.