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.

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.

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.

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.

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.

Filter-Based Protein Digestion (FPD): A Detergent-Free and Scaffold-Based Strategy for TMT Workflows.

High-throughput proteome profiling requires thorough optimization to achieve comprehensive analysis. We developed a filter aided sample preparation (FASP)-like, detergent-free method, termed Filter-Based Protein Digestion (FPD). We compared FPD to protein extraction methods commonly used in isobaric tag-based proteome profiling, namely trichloroacetic acid (TCA) and chloroform-methanol (C-M) precipitation. We divided a mammalian whole cell lysate from the SH-SY5Y neuroblastoma cell line for parallel protein processing with TCA (n = 3), C-M (n = 2), and FPD using either 10 kDa (n = 3) or 30 kDa (n = 3) molecular weight cutoff membranes. We labeled each sample with tandem mass tag (TMT) reagents to construct a TMT11-plex experiment. In total, 8654 proteins were quantified across all samples. Pairwise comparisons showed very little deviation for individual protein abundance measurements between the two FPD methods, whereas TCA and FPD showed the most difference. Specifically, membrane proteins were more readily quantified when samples were processed using TCA precipitation than other methods tested. However, globally, only 4% of proteins differed greater than 4-fold in the most divergent pair of protein extraction methods (i.e., FPD10 and TCA). We conclude that the detergent-free FPD strategy, particularly using the faster-flowing 30 kDa filter, is a seamless alteration to high-throughput TMT workflows.

Reversible polyphenylacetylene helix conversion driven by a thermoresponsive rotaxane switch in the solid state.

The reversible helical pitch change of polyphenylacetylenes by a thermoresponsive rotaxane switch inserted into the side chain was demonstrated through an accompanying color change. Similar to an ethynyl rotaxane monomer, the corresponding polyphenylacetylene having a rotaxane moiety in its side chain exhibited a reversible helical pitch change induced by the thermoresponsive rotaxane switch, i.e., the amine/ammonium salt was converted by treatment with trichloroacetic acid (TCA) and subsequent heating, which led to thermal decomposition of TCA to chloroform and carbon dioxide. Such a rotaxane switch caused the helical pitch change and the accompanying color change of the polymer main chain in both solution and solid state.

Probing Coagulation Behavior of Individual Aluminum Species for Removing Corresponding Disinfection Byproduct Precursors: The Role of Specific Ultraviolet Absorbance.

Coagulation behavior of aluminum chloride and polyaluminum chloride (PACl) for removing corresponding disinfection byproduct (DBP) precursors was discussed in this paper. CHCl3, bromine trihalomethanes (THM-Br), dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA) formation potential yields were correlated with specific ultraviolet absorbance (SUVA) values in different molecular weight (MW) fractions of humic substances (HS), respectively. Correlation analyses and principal component analysis were performed to examine the relationships between SUVA and different DBP precursors. To acquire more structural characters of DBP precursors and aluminum speciation, freeze-dried precipitates were analyzed by fourier transform infrared (FTIR) and C 1s, Al 2p X-ray photoelectron spectroscopy (XPS). The results indicated that TCAA precursors (no MW limits), DCAA and CHCl3 precursors in low MW fractions (MW<30 kDa) had a relatively good relations with SUVA values. These DBP precursors were coagulated more easily by in situ Al13 of AlCl3 at pH 5.0. Due to relatively low aromatic content and more aliphatic structures, THM-Br precursors (no MW limits) and CHCl3 precursors in high MW fractions (MW>30 kDa) were preferentially removed by PACl coagulation with preformed Al13 species at pH 5.0. Additionally, for DCAA precursors in high MW fractions (MW>30 kDa) with relatively low aromatic content and more carboxylic structures, the greatest removal occurred at pH 6.0 through PACl coagulation with aggregated Al13 species.

An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells.

PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and the breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the physiological levels of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radioimmunoprecipitation assay buffer was increased by 450-fold compared with trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells.

STIM1/ORAI1-mediated Ca2+ Influx Regulates Enolase-1 Exteriorization.

Tumor cells use broad spectrum proteolytic activity of plasmin to invade tissue and form metastatic foci. Cell surface-associated enolase-1 (ENO-1) enhances plasmin formation and thus participates in the regulation of pericellular proteolysis. Although increased levels of cell surface bound ENO-1 have been described in different types of cancer, the molecular mechanism responsible for ENO-1 exteriorization remains elusive. In the present study, increased ENO-1 protein levels were found in ductal breast carcinoma and on the cell surface of highly metastatic breast cancer cell line MDA-MB-231. Elevated cell surface-associated ENO-1 expression correlated with augmented MDA-MB-231 cell migratory and invasive properties. Exposure of MDA-MB-231 cells to LPS potentiated translocation of ENO-1 to the cell surface and its release into the extracellular space in the form of exosomes. These effects were independent of de novo protein synthesis and did not require the classical endoplasmic reticulum/Golgi pathway. LPS-triggered ENO-1 exteriorization was suppressed by pretreatment of MDA-MB-231 cells with the Ca(2+) chelator BAPTA or an inhibitor of endoplasmic reticulum Ca(2+)-ATPase pump, cyclopiazonic acid. In line with these observations, the stromal interaction molecule (STIM) 1 and the calcium release-activated calcium modulator (ORAI) 1-mediated store-operated Ca(2+) entry were found to regulate LPS-induced ENO-1 exteriorization. Pharmacological blockage or knockdown of STIM1 or ORAI1 reduced ENO-1-dependent migration of MDA-MB-231 cells. Collectively, our results demonstrate the pivotal role of store-operated Ca(2+) channel-mediated Ca(2+) influx in the regulation of ENO-1 exteriorization and thus in the modulation of cancer cell migratory and invasive properties.

Effect of dissolved oxygen concentration on iron efficiency: Removal of three chloroacetic acids.

The monochloroacetic, dichloroacetic and trichloroacetic acid (MCAA, DCAA and TCAA) removed by metallic iron under controlled dissolved oxygen conditions (0, 0.75, 1.52, 2.59, 3.47 or 7.09 mg/L DO) was investigated in well-mixed batch systems. The removal of CAAs increased first and then decreased with increasing DO concentration. Compared with anoxic condition, the reduction of MCAA and DCAA was substantially enhanced in the presence of O2, while TCAA reduction was significantly inhibited above 2.59 mg/L. The 1.52 mg/L DO was optimum for the formation of final product, acetic acid. Chlorine mass balances were 69-102%, and carbon mass balances were 92-105%. With sufficient mass transfer from bulk to the particle surface, the degradation of CAAs was limited by their reduction or migration rate within iron particles, which were dependent on the change of reducing agents and corrosion coatings. Under anoxic conditions, the reduction of CAAs was mainly inhibited by the available reducing agents in the conductive layer. Under low oxic conditions, the increasing reducing agents and thin lepidocrocite layer were favorable for CAA dechlorination. Under high oxic conditions, the redundant oxygen competing for reducing agents and significant lepidocrocite growth became the major restricting factors. Various CAA removal mechanisms could be potentially applied to explaining the effect of DO concentration on iron efficiency for contaminant reduction in water and wastewater treatment.

Urine sample preparation and fractionation for global proteome profiling by LC-MS.

Urine has garnered tremendous interest over the past decade as a potential source of protein biomarkers for various pathologies. However, due to its low protein concentration and the presence of interfering compounds, urine constitutes a challenging analyte in proteomics. In the context of a project aimed at the discovery and evaluation of new candidate biomarkers of bladder cancer in urine, our laboratory has implemented and evaluated an array of preparation techniques for urinary proteome analysis. We present here the protocol that, in our hands, yielded the best overall proteome coverage with the lowest analytical effort. It begins with protein precipitation using trichloroacetic acid, in solution digestion and RP-C18 cartridge desalting of the resulting peptides mixture, and is followed by peptide fractionation by gel-free isoelectric focusing, and nano-LC-MS/MS for database compilation.

Extraction and quantification of adenosine triphosphate in mammalian tissues and cells.

Adenosine 5'-triphosphate (ATP) is the "energy currency" of organisms and plays central roles in bioenergetics, whereby its level is used to evaluate cell viability, proliferation, death, and energy transmission. In this chapter, we describe an improved and efficient method for extraction of ATP from tissues and cells using phenol-based reagents. The chaotropic extraction reagents reported so far co-precipitate ATP with insoluble proteins during extraction and with salts during neutralization. In comparison, the phenol-based reagents extract ATP well without the risks of co-precipitation. The extracted ATP can be quantified by the luciferase assay or high-performance liquid chromatography.

Proteomic platform for the identification of proteins in olive (Olea europaea) pulp.

The nutritional and cancer-protective properties of the oil extracted mechanically from the ripe fruits of Olea europaea trees are attracting constantly more attention worldwide. The preparation of high-quality protein samples from plant tissues for proteomic analysis poses many challenging problems. In this study we employed a proteomic platform based on two different extraction methods, SDS and CHAPS based protocols, followed by two precipitation protocols, TCA/acetone and MeOH precipitation, in order to increase the final number of identified proteins. The use of advanced MS techniques in combination with the Swissprot and NCBI Viridiplantae databases and TAIR10 Arabidopsis database allowed us to identify 1265 proteins, of which 22 belong to O. europaea. The application of this proteomic platform for protein extraction and identification will be useful also for other proteomic studies on recalcitrant plant/fruit tissues.

Synthesis and electrocatalytic activity of haemin-functionalised iron(II, III) oxide nanoparticles.

Haemin-functionalised magnetic iron(II, III) oxide (Fe3O4) nanoparticles (Fe3O4/haemin) were synthesised by changing the acidity of a solution of the two compounds. The nanoparticles were characterised by transmission electron microscopy, UV-vis absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, measurement of magnetisation, and electrochemical techniques. The properties of both haemin and Fe3O4 were retained. Thus, Fe3O4/haemin nanoparticles exhibited pronounced electrocatalytic activity towards trichloroacetic acid (TCA) like haemin itself. Interestingly, electrocatalytic activity towards TCA was affected by detection temperature, which was controlled via electrically heated carbon paste electrodes. The maximal catalytic current was 5.8 times higher at 60°C than at room temperature (25°C). This proposed electrochemical sensor for TCA possessed a linear detection range of 5-80 µM and a detection limit of 0.3 µM at 60°C.

Haloacetic acid removal by sequential zero-valent iron reduction and biologically active carbon degradation.

An innovative haloacetic acid (HAA) removal process was developed. The process consisted of a zero-valent iron (Fe(0)) column followed by a biologically active carbon (BAC) column that were efficient in degrading tri- and di-HAAs, and mono- and di-HAAs, respectively. The merit of the process was demonstrated by its performance in removing trichloroacetic acid (TCAA). An empty bed contact time of 10 min achieved nearly complete removal of 1.2 µM TCAA and its subsequent products, dichloroacetic acid (DCAA) and monochloroacetic acid (MCAA). HAA removal was a result of chemical dehalogenation and biodegradation rather than physical adsorption. Preliminary kinetic analyses were conducted and the pseudo-first-order rate constants were estimated at ambient conditions for Fe(0) reduction of TCAA and biodegradation of DCAA and MCAA by BAC. This innovative process is highly promising in removing HAAs from drinking water, swimming pool water, and domestic or industrial wastewater.

Optimization of a microwave-coupled enzymatic digestion process to prepare peanut peptides.

The best enzyme to prepare peanut peptides, papain, coupled with microwave irradiation was selected from five common proteases according to the results of the yield of peanut peptides [nitrogen solution index (NSI) in trichloroacetic acid (TCA), TCA-NSI] and the degree of hydrolysis (DH). The main factors that influenced the microwave-coupled enzymatic digestion method were optimized by response surface analysis. The optimal conditions obtained were as follows: microwave extraction time, 9.5 min; power, 600 W; substrate concentration, 4%; enzymatic reaction temperature, 50 °C; enzyme quantity, 6,500 U/g; pH, 7.1 (phosphate buffer, 0.05 mol/L). Under these conditions, TCA-NSI was 62.00% and DH was 25.89%, which is higher than that obtained with either protease hydrolysis or microwave hydrolysis alone.

An efficient extraction method for quantitation of adenosine triphosphate in mammalian tissues and cells.

Firefly bioluminescence is widely used in the measurement of adenosine 5'-triphosphate (ATP) levels in biological materials. For such assays in tissues and cells, ATP must be extracted away from protein in the initial step and extraction efficacy is the main determinant of the assay accuracy. Extraction reagents recommended in the commercially available ATP assay kits are chaotropic reagents, trichloroacetic acid (TCA), perchloric acid (PCA), and ethylene glycol (EG), which extract nucleotides through protein precipitation and/or nucleotidase inactivation. We found that these reagents are particularly useful for measuring ATP levels in materials with relatively low protein concentrations such as blood cells, cultured cells, and bacteria. However, these methods are not suitable for ATP extraction from tissues with high protein concentrations, because some ATP may be co-precipitated with the insolubilized protein during homogenization and extraction, and it could also be precipitated by neutralization in the acid extracts. Here we found that a phenol-based extraction method markedly increased the ATP and other nucleotides extracted from tissues. In addition, phenol extraction does not require neutralization before the luciferin-luciferase assay step. ATP levels analyzed by luciferase assay in various tissues extracted by Tris-EDTA-saturated phenol (phenol-TE) were over 17.8-fold higher than those extracted by TCA and over 550-fold higher than those in EG extracts. Here we report a simple, rapid, and reliable phenol-TE extraction procedure for ATP measurement in tissues and cells by luciferase assay.

Proteomics of desiccation tolerance during development and germination of maize embryos.

Maize seeds were used to identify the key embryo proteins involved in desiccation tolerance during development and germination. Immature maize embryos (28N) during development and mature embryos imbibed for 72 h (72HN) are desiccation sensitive. Mature maize embryos (52N) during development are desiccation tolerant. Thiobarbituric acid reactive substance and hydrogen peroxide contents decreased and increased with acquisition and loss of desiccation tolerance, respectively. A total of 111 protein spots changed significantly (1.5 fold increase/decrease) in desiccation-tolerant and -sensitive embryos before (28N, 52N and 72HN) and after (28D, 52D and 72HD) dehydration. Nine pre-dominantly proteins, 17.4 kDa Class I heat shock protein 3, late embryogenesis abundant protein EMB564, outer membrane protein, globulin 2, TPA:putative cystatin, NBS-LRR resistance-like protein RGC456, stress responsive protein, major allergen Bet v 1.01C and proteasome subunit alpha type 1, accumulated during embryo maturation, decreased during germination and increased in desiccation-tolerant embryos during desiccation. Two proteins, Rhd6-like 2 and low-molecular-weight heat shock protein precursor, showed the inverse pattern. We infer that these eleven proteins are involved in seed desiccation tolerance. We conclude that desiccation-tolerant embryos make more economical use of their resources to accumulate protective molecules and antioxidant systems to deal with maturation drying and desiccation treatment.