Highly sensitive detection of plant growth regulators by using terahertz time-domain spectroscopy combined with metamaterials.

The rapid and sensitive detection of plant-growth-regulator (PGR) residue is essential for ensuring food safety for consumers. However, there are many disadvantages in current approaches to detecting PGR residue. In this paper, we demonstrate a highly sensitive PGR detection method by using terahertz time-domain spectroscopy combined with metamaterials. We propose a double formant metamaterial resonator based on a split-ring structure with titanium-gold nanostructure. The metamaterial resonator is a split-ring structure composed of a titanium-gold nanostructure based on polyimide film as the substrate. Also, terahertz spectral response and electric field distribution of metamaterials under different analyte thickness and refractive index were investigated. The simulation results showed that the theoretical sensitivity of resonance peak 1 and peak 2 of the refractive index sensor based on our designed metamaterial resonator approaches 780 and 720 gigahertz per refractive index unit (GHz/RIU), respectively. In experiments, a rapid solution analysis platform based on the double formant metamaterial resonator was set up and PGR residues in aqueous solution were directly and rapidly detected through terahertz time-domain spectroscopy. The results showed that metamaterials can successfully detect butylhydrazine and N-N diglycine at a concentration as low as 0.05 mg/L. This study paves a new way for sensitive, rapid, low-cost detection of PGRs. It also means that the double formant metamaterial resonator has significant potential for other applications in terahertz sensing.

UV-vis sensor array combining with chemometric methods for quantitative analysis of binary dipeptide mixture (Gly-Gly/Ala-Gln).

Many endogenous peptides are circulating in bodily fluids at micromole level, and accurate analysis of endogenous peptides at such low level is important. In this study, we presented an extensible, facile and sensitive sensor array based on UV-Vis spectroscopy of the AuNPs combined with chemometric methods for quantitative analysis of binary peptide mixture (Gly-Gly/Ala-Gln) using UV-Vis spectroscopy. High concentration arginine (Arg) and Cr3+ can induce aggregation of the AuNPs and DNA-AuNPs. However, the glycylglycine (Gly-Gly) and alanyl-glutamine (Ala-Gln) can prevent the AuNPs from aggregation. We investigated the prevention of AuNPs aggregation by using Gly-Gly and Ala-Gln mixtures and constructed sensor arrays for quantitative analyses of Gly-Gly and Ala-Gln mixtures. The color change of the solution is relevant to the dose of the target, and it can be visualized by the naked eyes or monitored by UV-Vis spectrometry. Results showed that the concentrations of Arg and Cr3+ are the key factors affecting the sensitivity of the sensor array. Whereas when Gly-Gly and Ala-Gln have to be analyzed simultaneously, concentrations of Arg and Cr3+ both for Gly-Gly and Ala-Gln are difficult to be optimized. Taking the advantages of multivariate analysis and data fusion, PLS models and backward interval PLS (BiPLS) models were built for fused dataset constructed by UV-Vis data obtained at different concentrations of Arg and Cr3+. The best results were obtained from the PLS models. The proposed method can be extended to analysis of other peptides in more complex mixture systems.

Improvement of ubiquitylation site detection by Orbitrap mass spectrometry.

Ubiquitylation is an important posttranslational protein modification that is involved in many cellular events. Immunopurification of peptides containing a K-ε-diglycine (diGly) remnant as a mark of ubiquitylation combined with mass spectrometric detection has resulted in an explosion of the number of identified ubiquitylation sites. Here, we present several significant improvements to this workflow, including fast, offline and crude high pH reverse-phase fractionation of tryptic peptides into only three fractions with simultaneous desalting prior to immunopurification and better control of the peptide fragmentation settings in the Orbitrap HCD cell. In addition, more efficient sample cleanup using a filter plug to retain the antibody beads results in a higher specificity for diGly peptides and less non-specific binding. These relatively simple modifications of the protocol result in the routine detection of over 23,000 diGly peptides from HeLa cells upon proteasome inhibition. The efficacy of this strategy is shown for lysates of both non-labeled and SILAC labeled cell lines. Furthermore, we demonstrate that this strategy is useful for the in-depth analysis of the endogenous, unstimulated ubiquitinome of in vivo samples such as mouse brain tissue. This study presents a valuable addition to the toolbox for ubiquitylation site analysis to uncover the deep ubiquitinome. SIGNIFICANCE: A K-ε-diglycine (diGly) mark on peptides after tryptic digestion of proteins indicates a site of ubiquitylation, a posttranslational modification involved in a wide range of cellular processes. Here, we report several improvements to methods for the isolation and detection of diGly peptides from complex biological mixtures such as cell lysates and brain tissue. This adapted method is robust, reproducible and outperforms previously published methods in terms of number of modified peptide identifications from a single sample. In-depth analysis of the ubiquitinome using mass spectrometry will lead to a better understanding of the roles of protein ubiquitylation in cellular events.

Metabolomic Characterizations of Liver Injury Caused by Acute Arsenic Toxicity in Zebrafish.

Arsenic is one of the most common metalloid contaminants in groundwater and it has both acute and chronic toxicity affecting multiple organs. Details of the mechanism of arsenic toxicity are still lacking and profile studies at metabolic level are very limited. Using gas chromatography coupled with mass spectroscopy (GC/MS), we first generated metabolomic profiles from the livers of arsenic-treated zebrafish and identified 34 significantly altered metabolite peaks as potential markers, including four prominent ones: cholic acid, glycylglycine, glycine and hypotaurine. Combined results from GC/MS, histological examination and pathway analyses suggested a series of alterations, including apoptosis, glycogenolysis, changes in amino acid metabolism and fatty acid composition, accumulation of bile acids and fats, and disturbance in glycolysis related energy metabolism. The alterations in glycolysis partially resemble Warburg effect commonly observed in many cancer cells. However, cellular damages were not reflected in two conventional liver function tests performed, Bilirubin assay and alanine aminotransferase (ALT) assay, probably because the short arsenate exposure was insufficient to induce detectable damage. This study demonstrated that metabolic changes could reflect mild liver impairments induced by arsenic exposure, which underscored their potential in reporting early liver injury.

Enhanced detection of ubiquitin isopeptides using reductive methylation.

Identification of ubiquitination (Ub) sites is of great interest due to the critical roles that the modification plays in cellular regulation. Current methods using mass spectrometry rely upon tryptic isopeptide diglycine tag generation followed by database searching. We present a novel approach to ubiquitin detection based upon the dimethyl labeling of isopeptide N-termini glycines. Ubiquitinated proteins were digested with trypsin and the resulting peptide mixture was derivatized using formaldehyde-D2 solution and sodium cyanoborohydride. The dimethylated peptide mixtures were next separated by liquid chromatography and analyzed on a quadrupole-TOF based mass spectrometer. Diagnostic b2' and a1' ions released from the isopeptide N-terminus upon collision-induced dissociation (CID) were used to spectrally improve the identification of ubiquitinated isopeptides. Proof of principle was established by application to a ubiquitinated protein tryptic digest spiked into a six-protein mix digest background. Extracted ion chromatograms of the a1' and b2' diagnostic product ions from the diglycine tag resulted in a significant reduction in signal complexity and demonstrated a selectivity towards the identification of diglycine branched isopeptides. The method was further shown to be capable of identifying diglycine isopeptides resulting from in-gel tryptic digests of ubiquitin enriched material from a His-Ub transfected cell line. We envisage that these ions may be utilized in global ubiquitination studies with post-acquisition MS/MS (or MSe) data interrogation on high resolution hybrid mass spectrometers. ᅟ

Effects of water content on volatile generation and peptide degradation in the maillard reaction of glycine, diglycine, and triglycine.

Peptides abundant in food and protein hydrolysates are known to be important to process flavors. The present study reports the volatile profile of the Maillard reactions of glycine, diglycine, and triglycine. The reaction with glucose was conducted at 0-100% water content in glycerol medium at 160 degrees C for 1 h. Volatile compounds were quantified by stir bar sorptive extraction-gas chromatography-mass spectrometry, and nonvolatile compounds were quantified by high-performance liquid chromatography-tandem mass spectrometry. The major volatiles produced from each of the reaction systems were trimethylpyrazine and 2,5-dimethylpyrazine. Volatile generation increased as water decreased, and the overall reactivity of the glycine and glycine peptides in volatile formation was glycine approximately triglycine > diglycine. Triglycine was very unstable and mainly degraded into cyclic Gly-Gly and glycine, whereas diglycine had a higher stability than triglycine toward hydrolytic cleavage of the peptide bond. The amounts of glycine, diglycine, cyclic (Gly-Gly), and triglycine in the peptide-glucose reaction mixtures at different water content were reported.

Effects on the peritoneal membrane of rabbits of a single bicarbonate solution containing glycylglycine.

So far, no one has devised ideal, on-line mixed bicarbonate solutions for hemodialysis and continuous ambulatory peritoneal dialysis (CAPD) because current preparations can lead to precipitation of calcium and magnesium carbonate during treatment. The author has prepared a new, single bicarbonate dialysis solution that contains glycylglycine (BiGG). With a bicarbonate/glycylglycine ratio of 30/10 mM/L, this solution has a stable pH of 7.35; sterilized through a 0.22 micron filter, it remains stable for over 15 months when stored at 10 degrees-40 degrees C. When infused (40 mL/kg bw) in rabbits daily for 25 days, the BiGG solution showed excellent peritoneal-membrane tolerance and biocompatibility. Comparative ultrafiltration (UF) studies with BiGG and standard lactate (La) solutions in rabbits showed that net UF with La solution peaked at 2 h and decreased significantly at 4 h and 6 h. Net UF with BiGG solution peaked at 4 h and was sustained at 6 h; in all instance it was greater than that of La solution by approximately 15%, 30%, and 40% at 2, 4, and 6 h, respectively. During the initial 2-h dwell period, the pH of La solution increased from an initial 5.5 to 7.18, whereas that of the BiGG solution remained stable at 7.35. In all rabbits, the peritoneal fluid contained phosphatidylcholine at all times. However, its concentration was significantly higher in animals receiving the BiGG solution than in those receiving the La solution. The author concludes that this new single BiGG PD solution is simple to use, stable, and of a normal pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced ultrafiltration in rabbits with bicarbonate glycylglycine peritoneal dialysis solution.

OBJECTIVE: The aim of the study was to investigate net ultrafiltration (NUF) with a new bicarbonate glycylglycine (BiGG) peritoneal dialysis solution compared to the standard lactate (La) solution. DESIGN: In six groups of 12 normal rabbits each we measured NUF after a 2-, 4-, and 6-hour peritoneal dialysis with a BiGG solution (pH 7.35) and a standard La solution (pH 5.5) of similar glucose, electrolyte, and osmolality formulation. Furthermore, we studied the phosphatidylcholine concentration in the effluent of the two solutions. RESULTS: NUF volume was significantly greater with the BiGG rather than with the La solution by approximately 15% (p < 0.05), 30% (p < 0.01), and 40% (p < 0.005) at 2, 4, and 6 hours, respectively. The glucose absorption rate was greater with the La solution than with the BiGG solution, but the difference was significant only at 2 hours (p < 0.05). pH was increased in the La solution from its initial value of 5.5 to 7.18, 7.32, and 7.40 at 2, 4 and 6 hours, respectively, while it remained almost unchanged in the BiGG solution. Phosphatidylcholine (PC) in the peritoneal effluent was significantly higher in the BiGG solution in all instances (p < 0.0001). CONCLUSION: It is concluded that the BiGG solution, which has a stable pH, 7.35, due to the potent buffering capacity of bicarbonate and glycylglycine, enhances peritoneal NUF by maintaining a higher osmotic gradient and retarding lymphatic absorption through an increase in PC concentration in the peritoneal cavity.

Estimation of catecholamines by ion-exchange chromatography on Asahipak ES-502C, using glycylglycine as the post-derivatizing agent.

The estimation of catecholamines in human urine was carried out by ion-exchange chromatography on a column of a weakly acidic ion exchanger with an hydrophilic matrix. The catecholamines were first adsorbed onto Amberlite CG-50 (buffered at pH 6.5 with 0.4 M phosphate buffer) and selectively eluted by 0.66 M boric acid solution. They were then separated from impurities that responded to fluorometric detection by isocratic elution from a column of Asahipak ES-502C, a cross-linked vinyl alcohol copolymer with carboxymethyl groups, at 60 degrees C. The mobile phase was 0.05 M sodium succinate buffer pH 5.25 containing 0.015 M borate and 0.5 mM ethylenediaminetetraacetate. Isoproterenol was used as the internal standard; epinephrine, norepinephrine, isoproterenol and dopamine were eluted in this order. One sample could be analyzed every 35 min. The detection limits were 0.2 ng for epinephrine and norepinephrine, 0.6 ng for dopamine. The elution pattern was quite reproducible; the elution volumes of the catecholamines had not changed after 500 determinations.

A scheme for determining the correct activity of the kinetic angiotensin-converting enzyme.

The absorbance difference measured when angiotensin-converting enzyme (EC 3.4.15.1) hydrolyzes the substrate N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine is the basis for measuring its activity. We show this difference to be instrument dependent, and describe a method for deriving it that is applicable to manual or automated procedures.