Vertical flow paper-based plasmonic device for cysteine detection.

Cystinuria, is an autosomal recessive genetic disorder involving increasingly high levels of poorly soluble cysteine in urine leading to formation of stones. Developing a facile, low-cost, point-of-care and selective sensor for diagnosis of cysteine is imperative. Accordingly, for the detection of cysteine, the present study demonstrates an inexpensive colorimetric, paper-based vertical flow plasmonic micro-well device with a two-minute turn-around time. The method encompasses the use of microbially-synthesized silver nanoparticles (AgNPs) that change from light brown / yellow to dark brown upon binding with Sulphur present in cysteine. This technique allows for visual detection up to 1 × 10-5 mM cysteine and can be easily offered as a rapid diagnostic test even at setups with minimal resources.

Systematic and Quantitative Assessment of Hydrogen Peroxide Reactivity With Cysteines Across Human Proteomes.

Protein cysteinyl residues are the mediators of hydrogen peroxide (H2O2)-dependent redox signaling. However, site-specific mapping of the selectivity and dynamics of these redox reactions in cells poses a major analytical challenge. Here we describe a chemoproteomic platform to systematically and quantitatively analyze the reactivity of thousands of cysteines toward H2O2 in human cells. We identified >900 H2O2-sensitive cysteines, which are defined as the H2O2-dependent redoxome. Although redox sites associated with antioxidative and metabolic functions are consistent, most of the H2O2-dependent redoxome varies dramatically between different cells. Structural analyses reveal that H2O2-sensitive cysteines are less conserved than their redox-insensitive counterparts and display distinct sequence motifs, structural features, and potential for crosstalk with lysine modifications. Notably, our chemoproteomic platform also provides an opportunity to predict oxidation-triggered protein conformational changes. The data are freely accessible as a resource at http://redox.ncpsb.org/OXID/.

Switch-on fluorescent strategy based on crystal violet-functionalized CdTe quantum dots for detecting L-cysteine and glutathione in water and urine.

The concentration of L-cysteine (Cys) and glutathione (GSH) is closely related to the critical risk of various diseases. In our study, a new rapid method for the determination of Cys and GSH in water and urine samples has been developed using a fluorescent probe technique, which was based on crystal violet (CV)-functionalized CdTe quantum dots (QDs). The original QDs emitted fluorescence light, which was turned off upon adding CV. This conjugation of CV and QDs could be attributed to electrostatic interaction between COO- of mercaptopropionic acid (MPA) on the surface of QDs and N+ of CV in aqueous solution. In addition, Förster resonance energy transfer (FRET) also occurred between CdTe QDs and CV. After adding Cys or GSH to the solution, Cys or GSH exhibited a stronger binding preference toward Cd2+ than Cd2+-MPA, which disturbed the interaction between MPA and QDs. Thus, most MPA was able to be separated from the surface of QDs because of the participation of Cys or GSH. Then, the fluorescence intensity of the CdTe QDs was enhanced. Good linear relationships were obtained in the range of 0.02-40 µg mL-1 and 0.02-50 µg mL-1, and the detection limits were calculated as 10.5 ng mL-1 and 8.2 ng mL-1, for Cys and GSH, respectively. In addition, the concentrations of biological thiols in water and urine samples were determined by the standard addition method using Cys as the standard; the quantitative recoveries were in the range of 97.3-105.8%, and relative standard deviations (RSDs) ranged from 2.5 to 3.7%. The method had several unique properties, such as simplicity, lower cost, high sensitivity, and environmental acceptability. Graphical abstract Crystal violet-functionalized CdTe quantum dots for detecting L-cysteine and glutathione with switch-on fluorescent strategy.

Natural collagenic skeleton of marine sponges in pharmaceutics: Innovative biomaterial for topical drug delivery.

The growing interest in the use of recyclable and biodegradable natural materials has become a relevant topic in pharmaceutics. In this work, we suggest the use and valorization of natural horny skeleton of marine sponges (Porifera, Dictyoceratida) as bio-based dressing for topical drug delivery. Biomaterial characterization focusing on morpho-functional traits, swelling behavior, fluid uptake performances, glycosaminoglycans content and composition and microbiological quality assessment was carried out to investigate the collagenic skeleton properties. After grinding and sieving processes, l-cysteine hydrochloride-loaded formulations were designed in form of powder or polymeric film by testing various drug concentrations and different drying parameters. Drug content, SEM analyses and in vitro permeation studies were performed to test the suitability of skeleton-based formulations. To this respect, drying time and temperature are key parameters for skeleton-mediated drug crystallization. Consequently, this behavior seems to influence drug loading and permeation profiles of formulations. The high percentages of drug are found after absorption into sponge powder and in vitro permeation studies demonstrate that cysteine is released more slowly than the pure drug within 1h. Such a system is attractive because it combines the known healing properties of cysteine with the advantageous potentials of the collagen/proteoglycan network, which can act as biocompatible carrier able to absorb the excess of the wound exudate while releasing the drug. Furthermore, due to its glycosaminoglycans content, natural sponge skeletal scaffold might act as bioactive-biomimetic carrier regulating the wound healing processes.

Simultaneous assessment of endogenous thiol compounds by LC-MS/MS.

Biological thiol compounds are very important molecules that participate in various physiological events. Alteration in levels of endogenous thiols has been suggested as a biomarker of early stage of pathological changes. We reported a chemical derivatization- and LC-MS/MS-based approach to simultaneously determine thiol compounds including glutathione (GSH), cysteine (Cys), N-acetyl cysteine (NAC), homocysteine (Hcy), and cysteinylglycine (CysGly) in biological samples. Thiol-containing samples were derivatized with monobromobimane (mBrB) at room temperature, followed by LC-MS/MS analysis. Assessment of the analytes with baseline separation was completed within 10min, using a gradient elution on a C18 reversed-phase column. Excellent linearity was observed for all analytes over their concentration ranges of 1-400µM. The lowest limits of detection (S/N=3) in a range from 0.31fmol (for NAC) to 4.98fmol (for CysGly) were achieved. The results indicate that this approach was sensitive, selective, and well suited for high-throughput quantitative determination of the biological thiols.

Thiazole orange as a fluorescent probe: Label-free and selective detection of silver ions based on the structural change of i-motif DNA at neutral pH.

Silver ions have been widely applied to many fields and have harmful effects on environments and human health. Herein, a label-free optical sensor for Ag(+) detection is constructed based on thiazole orange (TO) as a fluorescent probe for the recognition of i-motif DNA structure change at neutral pH. Ag(+) can fold a C-rich single stranded DNA sequence into i-motif DNA structure at neutral pH and that folding is reversible by chelation with cysteine (Cys). The DNA folding process can be indicated by the fluorescence change of TO, which is non-fluorescent in free molecule state and emits strong fluorescence after the incorporation with i-motif DNA. Thus, a rapid, sensitive, and selective method for the detection of Ag(+) and Cys is developed with a detection limit of 17 and 280nM, respectively. It is worth noting that the mechanism underlying the increase of the fluorescence of thiazole orange in the presence of i-motif structure is explained. Moreover, a fluorescent DNA logic gate is successfully designed based on the Ag(+)/Cys-mediated reversible fluorescence changes. The proposed detection strategy is label-free and economical. In addition, this system shows a great promise for i-motif/TO complex to analyze Ag(+) in the real samples.

In Vitro Assessment of Interaction Between Amino Acids and Copper in Neonatal Parenteral Nutrition.

BACKGROUND: The repeated blackening of in-line filters has been observed during the infusion of parenteral nutrition 2-in-1 mixtures (binary parenteral nutrition [BPN]) delivered in a neonatal intensive care unit. This study aimed to examine the elemental content of precipitates isolated from infused BPN bags and determine the main physicochemical interactions occurring in these bags. MATERIALS AND METHODS: The infusion of BPN mixtures was simulated in vitro following hospital practices. Filter membranes were examined by scanning electron microscopy and energy dispersion spectroscopy (EDS). Amino acid (AA) profiles were obtained from BPN mixtures to determine the concentrations of each AA. RESULTS: Analyzed filter membranes revealed conglomerates of particles on filter surfaces. An EDS analysis generated spectra from isolated particles, identifying copper and sulfur as the major chemical elements. AA mean concentrations were relatively close to the expected value for each AA, except cysteine. Cysteine concentrations were very significantly lower than the expected values. CONCLUSION: A specific interaction was identified between 1 AA (cysteine) and a trace element (copper) in our BPN mixtures.

A New, High Yield, Rapid, and Cost-Effective Protocol to Deprotection of Cysteine-Rich Conopeptide, Omega-Conotoxin MVIIA.

Almost all conopeptides purified from Conus venoms are cysteine-rich peptides. Among them, omega-conotoxin MVIIA, FDA approved peptide drug (Prialt(®)), selected as a cysteine-rich model that its protection from oxidation is critical during solid phase synthesis. Deprotection of cysteines is a crucial step after peptide synthesis. The current study aimed to set up a new highly efficient deprotection protocol for omega-conotoxin MVIIA. Deprotection was performed based on mercury acetate with significant major modification. The protocol accomplished based on the best molar ratio of peptide/mercury/2-ME that adjusted to 0.2 mm/3 mm/10 mm (50 µg/1 mg/10 µL). The yield and purity of omega-conotoxin MVIIA obtained at 93 and 95%, respectively. The total time of protocol shortened to 90 min instead of 6-20 h in routine methods. In this study, omega-conotoxin MVIIA was recovered in high yield and in the shortest time. Despite of other known protocols, molar ratio adjusted to minimum amount. In conclusion, this protocol would be suggested to cost-effective deprotection of thiol groups for similar cysteine-rich peptides.

Puzzling over protein cysteine phosphorylation--assessment of proteomic tools for S-phosphorylation profiling.

Cysteine phosphorylation has recently been discovered in both prokaryotic and eukaryotic systems, and is thought to play crucial roles in signaling and regulation of cellular responses. This article explores the topics of chemical stability of this type of structural modification and the resulting issues regarding affinity enrichment of S-phosphopeptides and their mass spectrometry-based detection in the course of general proteomics studies. Together, this work suggests that the current advances in phosphoproteomic methodologies provide adequate tools for investigating protein cysteine phosphorylation and appear to be immediately available for practical implementation. The article provides useful information necessary for designing experiments in the emerging cysteine phosphoproteomics. The examples of methodological proposals for S-linked phosphorylation detection are included herein in order to stimulate development of new approaches by the phosphoproteomic community.

Antioxidant changes of leek (Allium ampeloprasum var. porrum) during spontaneous fermentation of the white shaft and green leaves.

BACKGROUND: Leek is grown for its thickened cylindrical white shaft made up of long leaf bases. Despite the potentially valuable nutritional profile of the green leaves, a large portion remains unused owing its restricted culinary applications. This large quantity of this plant biomass could be valorized given an adequate stabilization method. In this study, we examined leek fermentation with regard to antioxidant changes. RESULTS: The oxygen radical absorbance capacity (ORAC) increased by 62% when the green leaves were fermented for 21 days, while 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity did not increase significantly. Fermentation resulted in an increase of endogenous polyphenolic compounds such as ferulic acid, astragalin, luteolin and naringenin. Moreover, fermentation stimulated the production of a series of polyphenolic compounds that were not present in the fresh leek. The flavour precursors in leek, i.e. methiin and isoalliin, were reduced by 91-93% and 100%, respectively, when spontaneous fermentation was allowed to occur on the white shaft and green leaves. CONCLUSION: Our results demonstrated that application of fermentation resulted in a higher ORAC value and polyphenol content of the leek plant, especially in the green leaves. These results indicate the nutritional relevance of fermentation, which hold promise as a stabilization technique.

A new strategy for improved glutathione production from Saccharomyces cerevisiae: use of cysteine- and glycine-rich chicken feather protein hydrolysate as a new cheap substrate.

BACKGROUND: Glutathione (GSH) is composed of the amino acids glutamic acid, cysteine and glycine. This study investigated the usability of chicken feather protein hydrolysate (chicken feather peptone, CFP) as a substrate for GSH production from Saccharomyces cerevisiae. RESULTS: CFP was found to be rich in ash (36.7 g per 100 g), protein (61.1 g per 100 g) and minerals (S, P, K, Ca, Fe, Na and Mg). It also had high contents of cysteine and glycine. CFP augmented biomass and GSH production by 53 and 115% respectively compared with the control medium. The highest biomass (17.4 g l(-1)) and GSH (271 mg L(-1)) concentrations were attained in CFP medium. The second highest biomass (16.8 g l(-1)) and GSH (255 mg L(-1)) concentrations were obtained in fish peptone medium. It was assumed that the high mineral, cysteine and glycine contents of CFP were related to cell growth and GSH synthesis in S. cerevisiae. CONCLUSION: This is the first report on the effect of cysteine- and glycine-rich protein hydrolysates on GSH production from S. cerevisiae. In this regard, CFP was tested for the first time as a GSH production substrate. As an additional contribution, a new hydrolysis process was developed for the preparation of protein hydrolysates.

Cysteinyl peptide capture for shotgun proteomics: global assessment of chemoselective fractionation.

The complexity of cell and tissue proteomes presents one of the most significant technical challenges in proteomic biomarker discovery. Multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based shotgun proteomics can be coupled with selective enrichment of cysteinyl peptides (Cys-peptides) to reduce sample complexity and increase proteome coverage. Here we evaluated the impact of Cys-peptide enrichment on global proteomic inventories. We employed a new cleavable thiol-reactive biotinylating probe, N-(2-(2-(2-(2-(3-(1-hydroxy-2-oxo-2-phenylethyl)phenoxy)acetamido)ethoxy)-ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (IBB), to capture Cys-peptides after digestion. Treatment of tryptic digests with the IBB reagent followed by streptavidin capture and mild alkaline hydrolysis releases a highly purified population of Cys-peptides with a residual S-carboxymethyl tag. Isoelectric focusing (IEF) followed by LC-MS/MS of Cys-peptides significantly expanded proteome coverage in Saccharomyces cerevisiae (yeast) and in human colon carcinoma RKO cells. IBB-based fractionation enhanced detection of Cys-proteins in direct proportion to their cysteine content. The degree of enrichment typically was 2-8-fold but ranged up to almost 20-fold for a few proteins. Published copy number annotation for the yeast proteome enabled benchmarking of MS/MS spectral count data to yeast protein abundance and revealed selective enrichment of cysteine-rich, lower abundance proteins. Spectral count data further established this relationship in RKO cells. Enhanced detection of low abundance proteins was due to the chemoselectivity of Cys-peptide capture, rather than simplification of the peptide mixture through fractionation.

A colorimetric sensor for determination of cysteine by carboxymethyl cellulose-functionalized gold nanoparticles.

A simple and sensitive colorimetric method for cysteine detection was established based on the carboxymethyl cellulose-functionalized gold nanoparticles (CMC-AuNPs). The nanoparticles were directly synthesized with sodium carboxymethyl cellulose by a simple approach, which would protect particles against salt-induced aggregation. Then the CMC-AuNPs solution exhibited a high colorimetric selectivity to cysteine. The assay results indicated that the introduction of cysteine could induce the aggregation of the colloidal solutions at the presence of sodium chloride, displaying changes in color and in UV-vis absorption spectra. Thus an exceptionally simple, rapid method for detecting cysteine was obtained at the linear range of 10.0-100.0 microM with the relative coefficient of 0.997. The proposed method possessed the advantages of simplicity and sensitivity, and was applied to real urine sample detection. The results were satisfying and the proposed method was especially appropriate for detection of cysteine in biological samples.

Methodology for determining disulfide linkage patterns of closely spaced cysteine residues.

We report the development and application of a method for determining bonding patterns in disulfide-linked peptides containing closely spaced cysteine residues. Through the utility of classic N-terminal sequencing chemistry coupled with facile liquid chromatography and mass spectrometric analysis of the cleavage products, we report the ability to demonstrate unambiguous assignment of paired cysteine residues, using human insulin as a model protein. The conditions of the technique were selected and optimized to maintain disulfide integrity. In a forthcoming article, we will present the results of this method as applied to the complete elucidation of linkages in disulfide variants of a therapeutic monoclonal antibody of the IgG2 subclass.

Simple and sensitive colorimetric detection of cysteine based on ssDNA-stabilized gold nanoparticles.

In this paper, we demonstrate a simple and sensitive colorimetric detection of cysteine based on the cysteine-mediated color change of ssDNA-stabilized gold nanoparticles (AuNPs). Cysteine is capable of absorbing onto AuNPs surfaces via the strong interaction between its thiol group and gold. ssDNA molecules which stabilize AuNPs against salt-induced aggregation are removed away by cysteine encapsulation on the AuNPs surfaces, resulting in a characteristic color change of AuNPs from red to blue as soon as salt is added. The ratio of absorptions at 640 to 525 nm (A(640)/A(525)) is linear dependent on the cysteine concentration in the range from 0.1 to 5 microM. Furthermore, amino acids other than cysteine cannot mediate the color change under the identical conditions due to the absence of thiol groups, suggesting the selectivity of the proposed method toward cysteine. The employment of complicated protocols and sophisticated processes such as the preparation of modified AuNPs are successfully avoided in design to realize the simple and low-cost cysteine detection; and the high sensitivity and low cost of the method is favorable for practical applications.

A simple and sensitive method for L-cysteine detection based on the fluorescence intensity increment of quantum dots.

In this contribution, a simple and sensitive method for L-cysteine detection was established based on the increment of the fluorescence intensity of mercaptoacetic acid-capped CdSe/ZnS quantum dots (QDs) in aqueous solution. Meanwhile, the fluorescence characteristics and the optimal conditions were investigated in detail. Under the optimized conditions, the linear range of QDs fluorescence intensity versus the concentration of L-cysteine was 10-800 nmol L(-1), with a correlation coefficient (R) of 0.9969 and a limit of detection (3sigma black) of 3.8 nmol L(-1). The relative standard deviation (R.S.D.) for 0.5 micromol L(-1) L-cysteine was 1.1% (n = 5). There was no interference to coexisting foreign substances including common ions, carbohydrates, nucleotide acids and other 19 amino acids. The proposed method possessed the advantages of simplicity, rapidity and sensitivity. Synthetic amino acid samples, medicine sample together with human urine samples were analyzed by the methodology and the results were satisfying.

Quantitative determination of S-alk(en)ylcysteine-S-oxides by micellar electrokinetic capillary chromatography.

A novel method for determination of S-alk(en)ylcysteine-S-oxides by capillary electrophoresis has been developed and validated. The method is based on extraction of these sulfur amino acids by methanol, their derivatization by fluorenylmethyl chloroformate and subsequent separation by micellar electrokinetic capillary chromatography. Main advantages of the new method are simplicity, sensitivity, high specificity and very low running costs, making it suitable for routine analysis of a large number of samples. Employing this method, the content of S-alk(en)ylcysteine-S-oxides was determined in 12 commonly consumed alliaceous and cruciferous vegetables (e.g. garlic, onion, leek, chive, cabbage, radish, cauliflower and broccoli). The total content of these amino acids in the Allium species evaluated varied between 0.59 and 12.3mg g(-1) fresh weight. Whereas alliin was found only in garlic, isoalliin was the major S-alk(en)ylcysteine-S-oxide in onion, leek, chive and shallot. On the other hand, the cruciferous species analyzed contained only methiin in the range of 0.06-2.45mg g(-1) fresh weight.

Assessment of epidermal growth factor receptor with 99mTc-ethylenedicysteine-C225 monoclonal antibody.

Epidermal growth factor receptor (EGFR) plays an important role in cell division and cancer progression, as well as angiogenesis and metastasis. Since many tumor cells exhibit the EGFR on their surface, functional imaging of EGFR provides not only a non-invasive, reproducible, quantifiable alternative to biopsies, but it also greatly complements pharmacokinetic studies by correlating clinical responses with biological effects. Moreover, molecular endpoints of anti-EGFR therapy could be assessed effectively. C225 is a chimeric monoclonal antibody that targets the human extracellular EGFR and inhibits the growth of EGFR-expressing tumor cells. Also, it has been demonstrated that C225, in combination with chemotherapeutic drugs or radiotherapy, is effective in eradicating well-established tumors in nude mice. We have developed 99mTc-labeled C225 using ethylenedicysteine (EC) as a chelator. This study aimed at measuring uptake of 99mTc-EC-C225 in EGFR+ tumor-bearing animal models and preliminary feasibility of imaging patients with head and neck carcinomas. In vitro Western blot analysis and cytotoxicity assays were used to examine the integrity of EC-C225. Tissue distribution studies of 99mTc-EC-C225 were evaluated in tumor-bearing rodents at 0.5-4 h. In vivo biodistribution of 99mTc-EC-C225 in tumor-bearing rodents showed increased tumor-to-tissue ratios as a function of time. In vitro and biodistribution studies demonstrated the possibility of using 99mTc-EC-C225 to assess EGFR expression. SPECT images confirmed that the tumors could be visualized with 99mTc-EC-C225 from 0.5 to 4 h in tumor bearing rodents. We conclude that 99mTc-EC-C225 may be useful to assess tumor EGFR expression. This may be useful in the future for selecting patients for treatment with C225.

Assessment of the number of free cysteines and isolation and identification of cystine-containing peptides from acetylcholine receptor.

The number of free cysteines in each polypeptide of acetylcholine receptor from the electric organ of Torpedo californica has been assessed by alkylating the native protein with N-ethylmaleimide and iodoacetamide during homogenization of the tissue and alkylating the polypeptides with N-ethylmaleimide as they were unfolded in solutions of dodecyl sulfate. The cysteines unavailable for alkylation could be accounted for as specific cystines, connecting positions in the amino acid sequences of the individual polypeptides. Unreduced, alkylated polypeptides of acetylcholine receptor were digested with thermolysin or trypsin. Cystine-containing peptides in the chromatograms of the digests were identified electrochemically by the use of a dual gold/mercury electrode. Three thermolytic peptides and three tryptic peptides have been isolated from these digests and shown to contain intact cystines that were originally present in the native protein. The majority of these peptides contained an intact, intramolecular cystine connecting two cysteines in locations homologous to cysteines 128 and 142 from the alpha polypeptide. Each of these cystines from each of the polypeptides of acetylcholine receptor was isolated in at least one peptide, respectively. Each of these cystine-containing peptides also contained glucosamine. It can be concluded that each asparagine in the sequence Asn-Cys-Thr/Ser, which occurs in the respective, homologous location in every polypeptide, is glycosylated even though a cystine sits between the asparagine and the threonine or serine. In addition, the existence of the cystine connecting the adjacent cysteines, alpha 192 and alpha 193, in the alpha subunit of acetylcholine receptor [Kao, P. N., & Karlin, A. (1986) J. Biol. Chem. 261, 8085-8088] has been confirmed.

A camel milk whey protein rich in half-cystine. Primary structure, assessment of variations, internal repeat patterns, and relationships with neurophysin and other active polypeptides.

The amino acid sequence of a recently isolated camel milk protein rich in half-cystine has been determined by peptide analyses. The 117-residue protein has 16 half-cystine residues, concluded to correspond to disulfide bridges and suggesting a tight conformation of the molecule. Comparisons of the structure with those of other proteins reveal several interesting relationships. The camel protein is clearly homologous with a previously reported rat whey phosphoprotein of possible importance for mammary gland growth regulation, and with a mouse protein of probable relationship to neurophysins. The camel, rat and mouse proteins may represent species variants from a rapidly evolving gene. Residue identities in pairwise comparisons are 40% for the camel/rat proteins and 33% for the camel/mouse proteins, with 38 positions conserved in all three forms. The camel protein also reveals an internal repeat pattern similar to that for the other two proteins. The homology between the three milk whey proteins has wide implications for further relationships. Thus, previously noticed similarities, involving either of the milk proteins, include limited similarities to casein phosphorylation sites for the camel protein, to neurophysins in repeat and half-cystine patterns for the mouse and rat proteins, and to an antiprotease for the rat protein. These similarities are reinforced by the camel protein structure and the recognition of the three whey proteins as related. Finally a few superficial similarities with the insulin family of peptides and with some other peptides of biological importance are noticed. Combined, the results relate the camel protein in a family of whey proteins, and extend suggestions of relationships with some binding proteins.