A general method to determine ionization constants of responsive polymer thin films

A general method has been developed to determine the ionization constants of polymer thin films based on the stimuli-responsiveness of the polymer. Robust polymer films were fabricated on silicon wafers and gold slides using perfluorophenyl azide (PFPA) as the coupling agent. The ionization constants were measured by a number of techniques including ellipsometry, dynamic contact angle goniometry, and surface plasmon resonance imaging (SPRi). Using poly(4-vinylpyridine) (P4VP) as the model system, P4VP thin films were fabricated and the ionization constants of the films were measured taking advantage of the pH responsive property of the polymer. The pK(a) determined by ellipsometry, ~4.0, reflects the swelling of the polymer film in response to pH. The pK(a) value calculated from the dynamic contact angle measurements, ~5.0, relies on the change in hydrophilicity/hydrophobicity of the films as the polymer undergoes protonation/deprotonation. The pK(a) value measured by SPRi, ~4.9, monitors in situ the change of refractive index of the polymer thin film as it swells upon protonation. This was the first example where SPRi was used to measure the ionization constants of polymers.     

A method to determine mixing enthalpies by DSC

A method has been developed that utilizes a special custom-made mixing device and HPLC micro liter syringe to perform mixing experiments of liquid systems directly in open measuring cells of differential scanning calorimeters. The present paper describes how to determine mixing enthalpies from time scans of the isothermal heat flux during an exothermal or endothermal process. Using ethylene glycol and the slightly volatile component water to calibrate the mixing calorimeter, the mixing enthalpy of the binary system poly(ethylene glycol) 400/water could be determined with sufficient precision compared to the results of measurements with a conventional flow calorimeter. This revised version was published online in July 2006 with corrections to the Cover Date.     

Desorption/ionization efficiency of peptides containing disulfide bonds in matrix-assisted laser desorption/ionization mass spectrometry

In order to elucidate the role of desorption/ionization efficiency of peptides in MALDI-MS, we focused on peptides with disulfide bonds, which form a rigid tertiary structure. We synthesized seven sets of peptides with one disulfide bond (oxytocin, somatostatin, [Arg(8)]-vasopressin, [Arg(8)]-vasotocin, cortistatin, melanin-concentrating hormone, urotensin II-related peptide) and five sets of peptides with two disulfide bonds (tertiapin, α-conotoxin GI, α-conotoxin ImI, α-conotoxin MI and α-conotoxin SI). Each peptide set consisted of three peptides: the oxidized form (S-S type), the reduced form (SH type), and an internal standard peptide in which all cysteine residues were substituted with alanine residues. In the case of urotensin II-related peptide, tertiapin, α-conotoxin ImI and α-conotoxin MI, the reduced form showed higher desorption/ionization efficiency than the oxidized form. In contrast, the other peptides revealed higher desorption/ionization efficiency in the oxidized form relative to the reduced form. These results imply that a rigid structure of peptides formed by disulfide bonds does not correlate with desorption/ionization efficiency in MALDI-MS.     

A simple mathematical method to determine the efficiencies of log-conical detectors

A new type of photon detector, log-conical, is proposed. The average path length traveled by an incident photon of arbitrary energy as well as the geometrical solid angle are calculated in a mathematical expression to determine the efficiencies of this detector for an arbitrarily positioned isotropic radiating point source. The off-axis effect of the source position was analyzed to demonstrate the powerful capability of the proposed method. The results are compared with those obtained using a standard 3″×3″ cylindrical detector of the same volume in order to show the enhanced efficiency of the log-conical detector.     

Derivatization of phosphorylated peptides with S- and N-nucleophiles for enhanced ionization efficiency in matrix-assisted laser desorption/ionization mass spectrometry

The identification of phosphorylation sites is essential for a full understanding of the cellular functions of proteins. However, mass spectrometric analysis is often hampered by the low abundance of phosphoproteins, the difficulty of obtaining full sequence coverage by specific proteolysis reactions, and the low ionization efficiency of phosphopeptides compared with their non-phosphorylated analogs. In the present work a beta-elimination/Michael addition was used to replace the phosphate groups of pSer or pThr by a group which gives rise to an enhanced ionization efficiency. In order to find optimum reaction conditions, beta-elimination/Michael addition was examined using phosphorylated model peptides. Whereas complete elimination of phosphate could be achieved by treatment with barium hydroxide in organic solvents such as ethanol or acetonitrile, the yield of the Michael adduct strongly depended on the nucleophile and the peptide sequence. Reaction with 2-phenylethanethiol, p-bromophenethylamine and ethylenediamine clearly resulted in products showing higher matrix-assisted laser desorption/ionization (MALDI) signal intensities compared with those of the corresponding phosphorylated precursors. The method was successfully used to identify phosphorylated sequences of ovalbumin and human Stat1 by in-gel derivatization with 2-phenylethanethiol and subsequent peptide mass fingerprint analysis of the trypsin digests.     

Development of a rapid method to determine phenolic and other polar compounds in walnut by capillary electrophoresis-electrospray ionization time-of-flight mass spectrometry

The aim of this work was to develop a capillary electrophoresis-mass spectrometry (CE-MS) method to identify and quantify phenolic and other related polar compounds in walnut samples. The extraction capacity of several solvent mixtures of phenolic compounds from walnut by conventional solid-liquid extractions was tested, and CE and electrospray ionization MS parameters were optimized. The finalized procedure is able to determine many well-known phenolic compounds present in walnuts and provide relevant information about the presence of minor polar compounds. A new compound in walnut ((2E,4E)-8-hydroxy-2,7-dimethyl-2,4-decadiene-1,10-dioic acid 6-O-beta-d-glucopiranosyl ester, [M-H](-) 403.161m/z) with a structure similar to glansreginins was also identified. Phenolic compounds correspond to 14-28% of total polar compounds quantified. Aglycone and glycosylated ellagic acid represent the principal components and account for 64-75% of total phenols in walnuts. However, the sum of glansreginins A, B and (2E,4E)-8-hydroxy-2,7-dimethyl-2,4-decadiene-1,10-dioic acid 6'-O-beta-d-glucopiranosyl ester was in the range of 72-86% of total quantified compounds. In addition, this is the first time that separation by CE with detection by electrospray ionization time-of-flight MS has been applied to the analysis of phenolic and other polar compounds in walnut samples, providing results in less than 15min.     

A method to determine and classify the chirality of the water medium

A new numerical method to determine the chirality of water configurations is developed. It consists in the comparison of matrices composed for both initial configuration and its mirror image based on the information of four bound water molecules. The method developed enables the rapid and unambiguous determination of the chirality of an aqueous system.     

A Gibbs sampling method to determine biomarkers for asthma

PurposeTo identify potential biomarkers and to uncover the mechanisms underlying asthma based on Gibbs sampling.MethodsThe molecular functions (MFs) with genes greater than 5 were determined using AnnotationMFGO of BAGS package, and the obtained MFs were then transformed to Markov chain (MC). Gibbs sampling was conducted to obtain a new MC. Meanwhile, the average probabilities of MFs were computed via MC Monte Carlo (MCMC) algorithm, followed by identification of differentially expressed MFs based on the probabilities of MF more than 0.6. Moreover, the differentially expressed genes (DEGs) and their correlated genes were screened and merged, called as co-expressed genes. Pathways enrichment analysis was implemented for the co-expressed genes.ResultsBased on the gene set more than 5, overall 396 MFs were determined. After Gibbs sampling, 5 differentially expressed MF were acquired according to alfa.pi > 0.6. Moreover, the genes in these 5 differentially expressed MF were merged, and 110 DEGs were identified. Subsequently, 338 co-expressed genes were gained. Based on the P value < 0.01, the co-expressed genes were significantly enriched in 6 pathways. Among these, ubiquitin mediated proteolysis contained the maximum numbers of 35 co-expressed genes, and cell cycle were enriched by the second largest number of 11 co-expressed genes, respectively.ConclusionsThe identified pathways such as ubiquitin mediated proteolysis and cell cycle might play important roles in the development of asthma and may be useful for developing the credible therapeutic approaches for diagnosis and treatment of asthma in future.     

A method to fast determine the coupling coefficients in CI calculation

A new algorithm for evaluating the coupling coefficients and the addresses of molecular integrals in configuration interaction (CI) calculations is presented, which leads to an improved CI calculation program CGUGA. The validity and efficiency of the new code are compared with other programs, such as MELD and GAUSSIAN-94.     

A method to identify and simultaneously determine the relative quantities of proteins isolated by gel electrophoresis

Gel electrophoresis is often used for the primary analysis and purification of proteins, and peptide mapping by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a widely used technique for the rapid identification of unknown proteins. The identification is usually obtained by digesting the protein with an enzyme and matching the masses of the proteolytic peptides with those of each protein in a sequence database. Another important aspect in many proteomic experiments is the determination of the relative protein quantities (e.g. comparison between control and altered states). Usually, this is obtained by comparing the spot intensities of two independent gels. This procedure is time-consuming and not very accurate. Recently, several methodologies using isotope labeling of proteins for quantitative proteomic studies have been introduced (e.g. using ICAT reagents or growing cells in isotopically enriched nutrients). However, none of these methodologies is foolproof and there is still the need for simple and inexpensive alternatives for determining the relative quantities of proteins. Previously, we showed that a mixture of acrylamide and deuterated acrylamide could be used as cysteine alkylating reagent prior to electrophoresis, improving the coverage and the confidence of the protein identification procedure (Sechi S, Chait BT. Anal. Chem. 1998; 70: 5150). Here we show that a similar approach can be used to obtain relative quantitation at the femtomole level of proteins isolated by gel electrophoresis. Deuterated acrylamide is used to alkylate the cysteines in one sample and regular acrylamide is used to alkylate the cysteines in the second sample. The two samples are then mixed together in a 1:1 ratio and the relative protein quantities are determined from the ion intensity ratios of the two cysteine-containing peptides isotopic envelopes (regular/deuterated). The analysis of several proteins mixed in different ratios is reported showing that this approach can reliably be used for protein identification and quantification. Briefly, a simple and inexpensive method for quantifying and simultaneously identifying proteins isolated by gel electrophoresis using MALDI-MS is presented.     

A radioactive tracer dilution method to determine the mass of molten salt

A new technique for molten salt mass determination, termed radioactive tracer dilution, that uses ²²Na as a tracer was validated at bench scale. It has been a challenging problem to determine the mass of molten salt in irregularly shaped containers, where a highly radioactive, high-temperature molten salt was used to process nuclear spent/used fuel during electrochemical recycling (pyro-processing) or for coolant/fuel salt from molten salt reactors. A radioactive source with known activity is dissolved into the salt. After a complete mixture, a small amount of the salt is sampled and measured in terms of its mass and radioactivity. By finding the ratio of the mass to radioactivity, the unknown salt mass in the original container can be precisely determined.

     

Capillary electrophoresis as a method to determine underivatized urinary lipoarabinomannans,a biomarker of active tuberculosis caused by Mycobacterium tuberculosis

Tuberculosis is a devastating contagious disease caused by Mycobacterium tuberculosis. This is the first report describing the development of novel capillary electrophoresis methods to detect lipoarabinomannans shed into the blood circulation by replicating bacteria. The novelty of the methods is the detection without derivatization. The lipoarabinomannan is detected owing to the ionization of the diverse functional groups of the structure, such as the multibranched mannan domain or the phosphatidyl group. Four alkaline solutions were used; normal polarity in three of them and reversed polarity in one. Urinary lipoarabinomannans by saccharide domains were identified with direct absorbance detection. The accuracy and the analytical sensitivity were then validated with cello‐, manno‐ and xylooligosaccharides. Lipoarabinomannan detection was feasible within 20 min (RSD 2.1%). This method worked at the dynamic range of 0.1–10 μg/mL. With reversed polarity, indirect absorbance detection, and pH 9.0 electrolyte were used, the analytes migrated already within 5 min (RSD 0.01%). Inorganic nonabsorbing ions were used for this method optimization. This improvement resulted in the detection limit of 1 pg/mL in water and in the linear dynamic range of 1 pg/mL to 10 ng/mL. In conclusion, the described method has great potential as a point‐of‐care assay for clinical use.     

A new strategy to determine the protein mutation site using matrix-assisted laser desorption ionization in-source decay: Derivatization by ionic liquid

Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) can be considered as state of the art in the field of proteins and peptides analysis. In this work, we have designed an ionic liquid derivative strategy to obtain abundant fragment ions in MALDI in-source decay (ISD) and used the analysis of angiogenin with mutation in the fortieth (K40I) as an instance. Firstly, we have synthesized two types of ionic liquids, 3-allyl-4-methyl-1H-imidazol-3-ium and 4-methyl-3-(pent-4-yn-1-yl)-1H-imidazol-3-ium. Then in the light-catalyzed reaction, the alkenyl ionic liquid can open the disulfide bond of K40I protein and add to the thiol. And the derived protein can process in-source decay under the effect of ionic liquid group to produce c–z type ions. Additionally this fragmentation is potentiated to support widely range of fragment ions which can cover the location of mutation. Our results have supplied a new top-down method about how to analyze the mutation or even post-translational modification of proteins in MALDI mass spectrometry.     

Fe+ chemical ionization of peptides

Laser-desorbed peptide neutral molecules were allowed to react with Fe⁺ in a Fourier transform mass spectrometer, using the technique of laser desorption/chemical ionization. The Fe⁺ ions are formed by laser ablation of a steel target, as well as by dissociative charge-exchange ionization of ferrocene with Ne⁺. Prior to reaction with laser-desorbed peptide molecules, Fe⁺ ions undergo 20–100 thermalizin collisions with xenon to reduce the population of excited-state metal ion species. The Fe⁺ ions that have not experienced thermalizing collisions undergo charge exchange with peptide molecules. Iron ions that undergo thermalizing collisions before they are allowed to react with peptides are found to undergo charge exchange and to form adduct species [M + Fe⁺] and fragment ions that result from the loss of small, stable molecules, such as H₂O, CO, and CO₂, from the metal ion-peptide complex.     

Application of the Gil-Av equation to determine the complexing constant by the gas-chromatographic method

Conclusion The complexing constants, determined by the gas-chromatographic method using the Gil-Av equation, can have either a true or an apparent value, depending on the concentration of the complexing agent and the basicity of the solvent (gas-chromatographic phase).Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 2, pp. 441–443, February, 1972.     

A simple method to determine the anomeric configuration of sialic acid and its derivatives by 13C-NMR

The anomeric configuration of sialic acid and its derivatives could be determined on the basis of the coupling pattern of C-1 in the gated proton-decoupled or selective proton decoupled ¹³C-NMR spectra; the α anomer gave a doublet C-1 signal while the β gave a singlet.The α-anomers gave a doublet C-1 signal while the β gave a singlet in their selective proton-decoupled spectra.