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PubMedDate not supplied

[Hydrolysis of peptides by immobilized bacterial peptide hydrolases].

The feasibility of hydrolysis of a mixture of peptides with an enzyme from the bacterium Xanthomonas rubrilineans, displaying a peptidase activity and immobilized on aluminum oxide, was studied. Kinetic schemes and equations allowing for approaching quantitative description of peptide hydrolysis in complex mixtures containing free amino acids and peptides were obtained. It was demonstrated that as a result of hydrolysis, the content of free amino acids in hydrolysates decreased 2.5- to 3-fold and the molecular weight of the constituent peptides, 2-fold.

Aluminum Oxide↗

PubMed2006

Tandem mass spectrometric analysis of a complex triterpene saponin mixture of Chenopodium quinoa.

A nano-HPLC electrospray ionization multi-stage tandem mass spectrometry (nLC-ESI-MS/MS) approach was applied to a complex crude triterpene saponin extract of Chenopodium quinoa seed coats. In ESI-MS/MS spectra of triterpene saponins, characteristic fragmentation reactions are observed and allow the determination of aglycones, saccharide sequences, compositions, and branching. Fragmentation of aglycones provided further structural information. The chemical complexity of the mixture was resolved by a complete profiling. Eighty-seven triterpene saponins comprising 19 reported and 68 novel components were identified and studied by MS. In addition to four reported, five novel triterpene aglycones were detected and characterized according to their fragmentation reactions in ESI-MS/MS and electron ionization mass spectrometry (EI-MS). As a novelty fragmentation pathways were proposed and analyzed based upon quantum chemical calculations using a hybrid Hartree-Fock density functional method. Accuracy of the assignment procedure was proven by isolation and structure determination of a novel compound. As the relative distribution and composition of saponins varies between different cultivars and soils, the presented strategy allows a rapid and complete analysis of Chenopodium quinoa saponin distribution and composition, and is particularly suitable for quality control and screening of extracts designated for pharmaceutical, agricultural, and industrial applications.

Chenopodium quinoa↗

PubMed2006

Real-life applications of the MULVADO software package for processing DOSY NMR data.

MULVADO is a newly developed software package for DOSY NMR data processing, based on multivariate curve resolution (MCR), one of the principal multivariate methods for processing DOSY data. This paper will evaluate this software package by using real-life data of materials used in the printing industry: two data sets from the same ink sample but of different quality. Also a sample of an organic photoconductor and a toner sample are analysed. Compared with the routine DOSY output from monoexponential fitting, one of the single channel algorithms in the commercial Bruker software, MULVADO provides several advantages. The key advantage of MCR is that it overcomes the fluctuation problem (non-consistent diffusion coefficient of the same component). The combination of non-linear regression (NLR) and MCR can yield more accurate resolution of a complex mixture. In addition, the data pre-processing techniques in MULVADO minimise the negative effects of experimental artefacts on the results of the data. In this paper, the challenges for analysing polymer samples and other more complex samples will also be discussed.

Magnetic Resonance Spectroscopy↗

PubMed1987

Rapid characterization of mucin oligosaccharides from rat small intestine with gas chromatography-mass spectrometry.

Mucin glycopeptides were isolated from rat small intestinal mucosa after reduction/alkylation, trypsin digestion and gel chromatography. The oligosaccharides were released by using alkaline-NaBH4, separated into neutral and acidic species and permethylated. The derivatized mixtures were analysed with fast atom bombardment mass spectrometry and gas chromatography-mass spectrometry using thin film columns. Permethylated neutral oligosaccharides with up to seven sugars could be chromatographed and detected with mass spectrometry. The complex mixture revealed was partly due to the linkage GalNAc being substituted at both position 3 and 6. The approach will be very useful when analysing small amounts of mucins and mucin fragments.

Animals↗

PubMed1987

Determination of eicosanoids, phospholipids and related compounds by thermospray liquid chromatography-mass spectrometry.

Thermospray mass spectrometry has proven to be a useful technique for analyzing various biological compounds including eicosanoids and phospholipids. Molecular ions as well as fragment ions which reveal useful structural information are produced for underivatized eicosanoids and phospholipids using filament-off or filament-on thermospray mass spectrometry, respectively. In conjunction with on-line chromatographic separation, complex mixtures of biological samples can be rapidly analyzed with great reliability. Data will be presented concerning the analysis of prostaglandins, other eicosanoids and molecular species of phospholipids as well as the application of these methodologies to complex biological samples.

Animals↗

PubMedDate not supplied

A review of analytical methods for the identification and quantification of hydrocarbons found in jet propellant 8 and related petroleum based fuels.

Jet propellant 8 (JP-8) is a complex mixture of compounds that varies from batch to batch. Quantification of various compound classes of JP-8, including BTEX, PAHs and VOCs, has been accomplished. Very few papers have tackled total JP-8 quantification because of its complexity. The components in JP-8 tend to co-elute and present at low concentrations, often nondetectable. JP-8 is the major source of chemical exposure for Department of Defense personnel and a potential hazard for civilians and marine animals. Some components of JP-8 have been identified as possible human carcinogens and have been studied extensively. Development of analytical methods to analyze the components of this fuel are essential to measure the extent of exposure, as well as the short-term and long-term exposure in rodents, humans and marine life. To date, JP-8 has been examined in urine, blood, contaminated water and fish tissue. This paper reviews methods currently utilized in the literature for the analysis of JP-8 and its components. This paper also discusses extraction methods and detectors commonly used in JP-8 and hydrocarbon analysis in general. Finally, the effects of exposure and the future of JP-8 and petroleum analysis with respect to human health are discussed.

Animals↗

PubMed1994

Prediction and optimization of the separation of metal cations by capillary electrophoresis with indirect UV detection.

The migration of metal cations in capillary electrophoresis with indirect UV detection was investigated. A mathematical model was used to relate the electrophoretic mobility of a metal cation with two background electrolyte factors: the pH and the concentration of a complexing agent. Imidazole, which has an electrophoretic mobility close to those of metal cations, was used as the visualization agent, and 2-hydroxyisobutyric acid (HIBA) was used as the complexing agent. Through computer modeling, the electrophoretic mobilities of fourteen metal cations were predicted on the basis of five initial experiments within the factor space of pH and HIBA concentration. Good agreement was observed between the predicted electrophoretic mobilities and the observed values of the metal cations. Subsequently, the separation of a complex mixture of metal cations was successfully optimized. A complete separation of 14 metal cations including alkali, alkaline earth and transition metal(II) cations was achieved within 4 min. In addition, the effectiveness of other visualization agents and complexing agents for the separation of metal cations was examined.

Cations↗

PubMed2002

Thermodynamics of cationic lipid-DNA complex formation as studied by isothermal titration calorimetry.

The detailed analysis of the cationic lipid-DNA complex formation by means of isothermal titration calorimetry is presented. Most experiments were done using 1,2-dioleyl-sn-glycero-3-ethylphosphocholine (EDOPC), but basic titrations were also done using DOTAP, DOTAP:DOPC, and DOTAP:DOPE mixtures. Complex formation was endothermic with less than 1 kcal absorbed per mole of lipid or DNA charge. This enthalpy change was attributed to DNA-DNA mutual repulsion within the lamellar complex. The exception was DOTAP:DOPE-containing lipoplex for which the enthalpy of formation was exothermic, presumably because of DOPE amine group protonation. Experimental conditions, namely, direction and titration increment as well as concentration of titrant, which dictate the structure of resulting lipoplex (whether lamellar complex or DNA-coated vesicle), were found to affect the apparent thermodynamics of complex formation. The structure, in turn, influences the biological properties of the lipoplex. If the titration of lipid into DNA was carried out in large increments, the DeltaH was larger than when the injection increments were smaller, a finding that is consistent with increased vesicle disruption under large increments and which is expected theoretically. Cationic lipid-DNA binding was weak in high ionic strength solutions, however, the effective binding constant is within micromolar range because of macromolecular nature of the interaction.

Animals↗

PubMed1999

Liquid chromatography-electrospray mass spectrometry as a tool for the analysis of sulfated oligosaccharides from mucin glycoproteins.

An approach for analyzing sulfated oligosaccharide alditol mixtures by liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) is described. Two columns, an amino-bonded column and a porous graphitized carbon column (PGC) were used. Oligosaccharides were eluted with linear gradients of acetonitrile and water, with 5 mM ammonium hydrogencarbonate or formate buffers at a basic pH. The methods were evaluated on a mixture of sulfated oligosaccharide alditols prepared from mucin glycoproteins from pig stomach. Results from LC-ESI-MS of the mixture were compared with the structural information obtained by high energy collision fragmentation using fast atom bombardment tandem mass spectrometry (FAB-MS-MS). The separation ability of the two columns was also tested using a more complex mixture of sulfated oligosaccharides from pig colon, where several isomers were detected. The potential use of in-source collision-induced dissociation (CID) to gain sequence information of sulfated oligosaccharides was also evaluated. The major fragment ions obtained by in-source CID of the trisaccharide Hex-3HexNAcol6-HexNAc6-SO3 were sufficient for assigning the oligosaccharide sequence and the position of the sulfate group within the monosaccharide moiety. The LC-ESI-MS approach should be a valuable tool for characterization of mucin glycosylation and alterations during pathological conditions.

Animals↗

PubMed2006

Past and future application of solid-state detectors in manned spaceflight.

The radiation exposure in space missions can be reduced by careful mission planning and appropriate measures, such as provision of a radiation shelter, but it cannot be eliminated. The reason for that is the high penetration capability of the radiation components owing to their high energies. Radiation is therefore an acknowledged primary concern for manned spaceflight and is a potentially limiting factor for long-term orbital and interplanetary missions. The radiation environment is a complex mixture of charged particles of solar and galactic origin and of the radiation belts, as well as of secondary particles produced in interactions of the galactic cosmic particles with the nuclei of atmosphere of the earth. The complexity even increases by placing a spacecraft into this environment owing to the interaction of the radiation components with the shielding material. Therefore it is a challenge to provide for appropriate measurements in this radiation field, coping with the limited resources on experiment power and mass. Solid-state dosemeters were already chosen for measurements in the first manned flights. Thermoluminescence dosemeters (TLDs) and plastic nuclear track detectors (PNTD) especially found a preferred application because they are light-weighted, need no power supply and they are tissue-equivalent. Most of the data available until 1996 were gathered by using these passive detectors; this especially holds for heavy ion particle spectra. The systems, supplemented by converter foils or fission detectors and bubble detectors, provide information on dose, particle flux-, energy- and linear energy transfer spectra of the ionising radiation and neutron fluxes and doses. From 1989, silicon detectors were used for dose and flux measurements and later on for particle spectrometry. Silicon detectors were demonstrated as a powerful tool for the description of space radiation environment. Optical simulated luminescence (OSL) detectors have now been introduced as a new system in space research. Both, OSL and superheated drop detectors are candidates for personal dosimetry systems. The article will summarise past results, and results of measurements performed recently on the ISS, and conclude with future aspects.

Cosmic Radiation↗

PubMed1977

Thin-layer chromatography of guanidino compounds.

Twelve guanidino compounds including, among others, glycocyamine, creatine, creatinine, arginine, guanidinosuccinic acid were submitted to thin-layer chromatography on plates precoated with silica gel or cellulose, respectively. Optimal resolution was obtained by two-dimensional chromatography on cellulose layers when the two solvent systems iso-propanol-acetic acid-water (volumes, 60 ml + 45 ml + 15 ml) and iso-propanol-dimethyl-formamide-water (volumes, 40 ml + 20 ml + 20 ml) were used for the first and second dimension, respectively. This procedure permitted the resolution of 10 distinct spots out of a mixture of the twelve guanidino compounds. Chloroform-methanol-ammonia (170 g/kg) (volumes, 40 ml + 20 ml + 20 ml) proved to be the most suitable solvent system for one-dimensional chromatography on cellulose layers of less complex mixtures of guanidino compounds.

Arginine↗

PubMed2005

Recent developments for the analysis of data obtained from isothermal calorimetry.

Isothermal calorimetry is rapidly becoming an indispensable tool for the quantitative determination of a variety of kinetic and thermodynamic parameters for a wide range of systems. In particular calorimetry is finding increased application to the investigation of stability and incompatibility of pharmaceutical materials. In order to draw meaningful conclusions and to predict behaviour in related systems it is necessary to have the means to calculate accurately parameters such as the rate constant and enthalpy. To this end several groups have been developing equations which describe calorimetric output in these terms. This paper will briefly outline some of these equations and discuss some of the limitations that currently exist in their application. A particular emphasis is placed on the recent developments relating to the application of these equations to flow calorimetric data. The main application of these equations is usually found in the pharmaceutical industry. Pharmaceutical formulations are usually extremely complex mixtures consisting of many different excipients as well as the active drug. Because of these large numbers of ingredients it is often observed that multiple chemical and physical process occur over the lifetime of the study. This complexity is then reflected in the calorimetric data rendering the application of the simple equations useless. Dealing with this complexity is a major issue amongst the calorimetric community and some of the recent advances in this field are also discussed.

Algorithms↗

PubMed1984

Mass action effects on competitive adsorption of fibrinogen from hemoglobin solutions and from plasma.

The strong effect of protein adsorption on blood and tissue compatibility is well known. Little is presently known about the mechanisms which control the composition of the adsorbed protein layer which forms upon exposure of surfaces to mixtures of proteins. Reexamination of the ability of hemoglobin to inhibit the adsorption of 125I fibrinogen to polyethylene revealed that the inhibition was strongly dependent on the fibrinogen concentration. These results suggested that fibrinogen adsorption from more complex mixtures such as plasma should also be strongly dependent on total concentration. Fibrinogen adsorption from plasma was found to be maximal at intermediate plasma concentrations, and was reduced at both low and high plasma concentrations. The plasma concentration at which this maximum occurred was 10% for polytetrafluoroethylene, 1% for polyethylene, and 0.1% for glass. The unusual concentration dependence is attributed to mass action effects on the competitive adsorption of proteins, specifically that competitive effectiveness is expected to increase as unoccupied surface adsorptive sites become less frequent. Analogous effects of adsorption time on competitive adsorption are also predicted due to the changing concentration at the interface as the buffer is gradually replaced by protein solution. These mass action effects are very similar to previous qualitative observations by Vroman and are therefore dubbed the " Vroman effect".

Adsorption↗

PubMed1992

Synthetic peptides as vaccines.

The economics of vaccines has been a major limitation in the commercial research and development of new approaches. This coupled with the natural scientific desire to simplify and define the composition of effective vaccines argues that the future of vaccines lies in novel approaches that will discover effective and less expensive components. Peptides, whether they are chemically synthesized or produced in bacteria, are an attractive possibility. To substitute linear peptides for complex mixtures of proteins would be a major technical advance and would stimulate tremendous commercial interest. However, at the present time I view this approach still unlikely to be of major practical importance. I conclude this because of the complexity of immunological responses to microorganisms. Even though, in some instances, a cytotoxic T-cell response or even the majority of the antibody response to a pathogen can be defined by a short linear peptide, most people believe that multiple effector functions of the immune system should be stimulated in optimal vaccines. For a small cocktail of peptides to reproduce the diversity of responses elicited by a virus, parasite, or bacterium is unlikely. However, I fully realize that remarkable progress has occurred towards understanding the structural requirements necessary to stimulate cellular and humoral immune responses, and peptides have been integral in the development of this field. Also, the success of several research groups in developing effective antiviral vaccines using short linear peptides argues that I might be painting too dark of a picture. As someone who has used this strategy to explore peptide-MHC and peptide-antibody interactions, I am a strong scientific supporter of the approach. In this forum I am purposely cautious in my optimism. As the details of the complex molecular and cellular interactions that control the immune system are elucidated, both the number of strategies and the possible applications of modulating the immune response will increase as well. In addition to protective immunity to pathogens, cancer therapy could be revolutionized if tumor-specific cytotoxic T-cells could be generated routinely. Novel therapeutic approaches to allergy, autoimmunity, and transplantation can be envisioned if the T-lymphocytes responsible for these syndromes could be modulated without total immune suppression. Consequently, I am confident that the experiments described in this chapter will be central to developing exciting new therapeutic and prophylactic compounds, but I am not sure that they will resemble naturally occurring peptides. The one aspect I am confident of is that the capacity of the immune response to protect the organism will continually surprise us.

Animals↗

PubMed1978

Polypeptide sequencing by liquid chromatography mass spectrometry.

Key steps in a proposed automated system for polypeptide sequencing utilizing a liquid chromatograph mass spectrometer computer system have been tested with mixtures containing up to six model oligopeptides. At the low nanomole level it was possible to obtain complete sequence information for all components in many, but not all, of the mixtures tried. Interpretation of the results is complicated by the presence of numerous side-products formed in the derivatization process. Minimization of such impurities will be necessary to reduce the ambiguity of the sequence information resulting from more complex mixtures, such as those expected from the degradation of larger polypeptides, and to reduce sample requirements to the subnanomole level. However, the present system appears to have unique advantages over other proposed automated methods.

Amino Acid Sequence↗

PubMed2005

Detection of the common RNA nucleoside pseudouridine in mixtures of oligonucleotides by mass spectrometry.

Pseudouridine, an isomer of uridine, is probably the most common of many posttranscriptional RNA modifications found in nature. Although mass spectrometry has become widely used in the characterization of modified nucleic acids, its application to the recognition and sequence placement of pseudouridine has not been straightforward, particularly in the case of complex mixtures such as those resulting from selective enzymatic hydrolysis of RNA into oligonucleotides. We report results of a study of the characteristic dissociation reactions of pseudouridine-containing oligonucleotides following ionization by electrospray and use of those pathways in an LC/MS-based method applicable to direct analysis of RNase digests of RNA. As a consequence of the C-C (rather than C-N) glycosidic bond of pseudouridine, the otherwise common dissociation paths involving base loss do not occur, resulting in characteristic formation of a set of low-mass negative ions containing the intact glycosidic bond (m/z 225, 207, 189, 165, 164, 139), which permit recognition of pseudouridine-containing oligonucleotides. Those components can subsequently be subjected to sequence analysis by MS/MS, in which enhancement of selective sequence-determining ions (a-, w-, y-types), and absence of a - base ions, are observed at the site of pseudouridylation. Also, selected reaction pathways can be monitored in the LC/MS/MS analysis that are indicative of pseudouridine at the 5' terminus (m/z 225 --> 165), internal positions (m/z 207 --> 164), and in the RNase T1-derived product Psi pGp (m/z 668 --> 207) arising from the RNA sequence ...G Psi G... These procedures can be effectively integrated into an existing suite of LC/ESI-MS-based methods designed for the analysis of posttranscriptionally modified sites in RNA.

Base Sequence↗

PubMed1998

Regulation and function of pulmonary surfactant protein B.

Pulmonary surfactant, a complex mixture of phospholipids and specific associated proteins, reduces the surface tension at the air-liquid interface of the distal conducting airways and gas exchanging alveoli of the lung. Lipids, primarily neutral and phospholipids, compose approximately 90% of the surfactant complex. The remaining 10% of surfactant is composed of at least three surfactant-specific proteins, designated surfactant protein A (SP-A), SP-B, and SP-C. These proteins contribute to the formation, stabilization, and function of organized surfactant structures. This article briefly reviews the normal composition and function of pulmonary surfactant and specifically reviews the structure, function, and regulation of surfactant protein B (SP-B). The recent identification of neonates with refractory respiratory failure due to a genetic absence of SP-B and the study of transgenic mice in which SP-B gene expression has been ablated highlight the importance of the protein to surfactant function, synthesis, and metabolism and to the maintenance of lung function. Gene reconstitution experiments in vitro and in SP-B-deficient transgenic mice suggest specific functions for the amino and carboxyl terminal domains of the protein. SP-B deficiency is a potential target for gene therapy in human patients.

Amino Acid Sequence↗

PubMed2003

Use of surfactant modified ultrafiltration for perchlorate (Cl(O)(4-)) removal.

Determinations of perchlorate anion (ClO(4)(-)) transport and rejection were performed using a surfactant modified ultrafiltration (UF) membrane. Perchlorate anion (at a concentration of 100 microg/L of ClO(4)(-), spiked with KClO(4)) was introduced to the membrane as a pure component, in binary mixtures with other salts, cationic and anionic surfactants, and at various ionic strength conditions (conductivity). Also, a natural source water was spiked with perchlorate in the presence of cationic and anionic surfactants and used to determine the effects of a complex mixture (including natural organic matter (NOM)) on the observed rejection. All filtration measurements were performed at approximately the same permeate flow rate in order to minimize artifacts from mass transfer at the membrane interface. The objective of this study was to modify a negatively charged UF membrane in terms of the fundamental mechanisms, steric/size exclusion and electrostatic exclusion and to enhance perchlorate rejection, with synthetic water and a blend of Colorado River water and State Project water (CRW/SPW). Previous work suggested that perchlorate was dominantly rejected by electrostatic exclusion for charged nanofiltration (NF) and UF membranes (Rejection of perchlorate by reverse osmosis, nanofiltration and ultrafiltration (UF) membranes: mechanism and modeling. Ph.D. dissertation, University of Colorado, Boulder, USA, 2001). In that research, perchlorate rejection capability was quickly lost in the presence of a sufficient amount of other ions. However, this study showed that ClO(4)(-) was excluded from a (negatively) charged UF membrane with pores large with respect to the size of the ion. Although perchlorate rejection capability due to apparent electrostatic force was reduced in the presence of a cationic surfactant, a desired amount of the ClO(4)(-) was excluded by steric exclusion. The steric exclusion was due to decreasing membrane pore size caused by the adsorption of the cationic surfactant.

Filtration↗