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PubMed2006

Radiotracer measurements of protein mass transfer: kinetics in ion exchange media.

We describe a method to measure protein mass transfer kinetics in ion exchange adsorbents for preparative chromatography based on the use of radioactively labeled protein. The method was developed and evaluated using lysozyme as a test protein with the three commercial strong-acid cation exchangers SP-Sepharose-FF, SP-Sepharose-XL, and S-HyperD. Iodination with 125I was used to label the protein, which was added in trace amounts (approximately 0.1%) to an unlabeled protein solution. The solution was recirculated through a shallow bed of the adsorbent particles and the radioactivity accumulated in the bed measured with a gamma-counter as a function of time. Radiotracer-based kinetics measurements were found to be in good agreement with results obtained with a conventional shallow-bed technique, provided that freshly labeled protein solutions were used. The method has advantages in terms of simplicity, ability to deal with adsorption from complex mixtures, and the potential for measurements under tracer diffusion conditions. Kinetics results obtained for the three different stationary phases were generally consistent with previous studies. Protein mass transfer can be described by a pore diffusion model with a nearly salt-independent pore diffusivity for SP-Sepharose-FF and by a homogeneous diffusion model with a saltindependent adsorbed phase diffusivity for S-HyperD. However, it appears that a more complex model, accounting for parallel pore and surface diffusion, is needed to describe protein mass transfer in SP-Sepharose-XL. The modeling results were found to be correlated with the apparent pore sizes determined by inverse SEC.

Chromatography, Ion Exchange↗

PubMed1997

Application of mixed mobile phases and a step gradient method in capillary electrochromatography for the separation of isomeric polycyclic aromatic hydrocarbon-deoxyribonucleoside adduct mixtures prepared in vitro.

Capillary electrochromatography (CEC) was used for the analysis of mixtures of neutral isomeric compounds derived from the reaction of carcinogenic hydrocarbon (benzo[g]chrysene and 5,6-dimethylchrysene) dihydrodiol epoxides with calf thymus deoxyribonucleic acid (DNA). The CEC analysis demonstrated higher resolution, greater speed and lower analyte consumption than high-performance liquid chromatography (HPLC) in the analysis of the same samples using the same type of stationary phase. Proper selection of the mixed mobile phases was critical for the separation of these complex mixtures with enhanced speed and selectivity. The use of a step gradient further improved the speed of the CEC analysis resulting in electrochromatograms that required only 25-70% of the corresponding HPLC analysis times.

DNA Adducts↗

PubMed2004

Automated Fourier transform infrared analysis of urinary stones: technical aspects and example of procedures applied to carbapatite/weddellite mixtures.

New software (ScanLith) was developed to provide an automated procedure for identifying and quantifying crystalline species in urinary calculi. The first step was to strictly define operative conditions of sample preparation because they have a significant influence on the stability of various crystalline phases. Second, we determined the quantification coefficients of a polynomial curve required to develop the automated procedure. This was illustrated by the study of carbapatite and weddellite, as both constituents represent one of the most frequent associations found in stones analyzed in our laboratory. The following quadratic equation was obtained with a correlation coefficient r2 = 0.9997: Y = -0.5144x2 + 1.5239x - 0.0141, where Y is the absorbance ratio carbapatite/weddellite and x is the percentage of carbapatite in the mixture. The absorbance of carbapatite and weddellite was measured at 1035 cm(-1) and 1325 cm(-1), respectively. The third step was to validate the ScanLith procedure in routine analysis by comparing computed results with those of an expert. Concordance (r2 = 0.9824) was better than that previously reported using various computerized systems with a mean deviation of 4%. Algorithms developed in ScanLith to identify main and minor components found in urinary stones, even in complex mixtures containing up to seven constituents, allowed us to lower the detection threshold down to 1 to 10% depending on the main component.

Algorithms↗

PubMed2004

Fractionation of peptides and identification of proteins from Saccharomyces cerevisiae in proteomics with the use of reversed-phase capillary liquid chromatography and pI-based approach.

The aim of the work was to explore the identification of proteins from Saccharomyces cerevisiae using combined capillary reversed-phase liquid chromatography (RPLC) and in-solution isoelectric focusing (sIEF) for fractionation of peptides prior to mass spectrometry analysis. That method was proved to be the alternative separation method for complex mixtures of protein tryptic digests in proteomics. Analysis of the identification of peptides was performed with the use of electrospray ionization-ion trap tandem mass spectrometry (ESI-IT-MS/MS). First, the sIEF fractionation was carried out prior to separation and mass spectrometry identification by nano-LC/ESI-MS/MS instrument. The proposed approach based on sIEF and nano-LC/ESI-MS/MS analysis was proved to be an efficient and accurate alternative fractionation method of complex protein digests and can be considered as the useful tool for identification of proteins. Moreover, analytical information from that approach can be considered as the additional source of database matching constraint and can be valuable tool for analytical and bioinformatics studies of peptides fractionation in proteomics. Based on the MS/MS results obtained with ESI-IT-MS/MS instrument, 851 proteins from S. cerevisiae were identified. However, after careful analysis of the data reduction in number of proteins to 126 was obtained. Those results are discussed and interpreted in the view of the evaluation method used.

Animals↗

PubMed2003

Comprehensive two-dimensional liquid chromatography x gas chromatography: evaluation of the applicability for the analysis of edible oils and fats.

Edible fats and oils are complex mixtures containing a wide range of (classes of) compounds. The most important group of compounds are the triglycerides (triacylglycerides, TAGs). Because of the large number of possible fatty acid combinations, an enormous number of TAGs is possible. In the present feasibility study, the applicability of different modes of comprehensive two-dimensional LCXGC for detailed oil and fat analysis is evaluated. Comprehensive LCXGC was found to be an extremely powerful analytical method for the analysis of complex TAG samples. Using the new comprehensive set-ups, TAGs can be separated according to two independent parameters: carbon number vs. number of double bonds, or fatty acid composition vs. number of double bonds. The information content of comprehensive separations by far exceeds that of the current generation of analytical methods. The quantitative results of the separations show a good agreement with data obtained from standard analytical methods. The comprehensive methods studied can also be used for fingerprinting of oil samples, as well as for the analysis of target compounds or compound groups. Highly detailed separations of olive oil samples were obtained. Zooming in on one region of the chromatogram allowed reliable analysis of wax esters without interferences of sterol esters.

Chromatography, Gas↗

PubMed2003

A novel in-vitro technique for studying percutaneous permeation with a membrane-coated fiber and gas chromatography/mass spectrometry: part I. Performances of the technique and determination of the permeation rates and partition coefficients of chemical mixtures.

PURPOSE: To develop a novel in-vitro technique for rapid assessment of percutaneous absorption of chemical mixtures. METHODS: A silastic membrane was coated on to a fiber to be used as a permeation membrane. The membrane-coated fiber was immersed in the donor phase to partition the compounds into the membrane. At a given partition time, the membrane-coated fiber was transferred into a GC injector to evaporate the partitioned compounds for quantitative and qualitative analyses. RESULTS: This technique was developed and demonstrated to study the percutaneous permeation of a complex mixture consisting of 30 compounds. Each compound permeated into the membrane was identified and quantified with GC/MS. The standard deviation was less than 10% in 12 repeated permeation experiments. The partition coefficients and permeation rates in static and stirred donor solution were obtained for each compound. The partition coefficients measured by this technique were well correlated (R2 = 0.93) with the reported octanol/water partition coefficients. CONCLUSIONS: This technique can be used to study the percutaneous permeation of chemical mixtures. No expensive radiolabeled chemicals are required. Each compound permeated into the membrane can be identified and quantified. The initial permeation rate and equilibrium time can be obtained for each compound, which could serve as characteristic parameters regarding the skin permeability of the compound.

Administration, Cutaneous↗

PubMed1998

Mass spectrometric mapping of ion channel proteins (porins) and identification of their supramolecular membrane assembly.

Mass spectrometric peptide mapping, particularly by matrix-assisted laser desorption-ionization (MALDI-MS), has recently been shown to be an efficient tool for the primary structure characterization of proteins. In combination with in situ proteolytic digestion of proteins separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometric peptide mapping permits identification of proteins from complex mixtures such as cell lysates. In this study we have investigated several ion channel membrane proteins (porins) and their supramolecular assembly in mitochondrial membranes by peptide mapping in solution and upon digestion in the gel matrix. Porins are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through bacterial and mitochondrial membranes. The well-characterized porin from Rhodobacter capsulatus (R.c.-porin) has been found to be a native trimeric complex by the crystal structure and was used as a model system in this study. R.c.-porin was characterized by MALDI-MS peptide mapping in solution, and by direct in situ-gel digestion of the trimer. Furthermore, in this study we demonstrate the direct identification of the noncovalent complex between a mitochondrial porin and the adenine nucleotide translocator from rat liver, by MALDI-MS determination of the specific peptides due to both protein sequences in the SDS-PAGE gel band. The combination of native gel electrophoresis and mass spectrometric peptide mapping of the specific gel bands should be developed as a powerful tool for the molecular identification of protein interactions.

Electrophoresis, Polyacrylamide Gel↗

PubMed2004

DNA-templated organic synthesis: nature's strategy for controlling chemical reactivity applied to synthetic molecules.

In contrast to the approach commonly taken by chemists, nature controls chemical reactivity by modulating the effective molarity of highly dilute reactants through macromolecule-templated synthesis. Nature's approach enables complex mixtures in a single solution to react with efficiencies and selectivities that cannot be achieved in conventional laboratory synthesis. DNA-templated organic synthesis (DTS) is emerging as a surprisingly general way to control the reactivity of synthetic molecules by using nature's effective-molarity-based approach. Recent developments have expanded the scope and capabilities of DTS from its origins as a model of prebiotic nucleic acid replication to its current ability to translate DNA sequences into complex small-molecule and polymer products of multistep organic synthesis. An understanding of fundamental principles underlying DTS has played an important role in these developments. Early applications of DTS include nucleic acid sensing, small-molecule discovery, and reaction discovery with the help of translation, selection, and amplification methods previously available only to biological molecules.

Chemistry, Organic↗

PubMed2001

Biodegradation of soluble aromatic compounds of jet fuel under anaerobic conditions: laboratory batch experiments.

Laboratory batch experiments were performed with contaminated aquifer sediments and four soluble aromatic components of jet fuel to assess their biodegradation under anaerobic conditions. The biodegradation of four aromatic compounds, toluene, o-xylene, 1,2,4-trimethylbenzene (TMB), and naphthalene, separately or together, was investigated under strictly anaerobic conditions in the dark for a period of 160 days. Of the aromatic compounds, toluene and o-xylene were degraded both as a single substrate and in a mixture with the other aromatic compounds, while TMB was not biodegraded as a single substrate, but was biodegraded in the presence of the other aromatic hydrocarbons. Substrate interaction is thus significant in the biodegradation of TMB. Biodegradation of naphthalene was not observed, either as a single substrate or in a mixture of other aromatic hydrocarbons. Although redox conditions were dominated by iron reduction, a clear relation between degradation and sulfate reduction was observed. Methanogenesis took place during the later stages of incubation. However, the large background of Fe(II) masked the increase of Fe(II) concentration due to iron reduction. Thus, although microbial reduction of Fe(III) is an important process, the evidence is not conclusive. Our results have shown that a better understanding of the degradation of complex mixtures of hydrocarbons under anaerobic conditions is important in the application of natural attenuation as a remedial method for soil and groundwater contamination.

Anaerobiosis↗

PubMed2003

Analysis of high-density lipoprotein apolipoproteins recovered from specific immobilized pH gradient gel pI domains by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The proteins associated with the circulating lipoproteins in the blood function not only for mediating lipid metabolism but also for maintaining structural stability of the micellelike structure. Any modifications of these proteins, by mutation or posttranslational modification, could compromise the function of these proteins and contribute to the development of cardiovascular disease. Because of the presence of extensive lipophilic domains, these proteins, when recovered from the lipoprotein particle (apolipoproteins) present an analytical challenge because of low solubility and proclivity toward aggregate formation. Our goal is to characterize these proteins by a combination of high-accuracy pI measurement coupled with MALDI analysis. In this report, we demonstrate the high resolution of immobilized pH gradient isoelectric focusing (IPG-IEF) for the analysis of these apolipoproteins isolated from serum HDL collected from a density gradient ultracentrifugation-based separation. The IPG separation of the HDL apolipoproteins was imaged and combined with digital analysis to produce a detailed pI profile of the apolipoproteins in the high-density lipoprotein (HDL) fraction. This is the first time that a high-resolution pI profile has been obtained for the HDL apolipoproteins. The feasibility of linking the pI profile to a MALDI-based molecular weight determination was achieved by incorporating passive elution of the intact proteins from the IPG gel with a four-component solvent mixture that solved the problem of recovery of the apolipoproteins from the IPG matrix. Twenty-five bands were observed in the pI profile. A survey analysis of 12 of these bands by MALDI indicated that they were associated with the known HDL apolipoproteins. While there is considerable overlap in the pI profiles of the apolipoproteins, linking the analysis with a MALDI-based second dimension in m/z is shown to be an efficient method to characterize this complex mixture of apolipoproteins.

Apolipoproteins↗

PubMed2007

Phase boundaries and biological membranes.

Bilayer mixtures of lipids are used by many researchers as chemically simple models for biological membranes. In particular, observations on three-component bilayer mixtures containing cholesterol show rich phase behavior, including several regions of two-phase coexistence and one region of three-phase coexistence. Yet, the relationship between these simple model mixtures and biological membranes, which contain hundreds of different proteins and lipids, is not clear. Many of the model mixtures have been chosen for study because they exhibit readily observed phase separations, not because they are good mimics of cell membrane components. If the many components of cell membranes could be grouped in some way, then understanding the phase behaviors of biological membranes might be enhanced. Furthermore, if the underlying interaction energies between lipids and proteins can be determined, then it might be possible to model the distributions of lipids and proteins in a bilayer membrane, even in complex mixtures.

Animals↗

PubMed1965

Effect of dietary factors upon the resistance of albino mice to experimental infection with Mycobacterium tuberculosis.

Layton, Herbert W. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Effect of dietary factors upon the resistance of albino mice to experimental infection with Mycobacterium tuberculosis. J. Bacteriol. 90:958-964. 1965.-Each of the major nutritional components of a synthetic diet was quantitatively altered to determine its effect upon the resistance of albino mice to infection with Mycobacterium tuberculosis. The animals were challenged after the first 2 weeks of feeding, and the percentage that survived acute death was determined statistically. The level of protein which provided the greatest percentage of survival was 30%; increases or decreases from this level were detrimental. The optimal fat level was found to be 5% for either corn oil or coconut oil. Survival decreased when greater amounts of oil were added, and this effect was especially marked for 40% coconut oil. Vitamin A enhanced survival when increased from the normal level of 20,000 units per kg of diet to 160,000 units, but further increases were harmful. An amount of 75 g/kg of a vitamin B complex mixture afforded optimal resistance, but 25-g shifts in either direction lowered this resistance. Vitamin K-free diets resulted in high levels of survival, and addition of the vitamin resulted in proportional decreases in resistance.

Animals↗

PubMed1984

Similarities in structure and function of calf thymus and Drosophila casein kinase II.

Both calf and Drosophila contain a type II casein kinase with similar molecular structure and catalytic activity. Purified calf thymus casein kinase II is composed of three subunits of Mr = 44,000 (alpha), 40,000 (alpha'), and 26,000 (beta) (Dahmus, M.E. (1981) J. Biol. Chem. 256, 3319-3325), whereas the Drosophila enzyme is composed of two subunits of Mr = 36,700 (alpha) and 28,200 (beta) (Glover, C. V. C., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 3258-3265). The native form of the enzyme is an alpha 2 beta 2 tetramer. Polyclonal antibodies prepared against each enzyme react with both the alpha and beta subunits of the homologous enzyme and cross-react with both subunits of the heterologous enzyme. Reaction of polyclonal antibodies with proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis establishes that no significant difference in subunit molecular weight exists between the purified enzymes and the enzyme present in initial cell extracts. Each antibody effectively inhibits the in vitro activity of the homologous enzyme and causes a slight inhibition in the activity of the heterologous enzyme. Peptide maps derived from purified subunits indicate that the alpha and beta subunits are unique and that there is extensive primary sequence homology between the corresponding subunits of the calf and Drosophila enzyme. Casein kinase II from both sources phosphorylates the same subunits of calf thymus RNA polymerase II and an identical set of proteins in a complex mixture of acid-soluble proteins from Drosophila tissue culture cells. The striking similarity in molecular structure and catalytic activity between the calf and Drosophila enzyme suggests that casein kinase II has been highly conserved in evolution.

Animals↗

PubMed1983

High-performance gel-permeation chromatography of polypeptides in a volatile solvent: rapid resolution and molecular weight estimations of proteins and peptides on a column of TSK-G3000-PW.

A gel-permeation column of TSK-G3000-PW that was equilibrated and developed with 36 or 45% acetonitrile in 0.1% trifluoroacetic acid fractionated mixtures of peptides with high resolving power. In addition, the elution volumes of 11 standard peptides and proteins were linearly related to the logarithms of their molecular weights in the acetonitrile-trifluoroacetic acid solvent at both low and high flow rates. Since the solvent is volatile and relatively transparent to short-wavelength ultraviolet light this high-performance gel-permeation system offers a rapid and highly sensitive method for the analysis, characterization, and purification of peptides and proteins from complex mixtures.

Acetonitriles↗

PubMed2005

Biomonitoring in occupational health: scientific, socio-ethical, and regulatory issues.

Biomonitoring is one of the best available tools for the prevention of deleterious effects resulting from occupational exposure to chemicals. The availability of analytical techniques having low detection limits allows for the measurement of numerous biomarkers. Complemented with quality control programs, our ability to collect validated information on exposure to toxicants improves. This is important as exposure doses tend to decrease in workplaces. Concurrently, there is an increasing preoccupation towards skin exposure, which cannot currently be reliably assessed through external measurements. Furthermore, as lower exposure doses are encountered, background concentrations of some biomarkers become a serious limitation to their use. This prompts researchers to seek for minor, more specific metabolites, that may however be produced through metabolic pathways that are prone to larger inter-individual variations. Assessment of exposure to complex mixtures of chemicals is another major challenge. There is a growing interest towards ethical issues in biomonitoring. The understanding of the advantages and of the limits of this preventive approach may be very different among occupational health professionals, but more importantly, between health professionals and those they are seeking to protect, i.e., the workers themselves. Many organizations have proposed guideline values for biomarker concentrations, but these seldom find their way in the various countries' bylaws. One underlying reason might be the greater complexity of the scientific aspects of biomarkers, whose understanding is required to set limit values, compared to the process of setting airborne limit concentrations. But the fact that the latter does not consider all aspects of biological complexity does not make it more reliable.

Animals↗

PubMed2003

Development of a LC-LC-MS complete heart-cut approach for the characterization of pharmaceutical compounds using standard instrumentation.

A total heart-cut LC-LC-MS technique is described for the analysis of pharmaceutical compounds. Utilizing ordinary LC-MS equipment, a standardized multi-dimensional, highly selective approach was developed. The system was developed on synthetic mixtures containing ten active pharmaceutical compounds. The methodology would be further deployed for the impurity characterization of batches of active chemical, process intermediates, and reference standards. Column-switching was employed to heart-cut sequential fractions from one set of LC conditions to another set of LC conditions. Two high efficiency columns, including the Merck Chromolith C18, for fast LC-LC analysis were used. The system provides a highly automated, turnkey, multi-dimensional approach applicable to a variety of complex mixtures with little user intervention. It is envisioned that this idea could be viewed as a platform for other types of LC-LC analyses including peptide and achiral-chiral analyses.

Chromatography, High Pressure Liquid↗

PubMed1990

Degradation pathways of PCB upon gamma irradiation.

In order to understand the modifications of the chromatographic profile of Aroclor 1260 upon gamma irradiation, a total of 14 pure polychlorinated biphenyl (PCB) congeners were separately irradiated in solution. Dechlorination was observed, and the generated products were investigated by gas chromatography and mass spectrometry. Degradation proceeds more rapidly in methanol/water mixture than in petroleum ether, but the relative amount of ortho-dechlorinated congeners formed upon irradiation was smaller in the former solvent. Ortho chlorines are preferentially lost in petroleum ether except when they are involved in a 2.5 (or 3.6) substitution pattern, in which case para dechlorination becomes predominant. The precursors of some toxicologically important congeners such as congeners 77, 118, 167, and 189 have been identified. These data are useful to rationalize the modifications of the chromatographic profile of PCB complex mixture upon gamma irradiation.

Aroclors↗

PubMed2005

Rapid and sensitive identification of major histocompatibility complex class I-associated tumor peptides by Nano-LC MALDI MS/MS.

Identification of major histocompatibility complex (MHC)-associated peptides recognized by T-lymphocytes is a crucial prerequisite for the detection and manipulation of specific immune responses in cancer, viral infections, and autoimmune diseases. Unfortunately immunogenic peptides are less abundant species present in highly complex mixtures of MHC-extracted material. Most peptide identification strategies use microcapillary LC coupled to nano-ESI MS/MS in a challenging on-line approach. Alternatively MALDI PSD analysis has been applied for this purpose. We report here on the first off-line combination of nanoscale (nano) LC and MALDI TOF/TOF MS/MS for the identification of naturally processed MHC peptide ligands. These peptides were acid-eluted from human leukocyte antigen (HLA)-A2, HLA-A3, and HLA-B/-C complexes separately isolated from a renal cell carcinoma cell lysate using HLA allele-specific antibodies. After reversed-phase HPLC, peptides were further fractionated via nano-LC. This additional separation step provided a substantial increase in the number of detectable candidate species within the complex peptide pools. MALDI MS/MS analysis on nano-LC-separated material was then sufficiently sensitive to rapidly identify more than 30 novel HLA-presented peptide ligands. Peptide sequences contained perfect anchor amino acid residues described previously for HLA-A2, HLA-A3, and HLA-B7. The most promising candidate for a T-cell epitope is an HLA-B7-binding nonamer peptide derived from the tumor-associated gene NY-BR-16. To demonstrate the sensitivity of our approach we characterized peptides binding to HLA-C molecules that are usually expressed at the cell surface at approximately only 10% the levels of HLA-A or HLA-B. In fact, multiple renal cell carcinoma peptides were identified that contained anchor amino acid residues of HLA-Cw5 and HLA-Cw7. We conclude that the nano-LC MALDI MS/MS approach is a sensitive tool for the rapid and automated identification of MHC-associated tumor peptides.

Amino Acid Sequence↗