Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Complex Mixtures”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,315 records · Page 73Linked to original sources
PubMed2001

Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: a non two-dimensional gel electrophoresis-based proteome analysis strategy.

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3-10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).

Chromatography, High Pressure Liquid↗
PubMed1991

Study on the mutagenic activity of 13 bioflavonoids with the Salmonella Ara test.

The mutagenicity of 13 flavonoids has been investigated with the L-arabinose forward mutation assay of Salmonella typhimurium. Each flavonoid was tested by both plate incorporation and preincubation mutagenesis protocols in the presence or the absence of mammalian metabolic activation (S9 mixture). All flavonoids gave a dose-response relationship and induced a number of AraR mutants considered statistically significant. Their minimum mutagenic doses (MMD) differed markedly in the Ara test, covering a 400-fold range: from 4 nmol for quercetin to 1626 nmol for taxifolin. Flavonols were the strongest mutagens, with mutagenic potencies (MMD-1) representing from 27 to approximately 2% that of quercetin. Comparatively, the mutagenicities of other flavonoids represented only less than or equal to 1%. The data reported in this paper for the Ara forward mutation test suggest structural requirements for mutagenicity of bioflavonoids like those previously reported for the His reverse mutation assay: (i) flavonols with a free hydroxyl at position 3 are the strongest mutagenic flavonoids, (ii) saturation of the 2,3 double bond diminishes the mutagenic potency, and (iii) free hydroxyl groups at positions 3' and 4' influence the non-requirement for metabolic activation. The mutagenic properties of quercetin and rutin in the Ara test support the idea that these flavonols are not the major putative mutagens in complex mixtures such as wine.

Arabinose↗
PubMed2000

Chromosome aberrations in pesticide-exposed greenhouse workers.

OBJECTIVES: The aim of this study was to investigate the possibility of subtoxic exposure to pesticides causing chromosome aberrations in greenhouse workers. METHODS: In a cross-sectional and prospective study design chromosome aberration frequencies in cultured lymphocytes were examined for 116 greenhouse workers exposed to a complex mixture of almost 50 insecticides, fungicides, and growth regulators and also for 29 nonsmoking, nonpesticide-exposed referents. RESULTS: The preseason frequencies of chromosome aberrations were slightly but not statistically significantly elevated for the greenhouse workers when they were compared with the referents. After a summer season of pesticide spraying in the greenhouses, the total frequencies of cells with chromosome aberrations were significantly higher than in the preseason samples (P=0.02) and also higher than for the referents (P=0.05). This finding was especially due to an increased number of cells with chromatid gaps between the first and second samples (P=0.001). The results may reflect an additive genotoxic effect of the spraying season, for which the use of insecticides and growth regulators (but not fungicides) culminates. The highest elevation in the risk of chromatid gaps was observed for persons who did not use gloves during re-entry activities such as nipping, cutting, pricking, and potting (risk ratio 2.88, 95% confidence interval 1.63-5.11). CONCLUSIONS: The present results suggest a genotoxic effect from a complex subtoxic occupational pesticide exposure. In general, the findings indicate the importance of personal protection, during high-exposure re-entry activities, in preventing pesticide uptake and genetic damage.

Adult↗
PubMed2000

The diversity of extradiol dioxygenase (edo) genes in cresol degrading rhodococci from a creosote-contaminated site that express a wide range of degradative abilities.

Analysis of the bacterial population of soil surface samples from a creosote-contaminated site showed that up to 50% of the culturable micro-organisms detected were able to utilise a mixture of cresols. From fifty different microbial isolates fourteen that could utilise more than one cresol isomer were selected and identified by 16S rRNA analysis. Eight isolates were Rhodococcus strains and six were Pseudomonas strains. In general, the Rhodococcus strains exhibited a broader growth substrate range than the Pseudomonas strains. The distribution of various extradiol dioxygenase (edo) genes, previously associated with aromatic compound degradation in rhodococci, was determined for the Rhodococcus strains by PCR detection and Southern-blot hybridization. One strain, Rhodococcus sp. II exhibited the broadest growth substrate range and possessed five different edo genes. Gene disruption experiments indicated that two genes (edoC and edoD) were associated with isopropylbenzene and naphthalene catabolism respectively. The other Rhodococcus strains also possessed some of the edo genes and one (edoB) was present in all of the Rhodococcus strains analysed. None of the rhodococcal edo genes analysed were present in the Pseudomonas strains isolated from the site. It was concluded that individual strains of Rhodococcus possess a wide degradative ability and may be very important in the degradation of complex mixtures of substrates found in creosote.

Base Sequence↗
PubMed2005

Snake C-type lectin-like proteins and platelet receptors.

Snake venoms are complex mixtures of biologically active proteins and peptides. Many affect haemostasis by activating or inhibiting coagulant factors or platelets, or by disrupting endothelium. Snake venom components are classified into various families, such as serine proteases, metalloproteinases, C-type lectin-like proteins, disintegrins and phospholipases. Snake venom C-type lectin-like proteins have a typical fold resembling that in classic C-type lectins such as the selectins and mannose-binding proteins. Many snake venom C-type lectin-like proteins have now been characterized, as heterodimeric structures with alpha and beta subunits that often form large molecules by multimerization. They activate platelets by binding to VWF or specific receptors such as GPIb, alpha2beta1 and GPVI. Simple heterodimeric GPIb-binding molecules mainly inhibit platelet functions, whereas multimeric ones activate platelets. A series of tetrameric snake venom C-type lectin-like proteins activates platelets by binding to GPVI while another series affects platelet function via integrin alpha2beta1. Some act by inducing VWF to bind to GPIb. Many structures of these proteins, often complexed with their ligands, have been determined. Structure-activity studies show that these proteins are quite complex despite similar backbone folding. Snake C-type lectin-like proteins often interact with more than one platelet receptor and have complex mechanisms of action.

Animals↗
PubMed1983

Mouse skin tumor initiation-promotion and complete carcinogenesis bioassays: mechanisms and biological activities of emission samples.

Extracts of soots obtained from various sources were applied to the skin of mice in an effort to identify carcinogens in these mixtures and to link these materials to the etiology of human cancer. Samples of coal chimney soot, coke oven materials, industrial carbon black, oil shale soot, and gasoline vehicle exhaust materials have been examined by this method. The studies reported here have been constructed to compare the carcinogenic and tumorigenic potency of extracts from various particulate emissions: coke ovens, diesel and gasoline vehicles and a roofing tar pot. Automobile emission samples were obtained by collecting the diluted and cooled exhaust on Teflon-coated glass fiber filters. Coke oven and roofing tar samples were particulate emission samples collected by impaction and filtration. The organic components associated with each of the particles were extracted with dichloromethane and dermally applied to SENCAR mice. All agents were applied as tumor initiators by using a five-dose protocol. Selected extracts were also applied as complete carcinogens and as tumor promotors. Statistical analyses of the resulting tumor data were performed by using nonlinear Poisson and probit models. The results from these experiments provide a suitable data base for comparative potency estimation of complex mixtures.

Air Pollutants↗
PubMed2006

Relations between PM10 composition and cell toxicity: a multivariate and graphical approach.

Previous studies have used particle mass and size as metrics to link airborne particles with deleterious health effects. Recent evidence suggests that particle composition can play an important role in PM-toxicity; however, little is known about the specific participation of components (individually or acting in groups) present in such a complex mixture that accounts for toxicity. This work explores relationships among PM(10) components in order to identify their covariant structure and how they vary in three sites in Mexico City. Relationships between PM(10) with cell toxicity and geographical location were also explored. PM(10) was analyzed for elemental composition, organic and elemental carbon, endotoxins and the induction of inhibition of cell proliferation, IL-6, TNFalpha and p53. PM(10) variables were evaluated with principal component analysis and one-way ANOVA. The inhibition of cell proliferation, IL-6 and TNFalpha were evaluated with factorial ANOVA and p53 with the Welch test. The results indicate that there is heterogeneity in particle mass, composition and toxicity in samples collected at different sites. Multivariate analysis identified three major groups: (1) S/K/Ca/Ti/Mn/Fe/Zn/Pb; (2) Cl/Cr/Ni/Cu; and (3) endotoxins, organic and elemental carbon. Groups 1 and 3 showed significant differences among sites. Factorial ANOVA modeling indicated that cell proliferation was affected by PM concentration; TNFalpha and IL-6 by the interaction of concentration and site, and p53 was different by site. Radial plots suggest the existence of complex interactions between components, resulting in characteristic patterns of toxicity by site. We conclude that interactions of PM(10) components determine specific cellular outcomes.

Air Pollutants↗
PubMed2004

Co-populated conformational ensembles of beta2-microglobulin uncovered quantitatively by electrospray ionization mass spectrometry.

Ordered assembly of monomeric human beta(2)-microglobulin (beta(2)m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis. Previously, we have shown that under acidic conditions (pH <5.0 at 37 degrees C), wild-type beta(2)m assembles spontaneously into fibrils with different morphologies. Under these conditions, beta(2)m populates a number of different conformational states in vitro. However, this equilibrium mixture of conformationally different species is difficult to resolve using ensemble techniques such as nuclear magnetic resonance or circular dichroism. Here we use electrospray ionization mass spectrometry to resolve different species of beta(2)m populated between pH 6.0 and 2.0. We show that by linear deconvolution of the charge state distributions, the extent to which each conformational ensemble is populated throughout the pH range can be determined and quantified. Thus, at pH 3.6, conditions under which short fibrils are produced, the conformational ensemble is dominated by a charge state distribution centered on the 9+ ions. By contrast, under more acidic conditions (pH 2.6), where long straight fibrils are formed, the charge state distribution is dominated by the 10+ and 11+ ions. The data are reinforced by investigations on two variants of beta(2)m (V9A and F30A) that have reduced stability to pH denaturation and show changes in the pH dependence of the charge state distribution that correlate with the decrease in stability measured by tryptophan fluorescence. The data highlight the potential of electrospray ionization mass spectrometry to resolve and quantify complex mixtures of different conformational species, one or more of which may be important in the formation of amyloid.

Amino Acid Substitution↗
PubMed1995

Structural analysis of the Bacillus subtilis delta factor: a protein polyanion which displaces RNA from RNA polymerase.

RNA polymerase from Bacillus subtilis is a complex mixture comprising a common core (beta beta' alpha 2), the 20.4 kDa delta (delta) protein, and of one of several sigma (sigma) specificity factors. The delta protein, together with several truncated variants, has been overproduced and purified from Escherichia coli. It is highly acidic (pI = 3.6) and contains two distinct regions, a 13 kDa amino-terminal domain with fairly uniform charge distribution and a glutamate and aspartate residue-rich carboxyl-terminal region. The purified amino-terminal domain (delta N) contains 32% alpha-helix and 16% beta-sheet, as judged by circular dichroism analysis. In contrast, an 8.5 kDa tryptic fragment containing the carboxyl-terminal region (delta C) is largely unstructured and highly charged (net charge of -47). RNA polymerase purified from a B. subtilis mutant with an insertion in the delta gene (rpoE::cat) contains a truncated delta protein, indicating that the amino-terminal domain is stable in vivo and contains a core-binding function. Addition of delta, but not sigma A or delta N, displaces RNA bound to RNA polymerase in a binary complex. The ability of delta to displace RNA efficiently requires the activities of both the amino-terminal core-binding domain and the polyanionic carboxyl-terminal region. Although delta C can also displace nucleic acids from RNA polymerase, this activity requires the addition of a large molar excess of protein and is relatively non specific in that both DNA and RNA are displaced. This suggests that the function of the amino-terminal domain is to bind and orient the carboxyl-terminal region on the surface of RNA polymerase.

Amino Acid Sequence↗
PubMed2006

Development and application of in vivo expression technology (IVET) for analysing microbial gene expression in complex environments.

Establishing the mechanisms by which microbes interact with their environment, including eukaryotic hosts, is a major challenge that is essential for the economic utilisation of microbes and their products. Techniques for determining global gene expression profiles of microbes, such as microarray analyses, are often hampered by methodological restraints, particularly the recovery of bacterial transcripts (RNA) from complex mixtures and rapid degradation of RNA. A pioneering technology that avoids this problem is In Vivo Expression Technology (IVET). IVET is a 'promoter-trapping' methodology that can be used to capture nearly all bacterial promoters (genes) upregulated during a microbe-environment interaction. IVET is especially useful because there is virtually no limit to the type of environment used (examples to date include soil, oomycete, a host plant or animal) to select for active microbial promoters. Furthermore, IVET provides a powerful method to identify genes that are often overlooked during genomic annotation, and has proven to be a flexible technology that can provide even more information than identification of gene expression profiles. A derivative of IVET, termed resolvase-IVET (RIVET), can be used to provide spatio-temporal information about environment-specific gene expression. More recently, niche-specific genes captured during an IVET screen have been exploited to identify the regulatory mechanisms controlling their expression. Overall, IVET and its various spin-offs have proven to be a valuable and robust set of tools for analysing microbial gene expression in complex environments and providing new targets for biotechnological development.

Anti-Bacterial Agents↗
PubMed2003

Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional electrophoresis and isotope-coded affinity tag technology.

Classical proteomics combined two-dimensional gel electrophoresis (2-DE) for the separation and quantification of proteins in a complex mixture with mass spectrometric identification of selected proteins. More recently, the combination of liquid chromatography (LC), stable isotope tagging, and tandem mass spectrometry (MS/MS) has emerged as an alternative quantitative proteomics technology. We have analyzed the proteome of Mycobacterium tuberculosis, a major human pathogen comprising about 4,000 genes, by (i) 2-DE and mass spectrometry (MS) and by (ii) the isotope-coded affinity tag (ICAT) reagent method and MS/MS. The data obtained by either technology were compared with respect to their selectivity for certain protein types and classes and with respect to the accuracy of quantification. Initial datasets of 60,000 peptide MS/MS spectra and 1,800 spots for the ICAT-LC/MS and 2-DE/MS methods, respectively, were reduced to 280 and 108 conclusively identified and quantified proteins, respectively. ICAT-LC/MS showed a clear bias for high M(r) proteins and was complemented by the 2-DE/MS method, which showed a preference for low M(r) proteins and also identified cysteine-free proteins that were transparent to the ICAT-LC/MS method. Relative quantification between two strains of the M. tuberculosis complex also revealed that the two technologies provide complementary quantitative information; whereas the ICAT-LC/MS method quantifies the sum of the protein species of one gene product, the 2-DE/MS method quantifies at the level of resolved protein species, including post-translationally modified and processed polypeptides. Our data indicate that different proteomic technologies applied to the same sample provide complementary types of information that contribute to a more complete understanding of the biological system studied.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

Comparison of indirect and direct approaches using ion-trap and Fourier transform ion cyclotron resonance mass spectrometry for exploring viperid venom proteomes.

In a sense, the field of snake venom proteomics has been under investigation since the very earliest biochemical studies where it was soon recognized that venoms are comprised of complex mixtures of bioactive molecules, most of which are proteins. Only with the re-emergence of 2D polyacrylamide gel electrophoresis (2D PAGE) and the recent developments in mass spectrometry for the identification/characterization of proteins coupled with venom gland transcriptomes has the field of snake venom proteomics began to flourish and provide exciting insights into the protein composition of venoms and subsequently their pathological activities. In this manuscript we will briefly discuss the state of snake venom proteomics followed by the presentation of several straightforward experiments designed to explore approaches to investigating venom proteomics. The first set of experiments used 1D gel electrophoresis (1D PAGE) of Crotalus atrox venom followed by slice-by-slice analysis of the proteins using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). In the second set of experiments, C. atrox and Bothrops jararaca venoms were subjected to in-solution digestion followed by Fourier transform ion cyclotron resonance (FTICR) LC/MS/MS. The peptide ion-maps of these venoms were compared along with the proteins identified. In addition, the results were compared to the results observed from the 1D PAGE approach. From these studies it is clear that sample de-complexation/fractionation before mass spectrometry is still the best approach for maximum proteome coverage. Furthermore, comparison of venom proteomes based on tryptic peptide identities between the proteomes is not particularly effective since there does not appear to be a sufficient number of such identical peptides, derived from related proteins, present in venoms. Finally, as has previously been recognized without either better databases of venom protein sequences or facile and rapid de novo sequencing technologies for mass spectrometry, snake venom proteome investigation will remain a laborious task.

Animals↗
PubMed1992

[Biochemical and clinical aspects of pulmonary surfactant proteins].

Pulmonary surfactant is a complex mixture of phospholipids and proteins which is synthesized and secreted by alveolar type II cells. Its presence is essential to prevent the collapse of alveoli at the end of expiration. Recently, it has been demonstrated that in addition to its reduction of surface tension of alveolar surfaces, pulmonary surfactant exhibits several other functions in the alveolar lining layer, and surfactant proteins are definitely involved in the expression of these functions. The present study first focused on the recent advances in basic research of hydrophilic surfactant apoproteins, SP-A and SP-D. Both are glycoproteins with C-type lectin structure at the C-terminal region and collagenous structure at the N-terminal half of the proteins. We revealed that SP-A binds specifically to dipalmitoylphosphatidylcholine and galactose-ceramide and asialo GM2, while SP-D binds specifically to phosphatidylinositol and glucose-ceramide. We discuss the physiologic and metabolic roles of the specific lipid binding with surfactant proteins of the surfactant system. We next studied changes in pulmonary surfactant in respiratory diseases using anti-human SP-A monoclonal antibodies. We demonstrated SP-A immunoglobulin complex in the sera of patients with idiopathic pulmonary fibrosis and pulmonary alveolar proteinosis.

Humans↗
PubMedDate not supplied

Residue organic mixtures from drinking water show in vitro mutagenic and transforming activity.

Indications of possible health effects of residue organics in drinking water have been sought using short-term tests of mutagenic and transforming activity. Ten percent or less of the total organic material in drinking water has been identified; the remainder is believed to include thousands of unknown nonvolatile compounds. Residual organics were concentrated from drinking water from representative U.S. cities by reverse osmosis followed by liquid-liquid extraction [yielding the reverse osmosis concentrate-organic extract (ROC-OE) fraction] and sorption-desorption on XAD-2 resin. Samples of these residue organics were provided by the Environmental Protection Agency for bioassay. They were examined for mutagenic activity by using Salmonella tester strains (primarily TA98 and TA100) and for transforming activity by using mouse fibroblasts (BALB/3T3 clone 1-13). City-specific patterns of dose-dependent bacterial mutagenesis and of bacterial toxicity were observed for these samples and for subfractions generated by sequential extractions with hexane, ethyl ether, and acetone. Mutagenic effects were essentially independent of a microsome activation system prepared from liver of Aroclor 1254-induced rats. On the basis of strain-specific effects in mutagenesis and differential distributions of mutagenic activity during liquid-liquid extraction, at least some of the active compounds are thought to be acidic, frameshift mutagens. The ROC-OE fraction of a New Orleans sample transformed BALB/3T3 cells in replicate experiments. By comparison with the bacterial mutagenesis data, cell transformation is a relatively sensitive method for detecting possible mutagenic and carcinogenic activity in this sample. The appropriateness of these systems for the assay of complex mixtures and the degree to which reverse osmosis concentrates contain the unaltered organic compounds in the original samples are discussed.

Animals↗
PubMed2003

Biofiltration of a mixture of volatile organic compounds on granular activated carbon.

The performance of a biofilter packed with Active Carbon (AC) was evaluated. The effluent (alcohol, ketones, esters, aromatic and chlorinated compounds) treated was a representative mixture of most common industrial emissions. To achieve a better knowledge of multicomponent adsorption mechanisms, and to underline the interest of inoculating AC, a control abiotic humidified filter had been operated in the same conditions as the biofilter. For a load of 110 g VOC m(-3) AC h(-1), after 55 days of operation, the removal efficiency was higher in the biotic than in the abiotic filter (85% vs 55%, respectively). Moreover, in the biofilter, at steady state, the elimination of all compounds was almost complete except for chlorinated compounds and p-xylene (removal efficiency of 25% and 64%, respectively). The microbial colonization of AC involved a decrease of the adsorption sites accessibility and enhanced the treatment of VOCs (volatile organic compounds) having a lower affinity for activated carbon. Moreover, while aromatic compounds and MIBK were eliminated along the overall height of the biofilter, pollutants with reduced affinity for AC, such as methanol, acetone, and halogenated compounds were only treated on the second half of the reactor. Thus, the affinity for activated carbon was an important parameter controlling the biodegradation process. Nevertheless, the use of AC as packing material in biofilters treating complex mixtures of VOCs is limited. Actually, similar removal efficiency could be reached, in the same conditions, for a biofilter packed with granular peat. Furthermore, for the biofilter packed with AC, the column height necessary to remove biodegradable compounds, with reduced affinity for the support, was important.

Adsorption↗
PubMed2006

Characterization of porphyrins, chlorins, and bacteriochlorins formed via allomerization of bacteriochlorophyll a. Synthesis of highly stable bacteriopurpurinimides and their metal complexes.

Allomerization of bacteriochlorophyll a (Bchl a) was studied under various reaction conditions. Bchl a on stirring with KOH/propanol produced an "unstable bacteriochlorin", which decomposed in acidic conditions to give a complex mixture containing bacteriopurpurin a as a principal component. The yields of other compounds varied and were found to be dependent on reaction condition. The structures of the isolated porphyrins, chlorins, and bacteriochlorins, related to Bchl a, were assigned on the basis of 1D, 2D NMR (ROESY), and mass spectroscopy analyses. The presence of fused anhydride rings in porphyrin, chlorin, and bacterichlorin systems showed a significant influence on their optical properties. Compared to bacteriochlorophyll a and bacteriopheophytin, the related structurally modified analogues, e.g., the bacteriopurpurin a, 13(1)/15(1)-N-alkyl isoimide, and the imide analogues were found to be more stable with a significant difference in spectroscopic properties. Bacteriochlorins containing anhydride, imide, or isoimide cyclic rings demonstrated a significant bathochromic shift of their Q bands in their electronic absorption spectra. Under basic conditions the formation of the 12-hydroxymethyl, 12-formyl, and 12-methylene analogues as byproducts from the 12-methyl-bacteriopurpurin-N-hexylimide could be due to subsequent oxidation of the vinylogous enolate intermediates. To investigate the effect of the central metal in the electronic spectra, the stable bacteriopurpurin-18-N-hexylimide was converted to a series of metal complexes [Zn(II), Cd(II), and Pd(II)] by following the direct or transmetalation approaches. Compared to the free-base analogue, these complexes showed a remarkable shift in their electronic absorption spectra.

Anhydrides↗
PubMed1993

Purification of membrane proteins using a micropreparative gel electrophoresis apparatus: purification of subunits of the integral membrane protein Bacillus subtilis aa3-type quinol oxidase for low level amino acid sequence analysis.

We have developed a continuous elution micropreparative gel electrophoresis system in which small amounts of proteins, e.g., 1-10 micrograms, are continuously eluted from one-dimensional polyacrylamide gel columns in either native or denatured form. Depending on the electrophoretical parameters proteins are separated according to their size and/or net charge. The system is based on a simple modification of the Mini-Protean II 2D unit from Bio-Rad Inc. The apparatus is connected on-line to a high-performance liquid chromatograph allowing accurate delivery of the elution solvent and direct analysis of the separated samples by uv detection. The high resolution of the apparatus allows the purification of individual proteins, even from complex mixtures, in only one step, with overall recoveries of approximately 70%. In addition to standard proteins the system has been applied to the purification of the membrane-bound and thus highly hydrophobic subunits I and II from Bacillus subtilis aa3-type quinol oxidase. The hydrophobic character of these subunits has hindered their purification by other conventional methods. Isolated subunits were collected directly into a buffer suitable for proteolytic digestion and automated amino acid sequence analysis. The internal amino acid sequences determined could all be found in the DNA sequence recently reported by Santana et al. (J. Biol. Chem. 267, 10225-10231, 1992), thereby confirming the expression of such an oxidase.

Amino Acid Sequence↗
PubMed2005

Probing anthocyanin profiles in purple sweet potato cell line (Ipomoea batatas L. Cv. Ayamurasaki) by high-performance liquid chromatography and electrospray ionization tandem mass spectrometry.

A purple line cell line (PL) generated from the storage root of purple-fleshed sweet potato (Ipomoea batatas L.) cv. Ayamurasaki produces a complex mixture of anthocyanins, and seven major anthocyanins have been isolated and identified to date. All these anthocyanins are exclusively cyanidin or peonidin 3-sophoroside-5-glucosides and their acylated derivatives. High-performance liquid chromatography (HPLC) coupled to photodiode array (PDA) detection and electrospray ionization tandem mass spectrometry (ESI-MS/MS) on a triple quadrupole instrument was employed to further investigate the anthocyanin composition of the PL extract. Precursor-ion analysis, product-ion analysis, and selected reaction monitoring (SRM) MS/MS experiments were conducted sequentially to screen and characterize anthocyanins in the aqueous extract of the PL cell line. Precursor-ion analysis specifically detected the molecular cations of each category of anthocyanins by scanning the precursors of anthocyanidins (cyanidin, peonidin, and pelargonidin). The detected molecular cation of each anthocyanin was fragmented using product-ion analysis by collisionally activated dissociation (CAD). MS/MS using SRM detection was conducted to further confirm the fragmentation observed during product-ion analysis. In comparison to the commonly used product-ion analysis technique, the combined use of precursor-ion analysis, product-ion analysis, and SRM is particularly useful for positive identification of anthocyanins in complex matrixes and provides important information to confirm the proposed structures. Twenty-six anthocyanins were detected and characterized in the aqueous extract of the PL cell line. Several anthocyanins, including two pelargonidin derivatives, were tentatively identified for the first time in these cells.

Anthocyanins↗