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PubMed1986

Separation and quantification of choline and acetylcholine by thermospray liquid chromatography/mass spectrometry.

The development of sensitive analytical techniques for use in the identification and qualification of molecules of biological origin remains a constant challenge. Often the compounds of interest are present in a complex mixture and require extensive sample preparation prior to analysis. Thermospray liquid chromatography/mass spectrometry (LC/MS) has been shown to be a method which allows for both separation of target molecules from complex mixtures and sensitive specific detection using a mass spectrometer. In this work thermospray LC/MS is used for the direct detection of intact, underivatized choline and acetylcholine from mouse brain homogenate. The results of our analysis corroborate previous analyses using a variety of indirect measurement techniques and thereby show that this simple direct analysis has wide potential applicability.

Acetylcholine↗
PubMed1988

Surface respreading after collapse of monolayers containing major lipids of pulmonary surfactant.

Isotherms have been obtained near 37 degrees C for a series of repetitive compressions and expansions of monolayers that contain major components of lung surfactant. The minimum surface tension or maximum surface pressure which could be achieved under conditions of dynamic compression, and the rate of return of lipid from excluded phase to the monolayers were measured. Monolayers of pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or of DPPC plus 10 or 30 mol% of the calcium salt of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG) (POPG-Ca) achieved very high surface pressures or low surface tensions (near 0 mN m-1), but they showed no return of material from the collapse phases under the test conditions. Monolayers of POPG-Ca alone collapsed at relatively low surface pressures (high surface tensions), but showed good return of material from the collapse phase into the monolayer. Monolayers containing more complex mixtures of lipids (DPPC, phosphatidylglycerol (PG), unsaturated phosphatidylcholine (PC), cholesterol (chol] in ratios similar to those found in surfactant achieved minimum surface tensions intermediate between those of monolayers with less complex compositions. These more complex mixtures showed a better rate of return of lipids from the collapse phases to the monolayer than did simple DPPC-POPG mixtures. 31P-NMR and differential scanning calorimetric investigations of the mixture DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POP G/DPPG/chol (10:4:2:1:3) showed that in the bulk phase at 37 degrees C, it was in bilayers in the liquid-crystalline state.

Lipids↗
PubMed1984

Estimating glycoprotein carbohydrate chain structures by lectin reactivities in polyacrylamide gels.

Complex mixtures of cellular glycoproteins contain a myriad of different carbohydrate chains that cannot be easily analyzed without rigorous purification of each individual glycoprotein. We have analyzed the carbohydrate chains in complex mixtures of cellular glycoproteins by separation using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and interacting the gels with several 125I-labeled lectins. By use of in situ chemical modifications of the glycoproteins after their electrophoretic separation together with the known carbohydrate-binding specifities of several lectins, it has been possible to estimate glycoprotein carbohydrate chain structures. As an example we have examined the cellular glycoproteins of a ovary-colonizing metastatic variant of B16 melanoma and report the types of carbohydrate chains that are found on various melanoma glycoproteins.

Animals↗
PubMed1999

Identification of proteins in complexes by solid-phase microextraction/multistep elution/capillary electrophoresis/tandem mass spectrometry.

A method to directly identify proteins in complex mixtures by solid-phase microextraction (micro-SPE)/multistep elution/capillary electrophoresis (CE)/tandem mass spectrometry (MS/MS) is described. A sheathless liquid-metal junction interface is used to interface CE and electrospray ionization MS/MS. A subfemtomole detection limit is achieved for protein identification through database searching using MS/MS data. The SPE serves as a semiseparation dimension using an organic-phase step-elution gradient in combination with the second separation dimension for increased resolving power of complex peptide mixtures. This approach improves the concentration detection limit for CE and allows more proteins in complex mixtures to be identified. A 75-protein complex from yeast ribosome is analyzed using this method and 80-90% of the proteins in the complex can be identified by searching the database using the MS/MS data from a complete analysis. This multidimensional CE/MS/MS methodology provides an alternative to multidimensional liquid chromatography/MS/MS for direct identification of small amounts of protein in mixtures.

Electrophoresis, Capillary↗
PubMed2005

GCxMS of diesel: a two-dimensional separation approach.

Comprehensive two-dimensional gas chromatography can be viewed as a traditional gas chromatography with a sophisticated "elution-resolved" flame ionization detector (GC/FID) or a detector with separation capability. The concept of multidimensional chromatography can be extended to other detectors that also have separation capability, such as ultraviolet, infrared, and mass spectrometer. Mass spectrometry, combined with gas chromatography, GC/MS, has been a powerful separation/identification device for many years. However, if one applies the multidimensional separation concept to this combination with a nonfragmentation ionization method, GC/MS not only can be used as a separation/identification tool, but also a two (multiple) dimensional separation device, GCxMS. In this study, a two-dimensional separation (GCxMS) study of diesel composition is demonstrated and compared with the GCxGC technique. The major advantage of GCxMS is the compound class separation. The compound groups within a compound class are also well-separated on the basis of their parent masses. Because of the exact mass operation, the specific element containing compound distribution can also be generated through the extraction of specific mass groups. For qualitative analysis, GCxMS is a technique where one experiment can generate a wide range of information. GCxMS may also perform quantitative analysis when appropriate response factors for various compound groups are available. From GCxGC to GCxMS, the power of two (multiple) dimensional separation has just started exposing its advantages for complex mixture analysis. To achieve multiple dimensional separation in different forms, many improvements remain to be made. The challenge now is to combine/accommodate two or more different techniques to solve a specific complex separation problem. The GCxMS experience has pushed this effort one step ahead toward complete application of this new concept in the analysis of complex mixtures.

Journal Article↗
PubMed1985

Combination of three techniques for the study of complex enzyme mixtures: polyacrylamide gel electrophoresis, immunodiffusion and detection of enzymatic activity.

A technique is presented in which two different separation methods are combined. The first separation is carried out in polyacrylamide gel electrophoresis, which is then followed by immunodiffusion in agarose against appropriate antisera. Soluble and insoluble macromolecular carbohydrates were used as substrates in the detection of the various enzymatic activities. These three methods in combination can provide new information on enzymatically active proteins in mixtures, such as isoenzymes, and multiple forms varying in molecular size.

Electrophoresis, Polyacrylamide Gel↗
PubMed1996

Characterization of combinatorial peptide libraries by electrospray ionization Fourier transform mass spectrometry.

The advantages of Fourier transform mass spectrometry (FTMS) are precision high mass accuracy measurements and the capability of high resolution, multistage mass spectrometry together with a number of other advanced features. These powerful facilities can be used to rapidly screen complex mixtures without the necessity of chromatographic separations. The example shown here illustrates the use of the high resolving power and accurate mass capabilities of FTMS for the rapid, direct analysis of a complex mixture, which had been ionized by direct infusion electrospray ionization.

Electrochemistry↗
PubMed1991

Evaluation of displacement chromatography for the recovery of lactate dehydrogenase from beef heart under scale-up conditions.

A complex mixture of proteins was subjected to displacement development from a Tris Acryl DEAE anion-exchange column. Lactate dehydrogenase was used as the target protein in the evaluation of the resolution and separation under scale-up conditions. The conditions of operation were scaled up in terms of column size and protein load. Column length was found to play an important role in resolving the mixture. The performance of the displacement mode run was compared with a conventional ion-exchange elution run in terms of fraction purity and specific activities. In general, displacement chromatography of the complex mixture yielded better results.

Animals↗
PubMed2002

Synthesis, coordination to Rh(I), and hydroformylation catalysis of new beta-aminophosphines bearing a dangling nitrogen group: an unusual inversion of a Rh-coordinated P center.

Variants of the beta-aminophosphine L(1) [Ph(2)PCH(2)CH(Ph)NHPh] containing additional nitrogen donor functions have been prepared. These functions are branched off the C atom adjacent to the P atom, or the P atom itself. Ligand [Ph(2)PCH(o-C(6)H(4)NMe(2))CH(Ph)NHPh] has been obtained as a mixture of two diastereomers L(3A) and L(3B) by lithiation of L(2) [Ph(2)PCH(2)(o-C(6)H(4)NMe(2))] with n-BuLi followed by PhCH=NPh addition and hydrolysis. The diastereomers have been separated by fractional crystallization from ethanol. Ligand Et(2)NCH(2)P(Ph)CH(2)CH(Ph)NHPh has been obtained as a mixture of two diastereomers L(5A) and L(5B)(starting with P-Ph reductive cleavage of L(1) by lithium and subsequent hydrolysis to give PhP(H)CH(2)CH(Ph)NHPh (mixture of two diastereomers L(4A) and L(4B)). The latter reacts with diethylamine and formaldehyde to afford the L(5) diastereomeric mixture. Complexes RhCl(CO)(L) (L = L(3A), 1(A); L(3B), 1(B); L(5A/B), 2(A/B)) were obtained by reaction of [RhCl(CO)(2)](2) and the appropriate ligand or ligand mixture. Complexes 1(A), 1(B), and 2(A) have been isolated in pure form and characterized by classical techniques and by single-crystal X-ray diffraction. All structures exhibit a bidentate kappa-P,kappa-N(NHPh) mode similar to the complex containing L(1). While complexes 1(A) or 1(B) are stable in CDCl(3) solution, complex 2(A) slowly converts to its diastereomer 2(B). This unexpected epimerization appears to take place by inversion at the Rh-coordinated P center, an apparently unprecedented phenomenon. A mechanism based on a reversible P-C bond oxidative addition is proposed. The influence of the pendant nitrogen function of the diaminophosphines L(3A) and L(5A/B) on the rhodium catalytic activity in styrene hydroformylation has been examined and compared to that of the aminophosphines L(1) or L(2). The observed trends are related to the basicity of the dangling amine function and to its proximity to the metal center.

Journal Article↗
PubMed2006

The anatomy of the exposures that occurred around the World Trade Center site: 9/11 and beyond.

The attack on the World Trade Center (WTC) resulted in a new era of awareness on terrorism in the United States and the issues surrounding the potential for acute and/or long-term health outcomes caused by personal exposures to toxicants released during a terrorist event or an accident. The aftermath of the collapse yielded a situation usually not encountered in environmental health science: a large population's exposure to a previously uncharacterized complex mixture of airborne gases and particles, and re-suspendable particles (>2.5 microm in diameter). This led to a series of rapidly changing potential and actual exposure categories, both in space and time that were associated with the complex mixture of heterogeneous composition and character; e.g., very large particles mixed with much smaller amounts of fine particles, and gases released by uncontrolled combustion. The four categories of outdoor exposure that were encountered will be discussed over the period from September 11 until the fires ended on December 20, 2001. Further, the complex issue of indoor exposure to deposited dust will be highlighted from the beginning through the residual exposure issues being examined today (Category 5 period). The strength of the information on the initial WTC dust and smoke, and the smoke plumes from the fires and the continuing (permanent) gaps in our knowledge within the exposure sciences will be discussed, as well as our attempt to reconstruct exposure for various segments of the population in southern Manhattan and the surrounding areas. This all will be tied to lessons that must be considered in response to future events, natural or otherwise.

Air Pollutants↗
PubMed2006

Selective isolation of multiple positively charged peptides for 2-DE-free quantitative proteomics.

A method for quantitative proteomic analysis based on the selective isolation of multiply charged peptides (RH peptides) containing arginine and histidine residues is described. Two pools of proteins are digested in tandem with lysyl-endopeptidase and trypsin and the primary amino groups of proteolytic peptides are separately labeled with d3- and d0-acetic anhydride. This reaction has a dual purpose: (i) to allow the relative protein quantification in two different conditions and (ii) to restrict the positive charges of peptides to the presence of arginine and histidine. The N-acylated peptides are separated by cation-exchange chromatography into two groups, neutral and singly charged peptides (R+H 1) are retained into the column and can be eluted in batch or further fractionated using a saline gradient before LC-MS/MS analysis. In silico analysis revealed that the selective isolation of RH peptides considerably simplifies the complex mixture of peptides (three RH peptides/protein) and at the same time they represent 84% of the whole proteomes. The selectivity, and recovery of the method were evaluated with model proteins and with a complex mixture of proteins extracted from Vibrio cholerae.

Acetic Anhydrides↗
PubMed1980

Mutagenicity of airborne particles.

The physical and chemical properties of airborne particles are important for the interpretation of their potential biologic significance as genotoxic hazards. For polydisperse particle size distributions, the smallest, most respirable particles are generally the most mutagenic. Particulate collection for testing purposes should be designed to reduce artifact formation and allow condensation of mutagenic compounds. Other critical factors such as UV irradiation, wind direction, chemical reactivity, humidity, sample storage, and temperature of combustion are important. Application of chemical extraction methods and subsequent class fractionation techniques influence the observed mutagenic activity. Particles from urban air, coal fly ash, automobile and diesel exhaust, agricultural burning and welding fumes contain primarily direct-acting mutagens. Cigarette smoke condensate, smoke from charred meat and protein pyrolysates, kerosene soot and cigarette smoke condensates contain primarily mutagens which require metabolic activation. Fractionation coupled with mutagenicity testing indicates that the most potent mutagens are found in the acidic fractions of urban air, coal fly ash, and automobile diesel exhaust, whereas mutagens in rice straw smoke and cigarette smoke condensate are found primarily in the basic fractions. The interaction of the many chemical compounds in complex mixtures from airborne particles is likely to be important in determining mutagenic or comutagenic potentials. Because the mode of exposure is generally frequent and prolonged, the presence of tumor-promoting agents in complex mixtures may be a major factor in evaluation of the carcinogenic potential of airborne particles.

Air Pollutants↗
PubMed2006

Identification and quantification of protein carbonylation using light and heavy isotope labeled Girard's P reagent.

Protein carbonyls are one of the most widely studied markers of oxidative stress. Determining increases in the concentration of protein carbonyls known to be associated with neurodegenerative diseases, heart disease, cancer and ageing. Identification of carbonylation sites in oxidized proteins has been a challenge. Even though recent advances in proteomics has facilitate the identification of carbonylation sites in oxidized proteins, confident identification remains a challenge due to the complicated nature of oxidative damage and the wide range of oxidative modifications. Here, we report the development of a multiplexing strategy that facilitates confident carbonylated peptide identification through a combination of heavy and light isotope coding and a multi-step filtering process. This procedure involves (1) labeling aliquots of oxidized proteins with heavy and light forms of Girard's reagent P (GPR) and combining them in a 1:1 ratio along with (2) LC/MS and MALDI-MS/MS analysis. The filtering process uses LC/MS and MALDI-MS/MS data to rule out false positives by rejecting peptide doublets that do not appear with the correct concentration ratio, retention time, tag number, or resolution. This strategy was used for the identification of heavily oxidized transferrin peptides and resulted in identification 13 distinct peptides. The competency of the method was validated in a complex mixture using oxidized transferrin in a yeast lysate as well as oxidized yeast. Twenty-five percent of the peptides identified in a pure oxidized sample of transferrin were successfully identified from the complex mixture. Analysis of yeast proteome stressed with hydrogen peroxide using this multiplexing strategy resulted in identification of 41 carbonylated peptides from 36 distinct proteins. Differential isotope coding of model peptides at different concentrations followed by mixing at different ratios was used to establish the linear dynamic range for quantification of carbonylated peptides using light and heavy forms of GPR.

Amino Acid Sequence↗
PubMed2004

Selective detection of proteins in mixtures using electrospray ionization mass spectrometry: influence of instrumental settings and implications for proteomics.

We studied the effects of electrospray mass spectrometric instrumental settings on the relative and absolute detection of individual proteins in a five-component mixture. Conditions that were effective for a given protein could be very poor for the others, and vice versa, such that to a good approximation it was possible to find conditions for selective detection of individual proteins in a complex mixture without prior analytical separation. Some of these could be rationalized on the basis of the known biophysical properties of the individual proteins. The ability to vary the conditions of a mass spectrometric detection method on-line provides an important degree of freedom for the selective detection, and hence discrimination, of individual proteins and peptides in complex mixtures and has implications in proteomics, in particular with respect to top-down strategies for proteomic characterizations.

Animals↗
PubMed2005

Tagged polymerase chain reaction subtractive hybridization for the enrichment of phage display random peptide libraries.

Affinity selection of phage display peptide libraries is routinely used for isolating peptides capable of binding a range of molecules, including antibodies and receptors. This process is most successful when the selecting molecule is relatively pure, for example, a monoclonal antibody. However, isolation of peptides able to bind to target molecules present in a complex mixture is more difficult because the affinity selection process isolates peptides capable of binding to all molecules present in the mixture. Here we describe the development of a tagged polymerase chain reaction (PCR) subtractive hybridization method that is universally applicable for the targeted isolation of peptides able to bind to unique molecules within a complex mixture. We also describe a discriminatory limiting dilution PCR method that can be used to optimize hybridization conditions.

Bacteriophages↗
PubMed1988

Menaquinone composition in the classification and identification of aerobic actinomycetes.

Menaquinones were the only isoprenoid quinones found in 36 strains representing different species of the genera Nocardia, Mycobacterium, Rhodococcus, Amycolatopsis, Saccharothrix, Streptomyces, Nocardiopsis and Actinomadura. Dihydrogenated menaquinones with nine isoprene units [MK-9(H2)] were the main components isolated from Mycobacterium. Dihydrogenated and tetrahydrogenated menaquinones with eight isoprene units were the predominant compounds identified in typical Rhodococcus and Nocardia strains, respectively. "Nocardia phenotolerans" differed from all of the other Nocardia species included in the study, in that it contained the MK-9(H2) [MK-8(H2)] menaquinone system. Nocardioform bacteria lacking mycolic acids contained tetrahydrogenated menaquinones with nine isoprene units as the main component. The Streptomyces strains studied exhibited complex mixtures of partially saturated menaquinones with nine isoprene units with the hexa- and/or octahydrogenated components predominating. Actinomadurae contained major amounts of hexahydrogenated menaquinones with nine isoprene units. In contrast, the single Nocardiopsis strain examined possessed complex mixtures of menaquinones with ten isoprene units, the dihydrogenated components being main constituents.

Actinomycetales↗
PubMed1994

Two-dimensional gel electrophoresis/liquid chromatography for the micropreparative isolation of proteins.

Although comprehensive, column-based two-dimensional separation techniques offer enormous resolving power for a complex mixture, they often lack the ability to isolate the separated species for further analysis (e.g., proteins for Edman sequencing). This paper describes a micropreparative two-dimensional separation system for the isolation of proteins from complex mixtures, such as cell lysates. The system is composed of commercially available equipment: continuous-elution tube gel electrophoresis as the first dimension followed by gradient elution, reversed-phase perfusion liquid chromatography as the second dimension with a two-loop sampling valve as the interface between dimensions. The two-dimensional electrophoresis/liquid chromatography system (2D-EP/LC) shows high resolution of proteins since each dimension has orthogonal separation mechanisms (electrophoresis, size/charge; LC, hydrophobicity). Identification of proteins for further analysis is accomplished by superimposing a grid on the computer-generated 3D image.

Animals↗
PubMed2004

Polybrominated diphenyl ether congeners and toxaphene in selected marine standard reference materials.

Polybrominated diphenyl ether (PBDE) congeners and components of the complex mixture toxaphene are stable in the environment and readily bioaccumulated into wildlife and human tissues. PBDEs are presently used in large quantities worldwide as flame retardants in textiles, furniture, computer equipment, and cables. Toxaphene is a complex mixture of chlorinated bornanes and bornenes that was the most heavily used pesticide in the United States until it was banned in 1982; however, some countries continue to use toxaphene. The National Institute of Standards and Technology has quantified PBDE congeners and toxaphene in several available Standard Reference Materials (SRMs) using methods of gas chromatography with electron impact mass spectrometry (GC-EI-MS) and GC negative chemical ionization (NCI) MS, respectively. SRM 1588a Organics in Cod Liver Oil and SRM 1945 Organics in Whale Blubber were examined for PBDE congeners 47, 99, 100, 153, and 154, total toxaphene, and toxaphene congeners 26, 50, and 62. SRM 1946 Lake Superior Fish Tissue was also examined for total toxaphene and toxaphene congeners. The sum of the PBDE congeners (mean, (1 SD) wet basis) for SRM 1945 was 150 ng g(-1) (7 ng g(-1)). The concentration of PBDE 47 in SRM 1588a was 82.7 ng g(-1) (2.8 ng g(-1)). Other PBDEs were detected in SRM 1588a but were not quantified due their low levels. The total toxaphene (wet mass basis) was 1,210 ng g(-1) (127 ng g(-1)), 1,960 ng g(-1) (133 ng g(-1)), and 3,980 ng g(-1) (248 ng g(-1)) in SRMs 1945, 1946, and 1588a, respectively. The values for PBDEs and toxaphene determined in the SRMs, while not certified, indicate that the SRMs will be suitable control materials for PBDE and toxaphene analyses.

Adipose Tissue↗