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1.
J Am Soc Mass Spectrom ; 26(12): 2096-104, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26266643

ABSTRACT

Electron capture dissociation (ECD), which generally preserves the position of labile post-translational modifications, can be a powerful method for de novo sequencing of proteins and peptides. In this report, ECD product-ion mass spectra of singly and doubly sodiated, nonphosphorylated, and phosphorylated peptides are presented and compared with the ECD mass spectra of their protonated counterparts. ECD of doubly charged, singly sodiated peptides yielded essentially the same sequence information as was produced by the corresponding doubly protonated peptides. The presence of several sodium binding sites on the polypeptide backbone, however, resulted in more complicated spectra. This situation is aggravated by the zwitterionic equilibrium of the free acid peptide precursors. The product-ion spectra of doubly and triply charged peptides possessing two sodium ions were further complicated by the existence of isomers created by the differential distribution of sodium binding sites. Triply charged, phosphorylated precursors containing one sodium, wherein the sodium is attached exclusively to the PO4 group, were found to be as useful for sequence analysis as the fully protonated species. Although sodium adducts are generally minimized during sample preparation, it appears that they can nonetheless provide useful sequence information. Additionally, they enable straightforward identification of a peptide's charge state, even on low-resolution instruments. The experiments were carried out using a radio frequency-free electromagnetostatic cell retrofitted into the collision-induced dissociation (CID) section of a hybrid quadrupole/time-of-flight tandem mass spectrometer. Graphical Abstract ᅟ.


Subject(s)
Peptides/chemistry , Sodium/chemistry , Amino Acid Sequence , Electrons , Equipment Design , Mass Spectrometry/instrumentation , Mass Spectrometry/methods , Models, Molecular
2.
J Am Soc Mass Spectrom ; 26(5): 752-61, 2015 May.
Article in English | MEDLINE | ID: mdl-25652934

ABSTRACT

Dissociation of peptides induced by interaction with (free) electrons (electron-induced dissociation, EID) at electron energies ranging from near 0 to >30 eV was carried out using a radio-frequency-free electromagnetostatic (EMS) cell retrofitted into a triple quadrupole mass spectrometer. The product-ion mass spectra exhibited EID originating from electronically excited even-electron precursor ions, reduced radical cations formed by capture of low-energy electrons, and oxidized radical cations produced by interaction with high-energy electrons. The spectra demonstrate, within the limits of the triple quadrupole's resolving power, that high-energy EID product-ion spectra produced with an EMS cell exhibit essentially the same qualitative structural information, i.e., amino acid side-chain (SC) losses and backbone cleavages, as observed in high-energy EID spectra produced with a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer. The levels of fragmentation efficiency evident in the product-ion spectra recorded in this study, as was the case for those recorded in earlier studies with FT ICR mass spectrometers, is currently at the margin of analytical utility. Given that this shortcoming can be remedied, EMS cells incorporated into QqQ or QqTOF mass spectrometers could make tandem high-energy EID mass spectrometry more widely accessible for analysis of peptides, small singly charged molecules, pharmaceuticals, and clinical samples.


Subject(s)
Oligopeptides/chemistry , Peptide Fragments/analysis , Peptides/chemistry , Amino Acid Sequence , Animals , Cyclotrons , Electrochemical Techniques , Electromagnetic Fields , Fourier Analysis , Humans , Mass Spectrometry/instrumentation , Peptide Fragments/chemistry , Protein Stability , Proteolysis , Spectrometry, Mass, Electrospray Ionization/instrumentation , Substance P/chemistry
3.
J Am Soc Mass Spectrom ; 25(10): 1730-8, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25037842

ABSTRACT

A radio frequency-free electromagnetostatic (EMS) cell devised for electron-capture dissociation (ECD) of ions has been retrofitted into the collision-induced dissociation (CID) section of a triple quadrupole mass spectrometer to enable recording of ECD product-ion mass spectra and simultaneous recording of ECD-CID product-ion mass spectra. This modified instrument can be used to produce easily interpretable ECD and ECD-CID product-ion mass spectra of tyrosine-phosphorylated peptides that cover over 50% of their respective amino-acid sequences and readily identify their respective sites of phosphorylation. ECD fragmentation of doubly protonated, tyrosine-phosphorylated peptides, which was difficult to observe with FT-ICR instruments, occurs efficiently in the EMS cell.


Subject(s)
Peptides/chemistry , Tandem Mass Spectrometry/instrumentation , Tandem Mass Spectrometry/methods , Tyrosine/chemistry , Amino Acid Sequence , Electromagnetic Fields , Phosphorylation
4.
J Am Soc Mass Spectrom ; 25(6): 903-4, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24733275
5.
J Am Soc Mass Spectrom ; 22(4): 607-11, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21472599

ABSTRACT

Recently, we demonstrated that a radio-frequency-free electromagnetostatic (rf-free EMS) cell could be retrofitted into a triple quad mass spectrometer to allow electron-capture dissociation (ECD) without the aid of cooling gas or phase-specific electron injection into the cell (Voinov et al., Rapid Commun Mass Spectrom 22, 3087-3088, 2008; Voinov et al., Anal Chem 81, 1238-1243, 2009). Subsequently, we used our rf-free EMS cell in the same instrument platform to demonstrate ECD occurring in the same space and at the same time with collision-induced dissociation (CID) to produce golden pairs and even triplets from peptides (Voinov et al., Rapid Commun Mass Spectrom 23, 3028-3030, 2009). In this report, we demonstrate that ECD and CID product-ion mass spectra can be recorded at high resolution with flexible control of fragmentation processes using a newly designed cell installed in a hybrid Q-TOF tandem mass spectrometer.


Subject(s)
Tandem Mass Spectrometry/methods , Electrons , Oligopeptides/chemistry
6.
Chemosphere ; 82(10): 1380-6, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21194725

ABSTRACT

Twenty-four fluorochemicals were quantified in landfill leachates recovered from municipal refuse using an analytical method based on solid-phase extraction, dispersive-carbon sorbent cleanup, and liquid chromatography/tandem mass spectrometry. The method was applied to six landfill leachates from four locations in the US as well as to a leachate generated by a laboratory bioreactor containing residential refuse. All seven leachates had the common characteristic that short-chain (C(4)-C(7)) carboxylates or sulfonates were greater in abundance than their respective longer-chain homologs (≥ C(8)). Perfluoroalkyl carboxylates were the most abundant (67 ± 4% on a nanomolar (nM) basis) fluorochemicals measured in leachates; concentrations of individual carboxylates reaching levels up to 2800 ng L(-1). Perfluoroalkyl sulfonates were the next most abundant class (22 ± 2%) on a nM basis; their abundances in each of the seven leachates derived from municipal refuse were greater for the shorter-chain homologs (C(4) and C(6)) compared to longer-chain homologs (C(8) and C(10)). Perfluorobutane sulfonate concentrations were as high as 2300 ng L(-1). Sulfonamide derivatives composed 8 ± 2.1% (nM basis) of the fluorochemicals in landfill leachates with methyl (C(4) and C(8)) and ethyl (C(8)) sulfonamide acetic acids being the most abundant. Fluorotelomer sulfonates (6:2 and 8:2) composed 2.4 ± 1.3% (nM basis) of the fluorochemicals detected and were present in all leachates.


Subject(s)
Environmental Monitoring/methods , Fluorocarbons/analysis , Refuse Disposal , Water Pollutants, Chemical/analysis , Alkanesulfonic Acids/analysis , Caprylates/analysis , Chromatography, Liquid , Fluorocarbons/chemistry , Solid Phase Extraction , Tandem Mass Spectrometry , Water Pollutants, Chemical/chemistry , Water Pollution, Chemical/statistics & numerical data
7.
ISME J ; 5(5): 856-65, 2011 May.
Article in English | MEDLINE | ID: mdl-21068774

ABSTRACT

Metaproteomics is one of a suite of new approaches providing insights into the activities of microorganisms in natural environments. Proteins, the final products of gene expression, indicate cellular priorities, taking into account both transcriptional and posttranscriptional control mechanisms that control adaptive responses. Here, we report the proteomic composition of the < 1.2 µm fraction of a microbial community from Oregon coast summer surface waters, detected with two-dimensional liquid chromatography coupled with electrospray tandem mass spectrometry. Spectra corresponding to proteins involved in protein folding and biosynthesis, transport, and viral capsid structure were the most frequently detected. A total of 36% of all the detected proteins were best matches to the SAR11 clade, and other abundant coastal microbial clades were also well represented, including the Roseobacter clade (17%), oligotrophic marine gammaproteobacteria group (6%), OM43 clade (1%). Viral origins were attributed to 2.5% of proteins. In contrast to oligotrophic waters, phosphate transporters were not highly detected in this nutrient-rich system. However, transporters for amino acids, taurine, polyamines and glutamine synthetase were among the most highly detected proteins, supporting predictions that carbon and nitrogen are more limiting than phosphate in this environment. Intriguingly, one of the highly detected proteins was methanol dehydrogenase originating from the OM43 clade, providing further support for recent reports that the metabolism of one-carbon compounds by these streamlined methylotrophs might be an important feature of coastal ocean biogeochemistry.


Subject(s)
Gammaproteobacteria/metabolism , Plankton/metabolism , Proteomics , Roseobacter/metabolism , Seawater/microbiology , Bacterial Proteins/analysis , Chromatography, High Pressure Liquid , Gammaproteobacteria/classification , Gammaproteobacteria/genetics , Mass Spectrometry , Oceans and Seas , Oregon , Plankton/classification , Plankton/genetics , Roseobacter/classification , Roseobacter/genetics
8.
J Phys Chem B ; 114(46): 15092-105, 2010 Nov 25.
Article in English | MEDLINE | ID: mdl-20973555

ABSTRACT

The gas-phase structures and fragmentation pathways of the singly protonated peptide arginylglycylaspartic acid (RGD) are investigated by means of collision-induced-dissociation (CID) and detailed molecular mechanics and density functional theory (DFT) calculations. It is demonstrated that despite the ionizing proton being strongly sequestered at the guanidine group, protonated RGD can easily be fragmented on charge directed fragmentation pathways. This is due to facile mobilization of the C-terminal or aspartic acid COOH protons thereby generating salt-bridge (SB) stabilized structures. These SB intermediates can directly fragment to generate b(2) ions or facilely rearrange to form anhydrides from which both b(2) and b(2)+H(2)O fragments can be formed. The salt-bridge stabilized and anhydride transition structures (TSs) necessary to form b(2) and b(2)+H(2)O are much lower in energy than their traditional charge solvated counterparts. These mechanisms provide compelling evidence of the role of SB and anhydride structures in protonated peptide fragmentation which complements and supports our recent findings for tryptic systems (Bythell, B. J.; Suhai, S.; Somogyi, A.; Paizs, B. J. Am. Chem. Soc. 2009, 131, 14057-14065.). In addition to these findings we also report on the mechanisms for the formation of the b(1) ion, neutral loss (H(2)O, NH(3), guanidine) fragment ions, and the d(3) ion.


Subject(s)
Arginine/chemistry , Gases/chemistry , Peptides/chemistry , Protein Conformation , Protons , Computer Simulation , Lactams/chemistry , Mass Spectrometry/methods , Models, Molecular , Molecular Structure , Oxazolone/chemistry , Peptides/genetics , Peptides/metabolism
9.
Environ Toxicol Chem ; 29(4): 770-8, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20821505

ABSTRACT

A model was used to predict the photodebromination of the BDE-203, 197, 196, and 153, the major components of the octa-polybrominated diphenyl ether (PBDE) technical mixture, as well as BDE-47, and the predicted results were compared to the experimental results. The predicted reaction time profiles of the photodebromination products correlate well with the experimental results. In addition, the slope of the linear regression between the measured product concentrations of the first step of the photodebromination products and their enthalpies of formation was found to be close to their theoretical value. The photodebromination results of the octa-BDE technical mixture were compared with anaerobic microbial debromination results and were found to be the same in both experiments. The debromination pathways of technical octa-BDE mixture were identified and BDE-154, 99, 47, and 31 were found to be the most abundant hexa-, penta-, tetra-, and tri-BDE debromination products, respectively. In addition to photodebromination and anaerobic biodebromination, the model prediction was also compared to the zero-valent iron reduction of BDE-209, 100, and 47 and the same debromination products were observed. Good correlation was observed between the photodebromination rate constants of fifteen PBDE congeners and their calculated lowest unoccupied molecular orbital (LUMO) energies, indicating that PBDE photodebromination is caused by electron transfer. Furthermore, the rate constants for the three different PBDE debromination processes are controlled by C-Br bond dissociation energy. With the model from the present study, the major debromination products for any PBDE congener released into the environment can be predicted.


Subject(s)
Bacteria/metabolism , Halogenated Diphenyl Ethers/chemistry , Halogenated Diphenyl Ethers/metabolism , Iron/chemistry , Anaerobiosis , Oxidation-Reduction , Photochemistry
11.
Toxicol Lett ; 191(1): 20-5, 2009 Dec 01.
Article in English | MEDLINE | ID: mdl-19666090

ABSTRACT

Pretreatment of male C57BL/6 mice with low doses of the persistent organochlorine pesticide, chlordecone (CD), stimulated biliary excretion of exogenous cholesterol (CH) up to 3-fold. Increased biliary excretion occurred without changes in hepatic ATP-binding cassette transporter G8 (ABCG8) of the bile canaliculus or scavenger receptor class B type I (SR-BI) of the sinusoidal surface. A variety of tissues express scavenger receptor class B type II (SR-BII) and this protein was identified as a splice variant from the SR-BI gene. Although the function of SR-BII has not been elucidated it may play a role in CH homeostasis and trafficking distinctly different than SR-BI. Western blotting demonstrated that a single dose of CD promoted subcellular distribution of SR-BII to murine hepatic microsomes about 2.2-fold when compared to controls without effect on liver crude membrane SR-BII content. This was consistent with increased vesicular CH trafficking. Relative quantification of hepatic cytosolic proteins in a fraction that sequestered [(14)C]CH by mass spectrometry (MS) indicated no role for cytosolic CH binding proteins in CD altered CH homeostasis. Western blotting verified no effect of CD on liver fatty acid-binding protein (L-FABP) in cytosol. MS detected a statistically significant increase in myosin-9, which was also consistent with increased vesicular trafficking.


Subject(s)
Carrier Proteins/metabolism , Chlordecone/toxicity , Cytosol/drug effects , Cytosol/metabolism , Insecticides/toxicity , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Scavenger Receptors, Class B/metabolism , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism , Animals , Bile/metabolism , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Fatty Acid-Binding Proteins/metabolism , Homeostasis/drug effects , Male , Mass Spectrometry , Mice , Mice, Inbred C57BL , Pancreatic Elastase
12.
Anal Chem ; 81(3): 1238-43, 2009 Feb 01.
Article in English | MEDLINE | ID: mdl-19117494

ABSTRACT

A radio frequency-free (RFF), analyzer-independent cell has been devised for electron-capture dissociation (ECD) of ions. The device is based on interleaving a series of electrostatic lenses with the periodic structure of magnetostatic lenses commonly found in a traveling wave tube. The RFF electrostatic/magnetostatic ECD cell was installed in a Finnigan TSQ700 ESI triple quadrupole (QqQ) spectrometer, and its performance was evaluated by recording product-ion spectra of doubly protonated substance P, doubly protonated gramicidin S, doubly protonated neurotensin, and triply protonated neurotensin. These spectra were readily obtained without recourse to a buffering gas or synchronizing electron injection with a specific phase of an RF field. The mass spectra produced with the modified instrument appear in all respects (other than resolution and mass accuracy, which were limited by the mass spectrometer used) to be at least as good for purposes of peptide identification as those recorded with Fourier transform ion cyclotron resonance (FT ICR) instruments; however, the effort and time to produce the mass spectra were much less than required to produce their FT ICR counterparts. The cell's design and compact construction should allow it to be incorporated at relatively little cost into virtually any type of tandem mass spectrometer, for example, triple quadrupole, hybrid quadrupole ion trap, hybrid quadrupole time-of-flight, or even FT-ICR.


Subject(s)
Peptides/chemistry , Tandem Mass Spectrometry/methods , Fourier Analysis
13.
ISME J ; 3(1): 93-105, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18769456

ABSTRACT

The northwestern Sargasso Sea undergoes annual cycles of productivity with increased production in spring corresponding to periods of upwelling, and oligotrophy in summer and autumn, when the water column becomes highly stratified. The biological productivity of this region is reduced during stratified periods as a result of low concentrations of phosphorus and nitrogen in the euphotic zone. To better understand the mechanisms of microbial survival in this oligotrophic environment, we used capillary liquid chromatography (LC)-tandem mass spectrometry to detect microbial proteins in surface samples collected in September 2005. A total of 2215 peptides that mapped to 236 SAR11 proteins, 1911 peptides that mapped to 402 Prochlorococcus proteins and 2407 peptides that mapped to 404 Synechococcus proteins were detected. Mass spectra from SAR11 periplasmic substrate-binding proteins accounted for a disproportionately large fraction of the peptides detected, consistent with observations that these extremely small cells devote a large proportion of their volume to periplasm. Abundances were highest for periplasmic substrate-binding proteins for phosphate, amino acids, phosphonate, sugars and spermidine. Proteins implicated in the prevention of oxidative damage and protein refolding were also abundant. Our findings support the view that competition for multiple nutrients in oligotrophic systems is extreme, but nutrient flux is sufficient to sustain microbial community activity.


Subject(s)
Bacterial Proteins/isolation & purification , Membrane Transport Proteins/isolation & purification , Proteome/isolation & purification , Seawater/microbiology , Chromatography, Liquid , Oceans and Seas , Prochlorococcus/chemistry , Seawater/chemistry , Synechococcus/chemistry , Tandem Mass Spectrometry
14.
Rapid Commun Mass Spectrom ; 22(20): 3137-46, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18798194

ABSTRACT

The use of isobaric tagging for relative and absolute quantification (iTRAQ) has increased dramatically over the past few years. Many factors can affect the accuracy of quantification. Some of these include the number of biological/technical replicates, sample complexity, instrumentation, method of peptide/protein identification and the statistical techniques used for data analysis. It has been observed that the low collision energies normally used in electrospray ionization quadrupole time-of-flight (ESI QTOF) can result in low iTRAQ reporter ion abundances. We used two-way analysis of variance (ANOVA) to compare the iTRAQ ratios that were generated on an ESI QTOF and a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI TOF/TOF). It appears that iTRAQ analyses performed on an ESI QTOF without any special modifications to instrumental parameters produce essentially the same protein ratios as those obtained on a MALDI TOF/TOF.


Subject(s)
Liver/chemistry , Proteome/chemistry , Amino Acid Sequence , Animals , Chlordecone/chemistry , Cholesterol/blood , Data Interpretation, Statistical , Databases, Factual , Indicators and Reagents , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Spectrometry, Fluorescence , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
15.
Environ Sci Technol ; 42(17): 6369-77, 2008 Sep 01.
Article in English | MEDLINE | ID: mdl-18800503

ABSTRACT

Fluorochemicals are persistent contaminants that are globally distributed in air, water, sediments, and biota. Wastewater treatment plants (WWTPs) play an important role in mitigating pollutant releases from municipalities to aquatic and terrestrial environments. However, because WWTPs are point sources of fluorochemicals, it is important to understand their contribution to fluorochemical burdens in the greater context of watersheds. To this end, over a 1 week period, the mass flows of 11 fluorochemicals from seven WWTPs that discharge effluent into the Glatt River in Switzerland were measured and compared to the measured mass flows within the Glatt River. Overall, the fluorochemicals were not removed efficiently during wastewater treatment. Effluents from WWTPs and Glatt River water were dominated by perfluorooctane sulfonate, which was detected in all samples, followed by perfluorohexane sulfonate and perfluorooctanoate. The mass flows of fluorochemicals emanating from WWTPs were found to be conserved within the 35 km Glatt River, which indicates that input from the WWTPs is additive and that removal within the Glatt River is not significant. Per capita discharges of fluorochemicals were calculated from the populations served by the WWTPs studied; the values determined also account for the fluorochemical content of Lake Greifen (Greifensee), which is a lake at the headwaters of the Glatt River that also receives treated wastewater.


Subject(s)
Environmental Pollutants/analysis , Fluorocarbons/analysis , Chromatography, Liquid , Quality Control , Reference Standards , Switzerland , Tandem Mass Spectrometry
17.
Environ Toxicol Chem ; 27(12): 2427-35, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18613751

ABSTRACT

With the phaseout of the manufacture of some polybrominated diphenyl ether (PBDE) formulations, namely penta-brominated diphenyl ether (BDE) and octa-BDE, and the continued use of the deca-BDE formulation, it is important to be able to predict the photodegradation of the more highly brominated congeners. A model was developed and validated to predict the products and their relative concentrations from the photodegradation of PBDEs. The enthalpies of formation of the 209 PBDE congeners were calculated, and the relative reaction rate constants were obtained. The predicted reaction rate constants for PBDEs show linear correlation with previous experimental results. Because of their large volume use, their presence in the environment, and/or importance in the photodegradation of the deca-BDE formulation, BDE-209, BDE-184, BDE-100, and BDE-99 were chosen for further ultraviolet photodegradation experiments in isooctane. The photodegradation model successfully predicted the products of the photochemical reactions of PBDEs in experimental studies. A gas chromatography retention time model for PBDEs was developed using a multiple linear regression analysis and, together with the photodegradation model and additional PBDE standards, provided a way to identify unknown products from PBDE photodegradation experiments. Based on the results of the photodegradation experiments, as well as the model predictions, it appears that the photodegradation of PBDEs is a first-order reaction and, further, that the rate-determining step is the stepwise loss of bromine. Our results suggest that, based on photodegradation, over time, BDE-99 will remain the most abundant penta-BDE, while BDE-49 and BDE-66 will increase greatly and will be comparable in abundance to BDE-47.


Subject(s)
Polybrominated Biphenyls/chemistry , Ethers , Models, Chemical , Photochemistry , Thermodynamics
18.
Appl Environ Microbiol ; 74(13): 4091-100, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18469119

ABSTRACT

"Candidatus Pelagibacter ubique," an abundant marine alphaproteobacterium, subsists in nature at low ambient nutrient concentrations and may often be exposed to nutrient limitation, but its genome reveals no evidence of global regulatory mechanisms for adaptation to stationary phase. High-resolution capillary liquid chromatography coupled online to an LTQ mass spectrometer was used to build an accurate mass and time (AMT) tag library that enabled quantitative examination of proteomic differences between exponential- and stationary-phase "Ca. Pelagibacter ubique" cells cultivated in a seawater medium. The AMT tag library represented 65% of the predicted protein-encoding genes. "Ca. Pelagibacter ubique" appears to respond adaptively to stationary phase by increasing the abundance of a suite of proteins that contribute to homeostasis rather than undergoing a major remodeling of its proteome. Stationary-phase abundances increased significantly for OsmC and thioredoxin reductase, which may mitigate oxidative damage in "Ca. Pelagibacter," as well as for molecular chaperones, enzymes involved in methionine and cysteine biosynthesis, proteins involved in rho-dependent transcription termination, and the signal transduction enzyme CheY-FisH. We speculate that this limited response may enable "Ca. Pelagibacter ubique" to cope with ambient conditions that deprive it of nutrients for short periods and, furthermore, that the ability to resume growth overrides the need for a more comprehensive global stationary-phase response to create a capacity for long-term survival.


Subject(s)
Alphaproteobacteria/growth & development , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Proteome , Seawater/microbiology , Adaptation, Physiological , Alphaproteobacteria/genetics , Alphaproteobacteria/metabolism , Bacterial Proteins/genetics , Chromatography, Liquid , Gene Expression Profiling , Gene Library , Mass Spectrometry
19.
Article in English | MEDLINE | ID: mdl-18042442

ABSTRACT

In this report, the effectiveness of high performance liquid chromatography (HPLC) in conjunction with electrospray ionization mass spectrometry (ESI-MS) is examined as a tool for identifying the sites of crosslinking in a protein that has been photoreacted with a non-photolabeled oligonucleotide. ESI-MS and MALDI-MS analyses preceded by off-line microflow and nanoflow HPLC, on-line microflow HPLC/ESI, and on-line nanoflow HPLC/ESI interfaces were performed in order to determine their relative effectiveness in separating mixtures of nucleopeptides and identifying sites of crosslinking on the individual components. The characteristics of these four techniques as well as possibilities for improving the analysis of nucleopeptides by ESI-MS are compared and discussed.


Subject(s)
Peptide Nucleic Acids/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Ultraviolet Rays , Chromatography, High Pressure Liquid , Nanotechnology
20.
Environ Microbiol ; 9(6): 1456-63, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17504483

ABSTRACT

Ecological studies indicate that aerobic anoxygenic phototrophic bacteria (AAP) that use bacteriochlorophyll to support phototrophic electron transport are widely distributed in the oceans. All cultivated marine AAP are alpha-3 and alpha-4 Proteobacteria, but metagenomic evidence indicates that uncultured AAP Gammaproteobacteria are important members of ocean surface microbial communities. Here we report the description of obligately oligotrophic, marine Gammaproteobacteria that have genes for aerobic anoxygenic photosynthesis. Three strains belonging to the OM60 clade were isolated in autoclaved seawater media. Polymerase chain reaction assays for the pufM gene show that these strains contain photosynthetic reaction centre genes. DNA sequencing and phylogenetic analysis indicate that the pufM genes are polyphyletic, suggesting multiple instances of lateral gene transfer. Peptide sequences from six photosynthesis genes (pufL, pufM, pufC, pufB, pufA and puhA) were detected by proteomic analyses of strain HTCC2080 cells grown aerobically in seawater. They closely match predicted peptides from an environmental seawater bacterial artificial chromosome clone of gammaproteobacterial origin, thus identifying the OM60 clade as a significant source of gammaproteobacterial AAP genes in marine systems. The cell yield and rate of growth of HTCC2080 in autoclaved, aerobic seawater increased in the light. These findings identify the OM60 clade as a source of Gammaproteobacteria AAP genes in coastal oceans, and demonstrate that aerobic, anoxygenic photosynthetic metabolism can enhance the productivity of marine oligotrophic bacteria that also grow heterotrophically in darkness.


Subject(s)
Bacterial Proteins/genetics , Gammaproteobacteria/genetics , Photosynthesis/genetics , Photosynthetic Reaction Center Complex Proteins/genetics , Seawater/microbiology , Gammaproteobacteria/classification , Gammaproteobacteria/isolation & purification , Marine Biology , Phylogeny
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