A gas chromatography/mass spectrometry assay to measure estradiol, catecholestradiols, and methoxyestradiols in plasma

We have developed a gas chromatography/mass spectrometry (GC/MS) assay to measure 17beta-estradiol (E) and its biologically active metabolites 2-hydroxyestradiol (2OHE) and 4-hydroxyestradiol (4OHE), and 2-methoxyestradiol (2MEOE) and 4-methoxyestradiol (4MEOE) in rat plasma. All analytes are well separated and show a linear relationship between concentration (0.25-5 pg/microl) and signal, and coefficients of variation (CVs) are low. Intra-assay CV for the lowest quality control samples (QCs) (0.375 pg/microl) were on average for 17beta-estradiol 20.5%, for 2-hydroxyestradiol 15.6%, for 4-hydroxyestradiol 16.5%, for 2-methoxyestradiol 16.5%, and for 4-methoxyestradiol 11.5%. The inter-assay CVs for the lowest QCs were for 17beta-estradiol 12.1%, for 2-hydroxyestradiol 7.1%, for 4-hydroxyestradiol 15.5%, for 2-methoxyestradiol 16.7%, and for 4-methoxyestradiol 9.7%. The highest sensitivity for this assay was observed for hydroxyestradiols followed by the methoxyestradiols and 17beta-estradiol. In summary, we describe a convenient, sensitive, and specific assay to measure 17beta-estradiol and its biologically active metabolites.     

Mechanisms of hydrazine toxicity in rat liver investigated by proteomics and multivariate data analysis

A proteomics approach combined with multivariate data analysis was used to examine the hepatotoxic effect of hydrazine in 30 male Sprague Dawley rats, assigned to four treatment groups and two control groups. Liver samples from the individual animals were resolved by two-dimensional differential gel electrophoresis (2-D DIGE) and protein patterns from the 2-D gels were analyzed by principal component analysis (PCA) and partial least squares regression (PLSR). The PCA plot was able to describe the variation in the protein expression related to dose and time, by separation or clustering of different animal groups. PLSR followed by variable selection (Jack-knifing) was used to select proteins that varied significantly in relation to the dose related response of the hydrazine treatment. The 10 up-regulated and 10 down-regulated proteins with highest rank in the PLSR model were identified by mass spectrometry. Hydrazine treatment induced altered expression of proteins related to lipid metabolism, Ca(2+) homeostasis, thyroid hormone pathways and stress response. Several of the identified proteins have not previously been implicated in hydrazine toxicity and may thus be regarded as new potential biomarkers of induced liver toxicity.     

Genetic analysis of the biosynthesis of non-ribosomal peptide- and polyketide-like antibiotics, iron uptake and biofilm formation by Bacillus subtilis A1/3

The Bacillus subtilis strain A1/3 shows exceptionally diverse antibiotic capacities compared to other B. subtilis strains. To analyze this phenomenon, mutants for the putative pantotheinyltransferase gene ( pptS), and for several genes involved in non-ribosomal peptide synthesis and polyketide synthesis were constructed and characterized, using bioassays with blood cells, bacterial and fungal cells, and mass spectrometry. Among at least nine distinct bioactive compounds, five antibiotics and one siderophore activity were identified. The anti-fungal and hemolytic activities of strain A1/3 could be eliminated by mutation of the fen and srf genes essential for the synthesis of fengycins and surfactins. Both pptS - and dhb -type mutants were defective in iron uptake, indicating an inability to produce a 2,3-dihydroxybenzoate-type iron siderophore. Transposon mutants in the malonyl CoA transacylase gene resulted in the loss of hemolytic and anti-fungal activities due to the inhibition of bacillomycin L synthesis, and this led to the discovery of bmyLD-LA-LB* genes. In mutants bearing disruption mutations in polyketide ( pksM - and/or pksR -like) genes, the biosynthesis of bacillaene and difficidins, respectively, was inactivated and was accompanied by the loss of discrete antibacterial activities. The formation of biofilms (pellicles) was shown to require the production of surfactins, but no other lipopeptides, indicating that surfactins serve specific developmental functions.Communicated by A. Kondorosi     

Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry

The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions.     

Sweet Substitute: a software tool for in silico fragmentation of peptide-linked N-glycans

A software tool, Sweet Substitute, is described, which assists tandem mass spectrometry (MS/MS)-based glycosylation characterization from within a tryptic digest. The algorithm creates a virtual nanoelectrospray-quadrupole time-of-flight style-MS/MS spectrum of any user-defined N-linked glycan structure. An empirical peak height modeling routine is implemented in the program. By comparing the theoretical MS/MS data with the deconvoluted and deisotoped experimental MS/MS data, the user is able to quickly assess whether a proposed candidate oligosaccharide structure is a plausible one.     

Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments

Despite recent progress in sequencing the complete genome of rice (Oryza sativa), the proteome of this species remains poorly understood. To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells. The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching. In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure. The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antifungal defense and signal systems in the membranes. Edman degradation revealed that 60–98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed. The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Jürgens     

Production of C<Subscript>35</Subscript> isoprenoids depends on H<Subscript>2</Subscript> availability during cultivation of the hyperthermophile<Emphasis Type="Italic"> Methanococcus jannaschii</Emphasis>

A series of five progressively saturated C₃₅ isoprenoids has been identified in cell-free extracts of the deep-sea methanogen Methanococcus jannaschii. Production and relative abundance of the isoprenoids were dependent on culture conditions; significant production occurred in a 16-l fermentor (12-l working volume) and a 2.5-l fermentor (2-l working volume) but could not be duplicated in serum bottles. Several factors were investigated and shown not to account for the different production levels, including medium composition, pH, and temperature. However, the interphase mass transfer rate was shown to significantly affect the production of C₃₅ isoprenoids in a fermentor. The structures of the novel isoprenoids were confirmed by hydrogenation reactions and mass spectra of the isoprenoids. Indirect evidence based on genomics and mass spectrometry data implicates head-to-head condensation of farnesyl pyrophosphate (C₁₅) with geranylgeranyl pyrophosphate (C₂₀) as the mechanism for C₃₅ synthesis.Communicated by J. WiegelB.P. Manquin and J.A. Morgan contributed equally to this work.     

Chemical identification of<Emphasis Type="Italic"> N</Emphasis>-acyl homoserine lactone quorum-sensing signals produced by<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis> strains in defined medium

The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and electrospray ionization tandem mass spectrometry (ESI MS/MS). Both strains synthesized several long-chain AHLs. Defined medium cultures of strain AK631 synthesized a complex mixture of AHLs with short acyl side chains. Strain 1021 produced no short-chain AHLs when grown on defined medium and made a somewhat different set of long-chain AHLs than previously reported for cultures in rich medium. While the two strains produced several AHLs in common, the differences in AHLs produced suggest that there may be significant differences in their patterns of quorum-sensing regulation.     

Analyzing bioterror response logistics: the case of smallpox

To evaluate existing and alternative proposals for emergency response to a deliberate smallpox attack, we embed the key operational features of such interventions into a smallpox disease transmission model. We use probabilistic reasoning within an otherwise deterministic epidemic framework to model the 'race to trace', i.e., attempting to trace (via the infector) and vaccinate an infected person while (s)he is still vaccine-sensitive. Our model explicitly incorporates a tracing/vaccination queue, and hence can be used as a capacity planning tool. An approximate analysis of this large (16 ODE) system yields closed-form estimates for the total number of deaths and the maximum queue length. The former estimate delineates the efficacy (i.e., accuracy) and efficiency (i.e., speed) of contact tracing, while the latter estimate reveals how congestion makes the race to trace more difficult to win, thereby causing more deaths. A probabilistic analysis is also used to find an approximate closed-form expression for the total number of deaths under mass vaccination, in terms of both the basic reproductive ratio and the vaccination capacity. We also derive approximate thresholds for initially controlling the epidemic for more general interventions that include imperfect vaccination and quarantine.