Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology

Systematic parallel analysis of the phosphorylation status of networks of interacting proteins involved in the regulatory circuitry of cells and tissues is certain to drive research in the post-genomics era for many years to come. Reversible protein phosphorylation plays a critical regulatory role in a multitude of cellular processes, including alterations in signal transduction pathways related to oncogene and tumor suppressor gene products in cancer. While fluorescence detection methods are likely to offer the best solution to global protein quantitation in proteomics, to date, there has been no satisfactory method for the specific and reversible fluorescent detection of gel-separated phosphoproteins from complex samples. The newly developed Pro-Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels and two-dimensional (2-D) gels. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. Other macromolecules, such as DNA, RNA, and sulfated glycans, fail to be detected with Pro-Q Diamond dye. The staining procedure is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Pro-Q Diamond dye technology can detect as little as 1-2 ng of beta-casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein. Fluorescence signal intensity correlates with the number of phosphorylated residues on the protein. Through combination of Pro-Q Diamond phosphoprotein stain with SYPRO(R) Ruby protein gel stain, Multiplexed Proteomics technology permits quantitative, dichromatic fluorescence detection of proteins in 2-D gels. This evolving discovery platform allows the parallel determination of protein expression level changes and altered post-translational modification patterns within a single 2-D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of changes over an unprecedented 500-1000-fold concentration range.     

Comparison of affinity tags for protein purification

Affinity tags are highly efficient tools for purifying proteins from crude extracts. To facilitate the selection of affinity tags for purification projects, we have compared the efficiency of eight elutable affinity tags to purify proteins from Escherichia coli, yeast, Drosophila, and HeLa extracts. Our results show that the HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, and the GST and MBP protein fusion tag systems differ substantially in purity, yield, and cost. We find that the HIS tag provides good yields of tagged protein from inexpensive, high capacity resins but with only moderate purity from E. coli extracts and relatively poor purification from yeast, Drosophila, and HeLa extracts. The CBP tag produced moderate purity protein from E. coli, yeast, and Drosophila extracts, but better purity from HeLa extracts. Epitope-based tags such as FLAG and HPC produced the highest purity protein for all extracts but require expensive, low capacity resin. Our results suggest that the Strep II tag may provide an acceptable compromise of excellent purification with good yields at a moderate cost.     

TMB-Hunt: An amino acid composition based method to screen proteomes for beta-barrel transmembrane proteins

Background  

Beta-barrel transmembrane (bbtm) proteins are a functionally important and diverse group of proteins expressed in the outer membranes of bacteria (both gram negative and acid fast gram positive), mitochondria and chloroplasts. Despite recent publications describing reasonable levels of accuracy for discriminating between bbtm proteins and other proteins, screening of entire genomes remains troublesome as these molecules only constitute a small fraction of the sequences screened. Therefore, novel methods are still required capable of detecting new families of bbtm protein in diverse genomes.     

Proteome of Aedes aegypti larvae in response to infection by the intracellular parasite Vavraia culicis

We report on the modification of the Aedes aegypti larval proteome following infection by the microsporidian parasite Vavraia culicis. Mosquito larvae were sampled at 5 and 15 days of age to compare the effects of infection when the parasite was in two different developmental stages. Modifications of the host proteome due to the stress of infection were distinguished from those of a more general nature by treatments involving hypoxia. We found that the major reaction to stress was the suppression of particular protein spots. Older (15 days) larvae reacted more strongly to infection by V. culicis (46% of the total number of spots affected; 17% for 5 days larvae), while the strongest reaction of younger (5 days) larvae was to hypoxia for pH range 5-8 and to combined effects of infection and hypoxia for pH range 3-6. MALDI-TOF results indicate that proteins induced or suppressed by infection are involved directly or indirectly in defense against microorganisms. Finally, our MALDI-TOF results suggest that A. aegypti larvae try to control or clear V. culicis infection and also that V. culicis probably impairs the immune defense of this host via arginases-NOS competition.     

Fluorescence assay for neurotoxin-modulated ion transport by the reconstituted voltage-activated sodium channel isolated from eel electric organ

A fluorescence assay for measuring Na channel activation in liposomes containing voltage-sensitive Na channels isolated from Electrophorus electricus is described. The assay is based on transport of a heavy-metal cation, T1+, through the activated channel to quench fluorescence of an internalized, water-soluble chromophore. The channel is "locked" in a chronically opened configuration with alkaloid neurotoxins such as veratridine or batrachotoxin. Diffusion potentials are used to amplify the signal, and enlarged liposomes (greater than 8000 A) result in time courses extended to the range of seconds. Analysis of the kinetics of quenching yields parameters that behave as linear functions of channel activation and reflect vesicle size and channel abundance. The k1/2's for activation by veratridine and batrachotoxin were 5 microM and 169 nM, respectively, and that for tetrodotoxin blockade was 4 nM. Externally applied QX-222 and tetrodotoxin each acted to partially block the stimulated signal, as expected for compounds that act on oppositely oriented channels in the membrane. Single-channel conductances estimated with either veratridine or batrachotoxin ranged between 0.6 and 40.7 pS, corresponding to transport numbers of (1.2 X 10(5)) to (8.1 X 10(6)) ions s-1 channel-1 under the conditions of assay. The assay is approximately 100-fold more sensitive than radiotracer influx assays, requiring 1 fmol of protein per time course.     

Alternate indices of electric and magnetic field exposures among Ontario electrical utility workers

Epidemiologic studies examining the risk of cancer among occupational groups exposed to electric fields (EF) and or magnetic fields (MF) have relied on traditional summaries of exposure such as the time weighted arithmetic or geometric mean exposure. Findings from animal and cellular studies support the consideration of alternative measures of exposure capable of capturing threshold and intermittent measures of field strength. The main objective of this study was to identify a series of suitable exposure metrics for an ongoing cancer incidence study in a cohort of Ontario electric utility workers. Principal components analysis (PCA) and correlational analysis were used to explore the relationships within and between series of EF and MF exposure indices. Exposure data were collected using personal monitors worn by a sample of 820 workers which yielded 4247 worker days of measurement data. For both EF and MF, the first axis of the PCA identified a series of intercorrelated indices that included the geometric mean, median and arithmetic mean. A considerable portion of the variability in EF and MF exposures were accounted for by two other principal component axes. The second axes for EF and MF exposures were representative of the standard deviation (standard deviation) and thresholds of field measures. To a lesser extent, the variability in the exposure variable was explained by time dependent indices which consisted of autocorrelations at 5 min lags and average transitions in field strength. Our results suggest that the variability in exposure data can only be accounted for by using several exposure indices, and consequently, a series of metrics should be used when exploring the risk of cancer owing to MF and EF exposure in this cohort. Furthermore, the poor correlations observed between indices of MF and EF reinforce the need to be take both fields into account when assessing the risk of cancer in this occupational group. Bioelectromagnetics 19:140–151, 1998. © 1998 Wiley-Liss, Inc.     

Isolation and characterization of bacteriophage BCJA1, a novel temperate bacteriophage active against the alkaliphilic bacterium, Bacillus clarkii

The isolation and characterization of a novel bacteriophage active against the obligately alkaliphilic bacterium Bacillus clarkii is described. The bacteriophage, designated BCJA1, is a member of the Siphoviridae family with a B1 morphology. It possesses an isometric head, which measures 65 nm between opposite apices, and a noncontractile tail of 195 nm length. It had a buoyant density of 1.518 g/ml and an estimated particle mass of 37 × 10⁷ daltons. BCJA1 was stable over the pH range of 6–11. A one-step growth experiment conducted at pH 10 demonstrated a latent period of about 40 min and a burst size of approximately 40. The purified bacteriophage appeared to consist of 10 proteins with the major head and tail proteins likely to be of molecular weight 36 500 and 28 000, respectively. The genome size was estimated to be between 32.1 and 34.8 kb. The percent G + C content of purified bacteriophage DNA was 45.6. The wildtype bacteriophage is temperate but a clear plaque mutant was isolated. Received: May 25, 1997 / Accepted: August 5, 1997     

Voltage activation of purified eel sodium channels reconstituted into artificial liposomes

We report here a characterization of the voltage-activated behavior of sodium channels purified from the electroplax of Electrophorus electricus. Single-channel activity in response to depolarizing pulses was recorded from patches excised from liposomes containing the reconstituted channel. Strong hyperpolarizations were required to elicit channel activity. Channels exhibited two typical gating patterns. They either would open in brief bursts upon depolarization and then inactivate (fast) or would stay opened for prolonged periods that frequently lasted several consecutive depolarizations and showed intense flickering (slow). The single-channel conductance estimated from the slope of the I-V curves ranged between 15 and 30 pS under several experimental conditions. Channels gating in either mode, fast or slow, were indistinguishable in terms of their sizes. No clear difference in their mean open times was observed. In addition to the two gating patterns, we also found a very clear tendency of the channels to stay quiet for long periods.