Expression and membrane integration of SARS-CoV M protein

SARS-CoV M gene fragment was cloned and expressed as a recombinant protein fused with a V5 tag at the C-terminus in Vero E6 cells. In addition to un-glycosylated and glycosylated proteins, one product with smaller size initiated in-frame from the third Met residues probably through ribosomal re-initiation was also detected. Translation initiated in-frame from the third Met is unusual since the sequence around the first Met of SARS-CoV M protein contains the optimal consensus Kozak sequence. The function of this smaller translated product awaits further investigation. Similar to other N-glycosylated proteins, glycosylation of SARS-CoV M protein was occurred co-translationally in the presence of microsomes. The SARS-CoV M protein is predicted as a triple-spanning membrane protein lack of a conventional signal peptide. The second and third trans-membrane regions (a.a. 46–68 and 78–100) are predicted to be the primary type helices, which will be able to penetrate into membrane by themselves, while the first trans-membrane region (a.a. 14–36) is predicted to be the secondary type helix, which is considered to be stabilized by the interaction with other trans-membrane segments. As expected, the second and third trans-membrane regions were able to insert a cytoplasmic protein into the endoplasmic reticulum membrane more efficiently than the first one. These results should be important for the study of SARS-CoV morphogenesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High-performance liquid chromatography-atmospheric pressure photoionization/tandem mass spectrometry for the detection of 17alpha-ethinylestradiol in hepatocytes

Atmospheric pressure photoionization (APPI) as an interface for the high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) system was employed for the direct determination of 17alpha-ethinylestradiol (EE(2)) in the incubation mixtures to support in vitro hepatic clearance studies. For the APPI source, the radical cation of the analyte via charge exchange with the dopant radical cation was used for the detection of EE(2) in the positive ion mode. It was demonstrated that the major signals of EE(2) in the acetonitrile/water mobile phase were substantially increased by replacing toluene with anisole as the dopant. The effects of several experimental conditions on the photoionization efficiency of EE(2) in the dopant-assisted APPI source were explored. Electrospray ionization (ESI) source was also suitable for the analysis of the analyte; however, ESI required a derivatization step prior to analysis. The applicability of the proposed HPLC-APPI-MS/MS approach following a protein precipitation procedure for the determination of EE(2) at low nano-mole levels was examined with respect to assay specificity and linearity. The assay results obtained by both HPLC-APPI-MS/MS and HPLC-ESI-MS/MS methods were in good agreement.     

A new meta-ANOVA approach for synthesizing information under signal-heterogeneity setting with application to nuclear magnetic resonance spectroscopic data

A new synthesizing statistical methodology is proposed to resolve issues of signal-heterogeneity in data sets collected through high-resolution ¹H nuclear magnetic resonance (NMR) spectroscopy. This signal-heterogeneity is typically caused by subjective operations for processing spectral profiles and measuring peak areas, non-homogeneous biological phases of experimental subjects, and variations of systems in multi-center. All these causes are likely to simultaneously impact signals of metabolic changes and their precision in a nonlinear fashion. As a combined effect, signal-heterogeneity chiefly manifests through non-homomorphic patterns of standardized treatment mean deviations spanning all experiments, and makes most remedial statistical models with linearity structure invalid. By avoiding a huge and very complex model, we develop a simple meta-ANOVA approach to synthesize many one-way-layout ANOVA analyses from individual experiments. A scale-invariant F-ratio statistic is taken as the summarizing sufficient statistic of a non-centrality parameter that supposedly captures the information about metabolic change from each experiment. Then a joint-likelihood function of a common non-centrality is constructed as the basis for maximum likelihood estimation and Chi-square likelihood ratio testing for statistical inference. We apply the meta-ANOVA to detect metabolic changes of three metabolites identified through pattern recognition on NMR spectral profiles obtained from muscle and liver tissues. We also detect effect differences among different treatments via meta-ANOVA multiple comparison.     

A simple and inexpensive on-column frit fabrication method for fused-silica capillaries for increased capacity and versatility in LC-MS/MS applications

A modified sol-gel method for a one-step on-column frit preparation for fused-silica capillaries and its utility for peptide separation in LC-MS/MS is described. This method is inexpensive, reproducible, and does not require specialized equipments. Because the frit fabrication process does not damage polyimide coating, the frit-fabricated column can be tightly connected on-line for high pressure LC. These columns can replace any capillary liquid transfer tubing without any specialized connections up-stream of a spray tip column. Therefore multiple columns with different phases can be connected in series for one- or multiple-dimensional chromatography.