Coupling liquid MALDI MS to liquid chromatography

Matrix-assisted laser desorption/ionisation (MALDI) coupled with time-of-flight (TOF) mass spectrometry (MS) is a powerful tool for the analysis of biological samples, and nanoflow high-performance liquid chromatography (nanoHPLC) is a useful separation technique for the analysis of complex proteomics samples. The off-line combination of MALDI and nanoHPLC has been extensively investigated and straightforward techniques have been developed, focussing particularly on automated MALDI sample preparation that yields sensitive and reproducible spectra. Normally conventional solid MALDI matrices such as α-cyano-4-hydroxycinnamic acid (CHCA) are used for sample preparation. However, they have limited usefulness in quantitative measurements and automated data acquisition because of the formation of heterogeneous crystals, resulting in highly variable ion yields and desorption/ ionization characteristics. Glycerol-based liquid support matrices (LSM) have been proposed as an alternative to the traditional solid matrices as they provide increased shot-to-shot reproducibility, leading to prolonged and stable ion signals and therefore better results. This chapter focuses on the integration of the liquid LSM MALDI matrices into the LC-MALDI MS/MS approach in identifying complex and large proteomes. The interface between LC and MALDI consists of a robotic spotter, which fractionates the eluent from the LC column into nanoliter volumes, and co-spots simultaneously the liquid matrix with the eluent fractions onto a MALDI target plate via sheath flow. The efficiency of this method is demonstrated through the analysis of trypsin digests of both bovine serum albumin (BSA) and Lactobacillus plantarum WCFS1 proteins.

Calcium Flux in Neuroblastoma Cells Is a Coupling Mechanism between Non-Genomic and Genomic Modes of Estrogens

Estrogens have been demonstrated to rapidly modulate calcium levels in a variety of cell types. However, the significance of estrogen-mediated calcium flux in neuronal cells is largely unknown. The relative importance of intra- and extracellular sources of calcium in estrogenic effects on neurons is also not well understood. Previously, we have demonstrated that membrane-limited estrogens, such as E-BSA given before an administration of a 2-hour pulse of 17beta-estradiol (E(2)), can potentiate the transcription mediated by E(2) from a consensus estrogen response element (ERE)-driven reporter gene. Inhibitors to signal transduction cascades given along with E-BSA or E(2) demonstrated that calcium flux is important for E-BSA-mediated potentiation of transcription in a transiently transfected neuroblastoma cell line. In this report, we have used inhibitors to different voltage-gated calcium channels (VGCCs) and to intracellular store receptors along with E-BSA in the first pulse or with E(2) in the second pulse to investigate the relative importance of these channels to estrogen-mediated transcription. Neither L- nor P-type VGCCs seem to play a role in estrogen action in these cells; while N-type VGCCs are important in both the non-genomic and genomic modes of estrogen action. Specific inhibitors also showed that the ryanodine receptor and the inositol trisphosphate receptor are important to E-BSA-mediated transcriptional potentiation. This report provides evidence that while intracellular stores of calcium are required to couple non-genomic actions of estrogen initiated at the membrane to transcription in the nucleus, extracellular sources of calcium are also important in both non-genomic and genomic actions of estrogens. Copyright (c) 2005 S. Karger AG, Basel.

Intercomparison of methods of coupling between convection and large-scale circulation. 2: Comparison over non-uniform surface conditions

As part of an international intercomparison project, the weak temperature gradient (WTG) and damped gravity wave (DGW) methods are used to parameterize large-scale dynamics in a set of cloud-resolving models (CRMs) and single column models (SCMs). The WTG or DGW method is implemented using a configuration that couples a model to a reference state defined with profiles obtained from the same model in radiative-convective equilibrium. We investigated the sensitivity of each model to changes in SST, given a fixed reference state. We performed a systematic comparison of the WTG and DGW methods in different models, and a systematic comparison of the behavior of those models using the WTG method and the DGW method. The sensitivity to the SST depends on both the large-scale parameterization method and the choice of the cloud model. In general, SCMs display a wider range of behaviors than CRMs. All CRMs using either the WTG or DGW method show an increase of precipitation with SST, while SCMs show sensitivities which are not always monotonic. CRMs using either the WTG or DGW method show a similar relationship between mean precipitation rate and column-relative humidity, while SCMs exhibit a much wider range of behaviors. DGW simulations produce large-scale velocity profiles which are smoother and less top-heavy compared to those produced by the WTG simulations. These large-scale parameterization methods provide a useful tool to identify the impact of parameterization differences on model behavior in the presence of two-way feedback between convection and the large-scale circulation.

Coupling an aerosol box model with one-dimensional flow: a tool for understanding observations of new particle formation events

Field observations of new particle formation and the subsequent particle growth are typically only possible at a fixed measurement location, and hence do not follow the temporal evolution of an air parcel in a Lagrangian sense. Standard analysis for determining formation and growth rates requires that the time-dependent formation rate and growth rate of the particles are spatially invariant; air parcel advection means that the observed temporal evolution of the particle size distribution at a fixed measurement location may not represent the true evolution if there are spatial variations in the formation and growth rates. Here we present a zero-dimensional aerosol box model coupled with one-dimensional atmospheric flow to describe the impact of advection on the evolution of simulated new particle formation events. Wind speed, particle formation rates and growth rates are input parameters that can vary as a function of time and location, using wind speed to connect location to time. The output simulates measurements at a fixed location; formation and growth rates of the particle mode can then be calculated from the simulated observations at a stationary point for different scenarios and be compared with the ‘true’ input parameters. Hence, we can investigate how spatial variations in the formation and growth rates of new particles would appear in observations of particle number size distributions at a fixed measurement site. We show that the particle size distribution and growth rate at a fixed location is dependent on the formation and growth parameters upwind, even if local conditions do not vary. We also show that different input parameters used may result in very similar simulated measurements. Erroneous interpretation of observations in terms of particle formation and growth rates, and the time span and areal extent of new particle formation, is possible if the spatial effects are not accounted for.