The quantitative and condition-dependent Escherichia coli proteome.

Measuring precise concentrations of proteins can provide insights into biological processes. Here we use efficient protein extraction and sample fractionation, as well as state-of-the-art quantitative mass spectrometry techniques to generate a comprehensive, condition-dependent protein-abundance map for Escherichia coli. We measure cellular protein concentrations for 55% of predicted E. coli genes (>2,300 proteins) under 22 different experimental conditions and identify methylation and N-terminal protein acetylations previously not known to be prevalent in bacteria. We uncover system-wide proteome allocation, expression regulation and post-translational adaptations. These data provide a valuable resource for the systems biology and broader E. coli research communities.

Evaluation of different two-dimensional chromatographic techniques for proteomic analysis of mouse cardiac tissue.

In proteomics experiments the first critical step after sampling is certainly sample preparation. Multidimensional chromatography techniques have emerged as a powerful tool for the large-scale analysis of such complex samples as biological samples. In order to evaluate these separation techniques, microgram quantities of protein extracted from mouse heart tissue were fractionated by four different chromatographic methods. Regarding peptide-level fractionation, the first dimension of separation was performed with high-pH reversed-phase chromatography (pH-RP) and strong cation exchange chromatography (SCX). Regarding protein-level fractionation, C(8) protein reversed-phase (C(8) -RP Prot) and high-recovery protein reversed-phase (hr-RP Prot) were used instead. The second dimension consisted of a reversed-phase nano-HPLC on-Chip coupled to an electrospray ionization quadrupole time-of-flight mass spectrometer for tandem mass spectrometric analysis. The performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The peptide-level pH-RP and the hr-RP Prot protein-level separations were the best methods, identifying 1338 and 1303 proteins, respectively. The peptide-level SCX, with 509 proteins identified, was the worst method.

A proteomic study of microgravity cardiac effects: feature maps of label-free LC-MALDI data for differential expression analysis.

We present a computational analysis of Mass Spectrometry (MS) data based on a proteomic study of mice cardiac tissue samples whose measured changes in peptide and protein abundance were obtained under normal (control or CTRL) and simulated microgravity conditions (hind-limb unloading or HLU). A pipeline consisting of experimental and computational steps has been designed to achieve, respectively, pre-fractionation to simplify mouse heart protein extracts and data synthesis by feature consensus maps. Both acid and neutral protein fractions obtained from differential solubility have been digested, and peptide mixtures have been resolved by LC-MALDI. The computational tools employed to analyze the MS data and reduce their complex dimensionality have included spectra alignment, denoising and normalization to obtain good-quality peak detection performance. In turn, features could be identified and further refined by searching patterns across repeated measurements obtained under differential conditions (HLU-CTRL, acid-neutral protein extracts). The assessment of reproducibility aspects for evaluating the efficacy of label-free comparative proteomic analysis, combined with the goal of identifying from HLU-CTRL consensus maps candidate proteins with differential expression, led to a computationally efficient approach delivering proteins related to the basic heart functions, cardiac stress and energy supply.

Phenilpropanoate identification in young wheat plants by liquid chromatography/tandem mass spectrometry: monomeric and dimeric compounds.

Metabolomic approach has become a very important tool for determining the actual gene expression and protein activity. Metabolism via the shikimate pathway in plant gives rise to a large number of aromatic compounds, including p-hydroxycinnamates, which are mainly associated with their cell walls in complex structures, ester-linked to heteroxilans. In this work, ferulate dehydrodimers and related compounds were extracted from the cell walls of young lyophilized wheat plants with 2 mol/l NaOH for 4 h at 25 °C under N(2). After solid phase extraction, samples were analysed by high-performance liquid chromatography-quadrupole-linear ion trap tandem mass spectrometry with the electrospray source operating in negative mode. Nineteen dehydrodiferulates, 2 hydrate forms, 3 oxo- forms, 2 decarboxilated forms and 1 dihydroxysinapate, were identified by mass spectra interpretation. In addition, five mixed dehydrodimers of ferulic-sinapic, ferulic-coumaric and ferulic-syringic acids were also identified. Reproducibility of peak areas was usually better than 10% but some minor components which may be procedural artefacts, although relatively mild conditions were used.

Immunoprecipitation on magnetic beads and liquid chromatography-tandem mass spectrometry for carbonic anhydrase II quantification in human serum.

In this study, a magnetic bead-based platform amenable to high-throughput protein carbonic anhydrase II (CA II) capture is presented. The key steps in this approach involved immunoaffinity purification of the target protein from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) operating in multiple reaction monitoring acquisition mode. Using a synthetic peptide standard and a structural analogue free-labeled internal standard, the resulting concentration was stoichiometrically converted to CA II serum concentration. The analytical steps, such as preparation of immunobeads, protein capture, proteolysis, and calibration, were optimized. The method was validated in terms of recovery (77%), reproducibility (relative standard deviation [RSD]<12%), and method detection limit (0.5 pmol ml(-1)). The developed method was applied to determining the CA II in eight healthy subjects, and the concentration measured was 27.3 pmol ml(-1) (RSD = 65%).

Surface adsorption of protein corona controls the cell internalization mechanism of DC-Chol-DOPE/DNA lipoplexes in serum.

Serum has often been reported as a barrier to efficient lipid-mediated transfection. Here we found that the transfection efficiency of DC-Chol-DOPE/DNA lipoplexes increases in serum. To provide insight into the mechanism of lipoplex-serum interaction, several state-of-the-art methodologies have been applied. The nanostructure of DC-Chol-DOPE/DNA lipoplexes was found to be serum-resistant as revealed by high resolution synchrotron small angle X-ray scattering, while dynamic light scattering measurements showed a marked size increase of complexes. The structural stability of DC-Chol-DOPE/DNA lipoplexes was confirmed by electrophoresis on agarose gel demonstrating that plasmid DNA remained well protected by lipids. Proteomics experiments showed that serum proteins competed for the cationic surface of lipid membranes leading to the formation of a rich a 'protein corona'. Combining structural results with proteomics findings, we suggest that such a protein corona can promote large aggregation of intact lipoplexes. According to a recently proposed size-dependent mechanism of lipoplex entry within cells, protein corona-induced formation of large aggregates most likely results in a switch from a clathrin-dependent to caveolae-mediated entry pathway into the cells which is likely to be responsible for the observed transfection efficiency boost. As a consequence, we suggest that surface adsorption of protein corona can have a high biological impact on serum-resistant cationic formulations for in vitro and in vivo lipid-mediated gene delivery applications.

Liquid chromatography-negative ion atmospheric pressure photoionization tandem mass spectrometry for the determination of brominated flame retardants in environmental water and industrial effluents.

We describe the development of a liquid chromatography with negative-ion atmospheric pressure photoionization tandem mass spectrometric (LC/NI-APPI/MS/MS) method for the simultaneous determination of tetrabromobisphenol A (TBBP-A) and five polybrominated diphenyl ethers (BDE-47, BDE-99, BDE-100, BDE-153 and BDE-154) in water. A mobile phase methanol/acetone/water was used, where acetone acts also as dopant. NI-APPI produced precursor ions corresponding to [M-H](-) for TBBP-A, [M-Br+O](-), and [M-2Br+O](-) for the BDE congeners studied. Each compound was quantified operating in multiple reaction monitoring mode. Linearity was observed in the range 0.025-10 ng injected for all compounds. Coefficients of determination R(2) ranged from 0.9934 to 0.9982. BDEs were poorly retained by solid-phase extraction (SPE) from river water and sewage treatment plant effluent, thus liquid-liquid extraction (LLE) by n-hexane should be used for these samples. The recoveries of TBBP-A and PBDEs from tap water (SPE), river water and industrial wastewater (LLE) were in the range of 81-88%, 78-92%, and 43-99%, respectively, with relative standard deviations below 17%. The limits of detection, based on signal-to-noise ratio of 3, ranged from 0.004 to 0.1 ng injected, and method quantification limits were 0.2-3.3 ng L(-1) but BDE47 (20.3 ng L(-1)). Only TBBP-A was found in a treated industrial sewage at 4 ng L(-1), while BDE-99 and BDE-100 were detected on suspended solids.

HPLC-CHIP coupled to a triple quadrupole mass spectrometer for carbonic anhydrase II quantification in human serum.

A method for carbonic anhydrase II (CA II) absolute quantification in human serum is presented. This method is based on high-performance liquid chromatography (HPLC)-Chip microfluidic device incorporating a nanoelectrospray source interfaced to a triple quadrupole mass spectrometer. The fraction containing CA II was isolated by preparative reversed-phase HPLC, and peptides obtained from the tryptic digest of the protein mixture were separated by the HPLC-Chip system. The multiple-reaction monitoring acquisition mode of a selected suitable CA II peptide and peptide internal standard allowed the selective and sensitive determination of a CA II. Absolute recovery of the method was 52 +/- 12%, while analytical recovery was 81 +/- 10%. For the eight samples analyzed, the matrix effect was found to be only -14 +/- 6%. A comparison among three regression lines type which were obtained by external calibration, matrix-matched calibration, and standard addition method, respectively, demonstrated that the first one is adequate in obtaining good accuracy and precision. Method quantification limit for CA II in serum was estimated to be 2 fmol/mL. CA II mean concentration in sera from eight healthy subjects was found to be 56 pmol/mL (relative standard deviation 24%).