Protein biomarkers for in vitro testing of embryotoxicity.

There are new challenges for hazard and risk assessment in the chemical industry with regard to REACH legislation in Europe and related activities in the U.S. and Japan, which require the development of novel in vitro models for the molecular characterization of drug- or chemical-related effects replacing conventional animal testing.

Unexpected common mechanistic pathways for embryotoxicity of warfarin and lovastatin.

Novel molecular content for fast in vitro strategies in the context of safety tests concerning developmental toxicity has a potential to substantially reduce animal experiments according to the "3R" concept (Reduce/Refine/Replace). Here we present and discuss data from a differential proteomic profiling of samples generated using embryonic stem cell derived in vitro models treated with a set of model substances. Among substance-dependent proteomic changes, potential surrogate markers were some isoforms of heat shock proteins and a component of the Ras pathway, present in several redundant isoforms due to posttranslational modifications.

Breast cancer proteomics reveals correlation between estrogen receptor status and differential phosphorylation of PGRMC1.

Introduction: Breast tumors lacking the estrogen receptor-alpha (ER-alpha) have increased incidence of resistance to therapy and poorer clinical prognosis.

Methods: Whole tissue sections from 16 cryopreserved breast cancer tumors that were either positive or negative for the ER (eight ER positive and eight ER negative) were differentially analyzed by multiplex imaging of two-dimensional PAGE gels using 54 cm isoelectric focusing. Differentially detected spots of Progesterone Receptor Membrane Component 1 (PGRMC1) were shown to differ in phosphorylation status by differential two dimensional polyacrylamide gel electrophoresis of phosphatase-treated tumor proteins.

A connection between the mitochondrial permeability transition pore, autophagy, and cerebral amyloidogenesis.

In a drug reprofiling attempt, we explored novel neuroprotective properties of 4-azasteroids by synthesizing chemical affinity tags capturing adenine nucleotide translocator-1, as a potential target. Dutasteride inhibits the mitochondrial transition pore and induces an increase of autophagosomal structures in human cell lines. In vivo, a surprising reduction of the beta-amyloid plaque load in a model for cerebral amyloidosis appears to connect release of neurotoxic peptides, mitochondrial apoptosis and autophagy.

Immunoproteomic identification and serological responses to novel Chlamydia pneumoniae antigens that are associated with persistent C. pneumoniae infections.

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C.

Differential proteomic profiling of mitochondria from Podospora anserina, rat and human reveals distinct patterns of age-related oxidative changes.

According to the 'free radical theory of ageing', the generation and accumulation of reactive oxygen species are key events during ageing of biological systems. Mitochondria are a major source of ROS and prominent targets for ROS-induced damage. Whereas mitochondrial DNA and membranes were shown to be oxidatively modified with ageing, mitochondrial protein oxidation is not well understood.

Metabolically stable isotope labeling prior to electrophoretic protein separation reveals differences in fractional synthesis rates between mitochondrial aldehyde dehydrogenase isoforms.

Living mice were subjected to whole body labeling by intravenous infusion of [(13)C]glucose as the sole carbon source. After 10 h infusion the mice were sacrificed, and liver proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Five spots were found to contain mitochondrial aldehyde dehydrogenase (ALDH2) by matrix assisted time of flight mass spectrometry protein identification.

Differential radioactive quantification of protein abundance ratios between benign and malignant prostate tissues: cancer association of annexin A3.

A differential quantitative protein expression study, comparing matched prostate cancerous and benign tissues from 31 patients, revealed proteins newly associated with prostate cancer. Average effects for 17 proteins whose abundance was significantly different (p<0.01) across patients ranged from 1.

Hexadecanedionic acid-sepharose 4B: A new tool for preparation of albumin-depleted plasma.

Serum and plasma are the major sources of human material for clinical molecular diagnostics and drug discovery. However, due to the high abundance of some proteins, of which serum albumin (SA) is most prominent, lower-abundance proteins often remain undetectable in proteomic analysis of these body fluids. We have used hexadecanedionic acid (HDA) immobilized to Sepharose 4B to develop an affinity resin that is effective in the removal of SA from plasma.

Comparison of different separation technologies for proteome analyses: isoform resolution as a prerequisite for the definition of protein biomarkers on the level of posttranslational modifications.

In this article we evaluate methods used to reveal the molecular complexity, which is generated in biological samples by posttranslational modifications (PTM) of proteins. We show how distinct molecular differences on the level of phosphorylation sites in a single protein (ovalbumin) can be resolved with different success using 1D and 2D gel-electrophoresis and reversed-phase liquid chromatography (LC) with monolithic polystyrol-divinylbenzol (PS-DVB) columns for protein separation, and matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) for protein identification. Phosphorylation site analysis was performed using enzymatic dephosphorylation in combination with differential peptide mass mapping.

Breast cancer proteomics by laser capture microdissection, sample pooling, 54-cm IPG IEF, and differential iodine radioisotope detection.

The presence of progesterone receptor (PR) in estrogen receptor (ER)-positive breast cancer is associated with a good prognosis, and indicates that tumors are likely to respond to tamoxifen. However, ER+/PR- tumors respond less well. To reveal the potential molecular mechanism of this phenomenon, we sought to identify differential protein abundances between invasive ductal carcinoma cells from cryopreserved ER+/PR+ and ER+/PR- mammary tumor specimens.

Comparative profiling of the mammalian mitochondrial proteome: multiple aconitase-2 isoforms including N-formylkynurenine modifications as part of a protein biomarker signature for reactive oxidative species.

The activity of mitochondria induces, as a byproduct, a variety of post-translational modifications in associated proteins, which have functional downstream consequences for processes such as apoptosis, autophagy, and plasticity; e.g., reactive oxygen species (ROS), which induce N-formyl-kynurenine from oxidized tryptophans in certain mitochondrial proteins which are localized in close spatial proximity to their source.

Differential radioactive proteomic analysis of microdissected renal cell carcinoma tissue by 54 cm isoelectric focusing in serial immobilized pH gradient gels.

We present a proof of principle study, using laser microdissection and pressure catapulting (LMPC) of two clinical tissue samples, each containing approximately 3.8 microg renal cell carcinoma protein and 3.8 microg normal kidney protein respectively from one patient.

Determination of fractional synthesis rates of mouse hepatic proteins via metabolic 13C-labeling, MALDI-TOF MS and analysis of relative isotopologue abundances using average masses.

Proteins of a liver extract taken from a metabolically (13)C-labeled mouse were separated by 2D-PAGE and identified after tryptic digestion by MALDI-TOF MS peptide mass fingerprinting. (13)C-Labeling of proteins was achieved by an infusion of U-(13)C-glucose, which is metabolized to labeled nonessential amino acids. The labeling was analyzed using the relative isotopologue abundances of the measured isotope pattern of tryptic peptides and quantified by their increase in the average molecular mass (DeltaAVM).

Molecular analysis of homocysteic acid-induced neuronal stress.

Hyperhomocysteinemia is a risk factor for vascular and neuronal lesions often observed with concomitant high levels of homocysteic acid. In contrast to homocysteine, homocysteic acid induces calcium influx into neurons, with characteristics of an excitotoxic glutamatergic agonist at elevated concentrations. On the molecular level this is correlated to fast modifications of proteins (phosphorylation and proteolysis).

Analysis of relative isotopologue abundances for quantitative profiling of complex protein mixtures labelled with the acrylamide/D3-acrylamide alkylation tag system.

The new method of analysis of relative isotopologue abundances (ARIA) applied here is based on the evaluation of total isotope patterns of tryptic protein fragments measured by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) to calculate the mixing ratios of composites consisting of stable isotope labelled and isotopically natural (unlabelled) proteins, as described in an accompanying paper in this issue. Recently, Sechi (Rapid Commun. Mass Spectrom.

Protein abundance quantification in embryonic stem cells using incomplete metabolic labelling with 15N amino acids, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and analysis of relative isotopologue abundances of peptides.

An isotope dilution method for protein quantification is presented in the context of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) and mass fingerprinting experiments, revealing an unappreciated high reproducibility and accuracy of relative peak intensity measurements. Labelled proteins were generated by growing cells in a medium containing (15)N-enriched amino acids, and were mixed with proteins of natural isotopic composition from control cells in ratios of approximately 0:1, 1:7, 1:2, 2:1, 7:1, and 1:0 (labelled/unlabelled). Mixtures were separated by two-dimensional gel electrophoresis and analysed by MALDI-TOFMS using typical experimental conditions.