ABSTRACT
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. In the frame of a European FP6 project on reproductive toxicology ( www.reprotect.eu ), we prepared protein samples from mouse embryonic stem cells differentiated into contracting cardiomyocytes according to the validated embryonic stem cell test (EST) protocol, which had been exposed to toxic substances selected by an expert committee from different in vivo categories of embryotoxicity. Lysates were used to carry out the following investigations: (i) identify optimal dose range conditions in the EST that are suitable for (ii) performing a differential quantitative proteomic study of underlying molecular pathways, (iii) define classes of substances with similar proteomic response patterns, (iv) relate these classes to the traditional in vivo categories of embryotoxicity with (v) the final goal to identify novel surrogate protein biomarker candidates for embryo toxicity. We found two distinct classes of toxic substances (Dinoseb, Ochratoxin-A, and Nitrofen vs ß-aminoproprionitril, Metoclopramide, Doxylamine succinate, and d-penicillamine) with clear pathway-related differences in their proteomic patterns. Most notably, different responses to cluster 1 and cluster 2 substances were observed for Heat shock protein ß-1, Ras-GTPase-activating protein SH3-domain binding protein, Ran binding protein 5, and Calreticulin, Dihydropyrimidinase-like 2 (Ulip2 protein). On the other hand, Heat shock protein 8 and Fscn1 protein were down-regulated by all compounds from both clusters.
Subject(s)
Animal Testing Alternatives/methods , Drug-Related Side Effects and Adverse Reactions , Embryonic Stem Cells/drug effects , Proteins/drug effects , Toxicity Tests , Animals , Biomarkers , Cell Differentiation , Cells, Cultured , Mice , Myocytes, CardiacABSTRACT
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. Site directed mutagenesis was used to create putative phosphorylation site point mutants in PGRMC1. Stable transfectants of these mutants in MCF7 cells were assayed for their survival after oxidative stress, and for AKT kinase phosphorylation. Immune fluorescence using anti-PGRMC1 monoclonal antibody 5G7 was performed on breast cancer tissue microarrays. RESULTS: Proteins significantly differentially abundant between estrogen receptor negative and estrogen receptor positive tumors at the 0.1% level were consistent with published profiles, suggesting an altered keratin pool, and increased inflammation and wound responses in estrogen receptor negative tumors. Two of three spots of PGRMC1 were more abundant in estrogen receptor negative tumors. Phosphatase treatment of breast tumor proteins indicated that the PGRMC1 isoforms differed in their phosphorylation status. Simultaneous mutation of PGRMC1 serine-56 and serine-180 [corrected] fully abrogated the sensitivity of stably transfected MCF7 breast cancer cells to peroxide-induced cell death. Immune fluorescence revealed that PGRMC1 was primarily expressed in ER-negative basal epithelial cells of mammary ductules. Even in advanced tumors, high levels of ER or PGRMC1 were almost mutually exclusive in individual cells. In five out of five examined ductal in situ breast cancers of comedo type, PGRMC1 was expressed in glucose transporter 1 negative or positive poorly oxygenated cells surrounding the necrotic core, surrounded by a more distal halo of ER-positive cells. CONCLUSIONS: PGRMC1 phosphorylation may be involved in the clinical differences that underpin breast tumors of differing ER status.
Subject(s)
Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Carcinoma, Intraductal, Noninfiltrating/metabolism , Carcinoma, Lobular/metabolism , Estrogen Receptor alpha/metabolism , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Protein Processing, Post-Translational , Proteomics , Receptors, Progesterone/metabolism , Amino Acid Substitution , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/pathology , Carcinoma, Lobular/pathology , Cell Line, Tumor , Electrophoresis, Gel, Two-Dimensional , Estrogens , Female , Humans , Membrane Proteins/genetics , Neoplasm Proteins/genetics , Neoplasms, Hormone-Dependent/metabolism , Neoplasms, Hormone-Dependent/pathology , Phosphorylation , Phosphoserine/metabolism , Protein Isoforms/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Progesterone/genetics , Recombinant Fusion Proteins/metabolism , Wound Healing/geneticsABSTRACT
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. The purpose of this study was an unbiased investigation of age-related changes in mitochondrial proteins and the molecular pathways by which ROS-induced protein oxidation may disturb cellular homeostasis. In a differential comparison of mitochondrial proteins from young and senescent strains of the fungal ageing model Podospora anserina, from brains of young (5 months) vs. older rats (17 and 31 months), and human cells, with normal and chemically accelerated in vitro ageing, we found certain redundant posttranslationally modified isoforms of subunits of ATP synthase affected across all three species. These appear to represent general susceptible hot spot targets for oxidative chemical changes of proteins accumulating during ageing, and potentially initiating various age-related pathologies and processes. This type of modification is discussed using the example of SAM-dependent O-methyltransferase from P. anserina (PaMTH1), which surprisingly was found to be enriched in mitochondrial preparations of senescent cultures.
Subject(s)
Aging/physiology , Mitochondria/chemistry , Mitochondrial Proton-Translocating ATPases/analysis , Protein Isoforms/analysis , Proteome , Animals , Cells, Cultured , Electrophoresis, Gel, Two-Dimensional , Fungal Proteins/analysis , Humans , Methyltransferases/analysis , Models, Biological , Oxidative Stress , Podospora/physiology , Protein Processing, Post-Translational , Rats , Reactive Oxygen Species/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
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. By measuring the isotopologue mass distributions of peptide ions, and modeling the (13)C content of the precursor amino acid pool, the fractional synthesis rate of ALDH2 molecules synthesized during the labeling period was determined. One of the five spots was observed to have a five-fold higher fraction of (13)C-containing newly synthesized ALDH2 than the spot with the highest ALDH2 content, and contained more than 60% of newly synthesized ALDH2 although it accounted for less than 20% of the total ALDH2 detected. The total range in the fraction of (13)C-containing proteins between different ALDH2 spots approached 50-fold. The ability to quantitatively characterize different protein isoforms of biological origin for ALDH2 and other proteins from living animals provides new avenues for the exploration of protein function.
Subject(s)
Aldehyde Dehydrogenase/biosynthesis , Electrophoresis, Gel, Two-Dimensional/methods , Isoenzymes/biosynthesis , Isotope Labeling , Mitochondria/enzymology , Aldehyde Dehydrogenase/isolation & purification , Animals , Isoenzymes/isolation & purification , Male , Mice , Mice, Inbred C57BL , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
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. Liquid chromatography-MALDI-TOF MS coupling with subsequent on-target tryptic protein digestion turned out to be the fastest method tested but yielded low resolution for the analysis of PTM, whereas 2D gel-electrophoresis, due to its unique capability of resolving highly complex isoform pattern, turned out to be the most suitable method for this purpose. The evaluated methods complement one another and in connection with efficient technologies for differential and quantitative analysis, these approaches have the potential to reveal novel molecular details of protein biomarkers.
Subject(s)
Biomarkers/analysis , Ovalbumin/isolation & purification , Protein Isoforms/isolation & purification , Protein Processing, Post-Translational , Proteome/analysis , Amino Acid Sequence , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid/methods , Electrophoresis, Gel, Two-Dimensional/methods , Electrophoresis, Polyacrylamide Gel/methods , Isoelectric Focusing/methods , Molecular Sequence Data , Ovalbumin/metabolism , Phosphorylation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Trypsin/metabolismABSTRACT
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. Both proteins are implicated in cell migration, cell survival, growth and embryonic development. Using the examples of warfarin and lovastatin, two substances with entirely different primary targets, the surrogate marker signature nevertheless indicates a common embryotoxic mode of action. We discuss these findings observed in in vitro toxicity tests, in a context of clinical validation and evidence-based toxicology.
Subject(s)
Animal Testing Alternatives , Embryonic Stem Cells/drug effects , Lovastatin/toxicity , Teratogens/toxicity , Toxicity Tests/methods , Warfarin/toxicity , 3T3 Cells , Animals , Cell Differentiation/drug effects , Cell Survival/drug effects , Electrophoresis, Polyacrylamide Gel , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Endpoint Determination , Heat-Shock Proteins/biosynthesis , Inhibitory Concentration 50 , Mice , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Reproducibility of Results , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Toxicity Tests/standards , ras Proteins/biosynthesisABSTRACT
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. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.
Subject(s)
Antibodies, Bacterial/immunology , Antigens, Bacterial/immunology , Chlamydia Infections/immunology , Chlamydophila pneumoniae/immunology , Antibodies, Bacterial/blood , Antigens, Bacterial/isolation & purification , Blotting, Western , Chlamydia Infections/microbiology , Electrophoresis, Gel, Two-Dimensional , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Humans , ProteomicsABSTRACT
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.
Subject(s)
Amyloidosis/drug therapy , Autophagy/drug effects , Azasteroids/pharmacology , Brain Diseases/drug therapy , Mitochondrial Membrane Transport Proteins/metabolism , Amyloid beta-Peptides/pharmacology , Amyloid beta-Protein Precursor/genetics , Amyloidosis/metabolism , Amyloidosis/pathology , Animals , Apoptosis/drug effects , Brain Diseases/metabolism , Brain Diseases/pathology , Disease Models, Animal , Dutasteride , Enzyme Inhibitors/pharmacology , Finasteride/pharmacology , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondrial ADP, ATP Translocases/metabolism , Mitochondrial Permeability Transition Pore , N-Methylaspartate/pharmacology , Neocortex/drug effects , Neocortex/metabolism , Neocortex/pathology , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Neuroprotective Agents/pharmacology , Plaque, Amyloid/drug effects , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Potassium Cyanide/pharmacology , Presenilins/genetics , Sex Factors , Testosterone/pharmacologyABSTRACT
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 1.5- to 6.1-fold, and included a number of known cancer markers. The most differentially abundant proteins between cancer and benign samples were isopeptidase T, serum amyloid P (SAP), annexin A3 (ANXA3) and mitochondrial enoyl coenzyme-A hydratase. SAP is restricted to stroma in healthy tissue, and the lower abundance in tumours may be explained by the reduced stromal content. ANXA3 is present in healthy epithelial cells, exhibits strong staining in precancerous prostatic intraepithelial neoplasia, and is relatively less abundant in individual tumour cells of increasing Gleason pattern (GP), despite exhibiting higher overall tissue abundance in tumours. ANXA3 staining was predominantly cytoplasmic, yet nuclear localization was also observed. Strongly staining single cells, possibly phagocytes, were interspersed in highly dedifferentiated GP5 tumour areas among tumour cells without measurable ANXA3. Local recurrent androgen ablation therapy-resistant tumours exhibit heterogenous low levels of ANXA3 staining. Results are discussed focussing on the potential implications for tumour tissues.
Subject(s)
Annexin A3/metabolism , Biomarkers, Tumor , Prostatic Hyperplasia/metabolism , Prostatic Neoplasms/metabolism , Radioisotopes , Adult , Aged , Diagnosis, Differential , Humans , Male , Middle Aged , Prostatic Hyperplasia/diagnosis , Prostatic Neoplasms/diagnosisABSTRACT
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. Two-dimensional gel analysis of the SA-depleted samples shows a significant enhancement of the low-abundance proteins and highly specific capture of serum albumin. The HDA resin shows better performance in terms of specificity than dye-based resins.
Subject(s)
Affinity Labels/chemistry , Palmitic Acids , Sepharose , Serum Albumin/chemistry , Serum/chemistry , Computational Biology , Electrophoresis, Gel, Two-Dimensional , Humans , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
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. This type of fast molecular changes has profound influence on cell death and survival with implications in a number of pathologies. The quantitative and differential analysis of bovine heart mitochondria by four 2D-PAGE methods, including 2D-PAGE with high-resolution IEF as first dimension, revealed that due to limited resolution, those methods employing blue native-, tricine-urea-, and 16-BAC-PAGE as the first dimension are less applicable for the differential quantitative analysis of redundant protein spots which might give insight into post-translational modifications that are relevant in age- and stress-related changes. Moreover, 2D-PAGE with high resolution IEF was able to resolve a surprisingly large number of membrane proteins from mitochondrial preparations. For aconitase-2, an enzyme playing an important role in mitochondrial aging, a more thorough molecular analysis of all separable isoforms was performed, leading to the identification of two particular N-formylkynurenine modifications. Next to protein redundancy, native protein-protein interactions, with the potential of relating certain post-translational modification patterns to distinct oligomeric states, e.g., oxidative phosphorylation super complexes, might provide novel and (patho-) physiologically relevant information. Among proteins identified, 14 new proteins (GenBank entries), previously not associated with mitochondria, were found.
Subject(s)
Aconitate Hydratase/biosynthesis , Aconitate Hydratase/chemistry , Kynurenine/analogs & derivatives , Mitochondria, Heart/enzymology , Reactive Oxygen Species/metabolism , Aconitate Hydratase/genetics , Animals , Biomarkers/metabolism , Cattle , Gene Expression Profiling , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Kynurenine/chemistryABSTRACT
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. Because current proteomics methods are hampered in the examination of most primary human tumor samples by the extreme tissue heterogeneity, we used laser capture microdissection (LCM) to isolate tumor cells and developed a sample pooling strategy to analyze small sample protein lysates. Proteins from LCM-harvested tumors were pooled into four sub-pools from each condition of three tumors/sub-pool, and proteins from respective paired sub-pools were co-electrophoresed by 2-DE using 54-cm IEF over pH 4-9. Abundance ratios were accurately quantified by a differential multiplex radioactive ProteoTope method at low attomole levels ( approximately 3.6 microg protein per labeling reaction, <180 ng per multiplex protein sample per 54-cm gel). Applying this approach, differentially displayed proteins were identified by MS using comigrating non-radioactively labeled tumor proteins. They include decreased cytochrome b5 and transgelin, and more abundant CRABP-II, cyclophilin A, Neudesin, and hemoglobin in ER+/PR+ tumors versus ER+/PR- providing a possible explanation for differential susceptibility against tamoxifen as a result of deregulated cytochrome b5-dependent metabolism. This study demonstrates the potential of ProteoTope and LCM to enable extremely sensitive and precise differential analyses from well-defined primary clinical specimen.
Subject(s)
Breast Neoplasms/chemistry , Electrophoresis, Gel, Two-Dimensional/methods , Lasers , Microdissection/methods , Neoplasm Proteins/analysis , Proteomics/methods , Antineoplastic Agents, Hormonal/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Cell Extracts/chemistry , Cryopreservation , Drug Resistance, Neoplasm , Female , Humans , Hydrogen-Ion Concentration , Iodine Radioisotopes/analysis , Mutation , Receptors, Estrogen/genetics , Receptors, Progesterone/genetics , Tamoxifen/therapeutic useABSTRACT
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. The study involved separate radio-iodination of each sample with both (125)I and (131)I, dual inverse replicate sample loading to high resolution 54 cm "daisy chain" serial immobilized pH gradient isoelectric focusing (IPG-IEF) 2D-PAGE gels, co-electrophoretic separation of cross-labeled proteins from different samples, and precision multiplex differential radioactive imaging to obtain signals specific for each sample coelectrophoresed within single gels but labeled with different isotopes of iodine, providing extremely precise intra-gel estimates of the abundance ratio for protein spots from both samples. Twelve multiplexed analytical radioactive SDS-gels from 4 serial IPG-IEF gels provided 24 individual radioactive images for a comprehensive analytical protein multiplex quantification study. A further 12 SDS gels containing (125)I-labeled sample were coelectrophoresed with preparative protein amounts obtained from whole tissue sections for the mass spectrometric identification of comigrating proteins. This consumed <40% of the (125)I-labeled sample, and <20% of the (131)I-labeled sample from the respective original 3.8 microg samples. Twenty-nine proteins were identified by mass spectrometry with PMF scores >70 that were >2-fold differentially abundant between the samples and t-test probabilities <0.05. We conclude that this combination of technologies provides excellent quality protein multiplex data for the differential abundance analysis of large numbers of proteins from extremely small samples, and is applicable to a broad range of clinical and related applications.
Subject(s)
Carcinoma, Renal Cell/metabolism , Gene Expression Regulation, Neoplastic , Isoelectric Focusing/methods , Liver Neoplasms/metabolism , Proteomics/methods , Cell Line, Tumor , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Gels , Humans , Hydrogen-Ion Concentration , Lasers , Mass Spectrometry , Odds Ratio , Proteins/chemistry , Proteome , Proton-Motive Force , Radioisotopes/therapeutic useABSTRACT
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). Fractional synthesis rates (FSR) of proteins were determined from corresponding peptides using measured DeltaAVM values as well as DeltaAVM values deduced from tRNA-precursor amino acid labeling, which in turn was derived from proteins showing high (13)C enrichments. The 8-h FSR values of 43 proteins were determined to range from 0 +/- 0.6 to 95 +/- 1%/8 h, with typical errors given as SEM values, which depend on the number of peptides of a specific protein usable for calculation. The method demonstrates that FSR values as an indicator for protein turnover in the liver proteome can be estimated within narrow error margins, providing baseline values from which treatment-dependent deviations could be detected with high statistical certainty.
Subject(s)
Liver/metabolism , Protein Biosynthesis , Proteins/analysis , Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Albumins/metabolism , Animals , Carbon Isotopes , Chemical Fractionation , Electrophoresis, Gel, Two-Dimensional , Glucose/pharmacokinetics , Isotope Labeling/methods , Male , Mice , Mice, Inbred C57BL , Peptide Mapping , Protein Precursors/analysis , Protein Precursors/metabolism , Reproducibility of ResultsABSTRACT
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). Within the homocysteic acid induced molecular signature we focused in more detail on phosphorylation of two proteins implicated as risk factors in schizophrenia and neurodegeneration: Dihydropyrimidinase related protein and 14-3-3 protein isoforms. Among the identified proteins there are known chaperones and oxidative metabolism enzymes, but a few are new in context of neuronal stress: Lasp-1, a vitamin D associated factor and an expressed sequence with features of a Rho GDP dissociation inhibitor. Moreover, we detect a specific proteolytic processing of heat shock protein 70 and proteindisulfide isomerase, which is abolished by vitamins (folic acid, vitamin B12, and vitamin B6), which also decrease elevated intracellular calcium levels induced by homocysteic acid.
Subject(s)
Cell Differentiation/drug effects , Homocysteine/analogs & derivatives , Homocysteine/pharmacology , Neurons/cytology , Pluripotent Stem Cells/cytology , Proteome , Amino Acid Sequence , Animals , Calcium/metabolism , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Hyperhomocysteinemia/metabolism , Mass Spectrometry , Mice , Molecular Sequence Data , Neurons/metabolism , Pluripotent Stem Cells/metabolismABSTRACT
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. A linear relationship is demonstrated between the relative isotopologue abundances (RIA values) for particular peaks in the isotopic distribution of tryptic peptide fragments of the proteins, and the mole fractions of labelled proteins in the mixture. Analysis of RIA values (ARIA quantification) for peptides of six typical silver-stained protein spots for the various mixtures could reproduce the experimentally contrived ratios with approximate errors between 4% (2:1 mixture) and about 18% (1:7 mixture). A consideration of error and its propagation is discussed. ARIA does not require complete separation of the isotope patterns of labelled and unlabelled peptides, and is therefore advantageous in combination with all kinds of labelling experiments in biological systems, because it is compatible with minimal metabolic incorporation of labelling reagent. Simulations indicate that the minimum required (15)N enrichment of the total amino acid pool sufficient for ARIA is less than 4%. In an accompanying paper in this issue, we apply ARIA to proteins differentially labelled with isotope-coded alkylation reagents.
Subject(s)
Embryo, Mammalian/cytology , Isotope Labeling , Peptides/analysis , Proteins/analysis , Stem Cells/chemistry , Amino Acids/analysis , Amino Acids/metabolism , Animals , Cells, Cultured , Electrophoresis, Gel, Two-Dimensional , Mass Spectrometry , Mice , Nitrogen Isotopes/analysis , Peptides/chemistry , Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
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. 2002; 16: 1416-1424) and Gehanne et al. (Rapid Commun. Mass Spectrom. 2002; 16: 1692-1698) introduced the use of differential quantitative mass analysis by MALDI-TOFMS using mixtures of standard proteins alkylated prior to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with either acrylamide (AA) or deuterium-labelled [2,3,3'-D(3)]-acrylamide (D3AA). In the present study we validate the AA/D3AA system, firstly by measuring the yield of proteins alkylated with AA, and secondly by using differential radioactive labels ((125)I and (131)I) to quantitatively establish that non-comigration in 2D-PAGE is negligible. ARIA is then applied to quantitatively estimate the relative proportions of peptides labelled with AA or D3AA in the validated system, using typical silver-stained 2D-PAGE protein spots from 2D gels loaded with 150 microg of total liver protein. The precision and limitations of ARIA quantification of peptides differentially alkylated with isotopomeric reagents are discussed.
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. 2002; 16: 1416-1424) and Gehanne et al. (Rapid Commun. Mass Spectrom. 2002; 16: 1692-1698) introduced the use of differential quantitative mass analysis by MALDI-TOFMS using mixtures of standard proteins alkylated prior to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with either acrylamide (AA) or deuterium-labelled [2,3,3'-D3 ]-acrylamide (D3AA). In the present study we validate the AA/D3AA system, firstly by measuring the yield of proteins alkylated with AA, and secondly by using differential radioactive labels (125 I and 131 I) to quantitatively establish that non-comigration in 2D-PAGE is negligible. ARIA is then applied to quantitatively estimate the relative proportions of peptides labelled with AA or D3AA in the validated system, using typical silver-stained 2D-PAGE protein spots from 2D gels loaded with 150 µg of total liver protein. The precision and limitations of ARIA quantification of peptides differentially alkylated with isotopomeric reagents are discussed.