RESUMO
Neuregulin-1 (Nrg1) is genetically linked to schizophrenia, a disease caused by neurodevelopmental imbalance in dopaminergic function. The Nrg1 receptor ErbB4 is abundantly expressed on midbrain dopaminergic neurons. Nrg1 has been shown to penetrate blood-brain barrier, and peripherally administered Nrg1 activates ErbB4 and leads to a persistent hyperdopaminergic state in neonatal mice. These data prompted us to study the effect of peripheral administration of Nrg1 in the context of Parkinson's disease, a neurodegenerative disorder affecting the dopaminergic system in the adult brain. We observed that systemic injections of the extracellular domain of Nrg1ß(1) (Nrg1ß(1)-ECD) increased dopamine levels in the substantia nigra and striatum of adult mice. Nrg1ß(1)-ECD injections also significantly protected the mouse nigrostriatal dopaminergic system morphologically and functionally against 6-hydroxydopamine-induced toxicity in vivo. Moreover, Nrg1ß(1)-ECD also protected human dopaminergic neurons in vitro against 6-hydroxydopamine. In conclusion, we have identified Nrg1ß(1)-ECD as a neurotrophic factor for adult mouse and human midbrain dopaminergic neurons with peripheral administratability, warranting further investigation as therapeutic option for Parkinson's disease patients.
Assuntos
Dopamina/metabolismo , Neuregulina-1/uso terapêutico , Neurônios/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologiaRESUMO
PURPOSE: In prostate cancer cases the early diagnosis of tumors carrying a high risk of progression is of the utmost importance. There is an urgent clinical need to avoid unnecessary biopsies and subsequent overtreatment. We validated annexin A3 as a diagnostic marker for prostatic disease in typical clinical populations and relevant segments, such as patients with a negative digital rectal examination and low prostate specific antigen. MATERIALS AND METHODS: We performed a blinded clinical study (ClinicalTrials.gov Identifier NCT00400894) from September 2005 to January 2007 in 591 patients who were continuously recruited from 4 European urological clinics. Urine was obtained directly after digital rectal examination and the annexin A3 concentration in urine was quantified by Western blot. Statistical analysis included combinations of annexin A3 with total, percent free, complexed and percent complexed prostate specific antigen. RESULTS: Combined readouts of prostate specific antigen and urinary annexin A3 were superior to all others with an area under the ROC curve of 0.82 for a total prostate specific antigen range of 2 to 6 ng/ml, 0.83 for a total prostate specific antigen range of 4 to 10 ng/ml and 0.81 in all patients. The best performing prostate specific antigen derivative was percent free prostate specific antigen with an area under the ROC curve of 0.68 for a total prostate specific antigen range of 2 to 6 ng/ml, 0.72 for a total prostate specific antigen range of 4 to 10 ng/ml and 0.73 in all patients. Annexin A3 has an inverse relationship to cancer and, therefore, its specificity was much better than that of prostate specific antigen. CONCLUSIONS: Annexin A3 quantification in urine provides a novel noninvasive biomarker with high specificity. Annexin A3 is complementary to prostate specific antigen or to any other cancer marker. It has a huge potential to avoid unnecessary biopsies with a particular strength in the clinically relevant large group of patients who have a negative digital rectal examination and prostate specific antigen in the lower range of values (2 to 10 ng/ml).
Assuntos
Anexina A3/urina , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/urina , Biomarcadores/urina , Detecção Precoce de Câncer , Humanos , Masculino , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/sangue , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Lobular/metabolismo , Receptor alfa de Estrogênio/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Receptores de Progesterona/metabolismo , Substituição de Aminoácidos , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/patologia , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Estrogênios , Feminino , Humanos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Fosforilação , Fosfosserina/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Progesterona/genética , Proteínas Recombinantes de Fusão/metabolismo , Cicatrização/genéticaRESUMO
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.
Assuntos
Envelhecimento/fisiologia , Mitocôndrias/química , ATPases Mitocondriais Próton-Translocadoras/análise , Isoformas de Proteínas/análise , Proteoma , Animais , Células Cultivadas , Eletroforese em Gel Bidimensional , Proteínas Fúngicas/análise , Humanos , Metiltransferases/análise , Modelos Biológicos , Estresse Oxidativo , Podospora/fisiologia , Processamento de Proteína Pós-Traducional , Ratos , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Aldeído Desidrogenase/biossíntese , Eletroforese em Gel Bidimensional/métodos , Isoenzimas/biossíntese , Marcação por Isótopo , Mitocôndrias/enzimologia , Aldeído Desidrogenase/isolamento & purificação , Animais , Isoenzimas/isolamento & purificação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The use of unmodified starches in frozen foods is severely limited by the undesirable textural changes that occur after freezing and thawing. Retrogradation of glucan chains leads to syneresis, a separation of the starch gel and water phases. Stabilization of the starch structure is normally achieved by chemical modification to prevent these changes from occurring. We have now created a freeze-thaw-stable potato starch by alteration of starch composition and structure by genetic modification. An amylose-free starch with short-chain amylopectin was produced by simultaneous antisense downregulation of three starch synthase genes. This starch is extremely freeze-thaw-stable and shows no syneresis even after five freeze-thaw cycles. The use of this starch has potential for environmental and consumer benefits because its production requires no chemical modification.
Assuntos
Oligonucleotídeos Antissenso/farmacologia , Sintase do Amido/química , Amido/química , Amilopectina/química , Amilose/química , Cromatografia , Congelamento , Plantas Geneticamente Modificadas , Solanum tuberosum/enzimologia , Fatores de TempoRESUMO
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.
Assuntos
Biomarcadores/análise , Ovalbumina/isolamento & purificação , Isoformas de Proteínas/isolamento & purificação , Processamento de Proteína Pós-Traducional , Proteoma/análise , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Focalização Isoelétrica/métodos , Dados de Sequência Molecular , Ovalbumina/metabolismo , Fosforilação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tripsina/metabolismoRESUMO
OBJECTIVES: Annexin A3 (ANXA3) is a potential marker for prostate cancer (PCa). We aimed to develop robust immunoassays suitable for quantifying ANXA3 in urine samples obtained following digital rectal examination (DRE) in order to facilitate the diagnostic performance evaluation of this marker. DESIGN AND METHODS: Anti-ANXA3 monoclonal antibodies were generated and their epitopes mapped. Two different ANXA3 assay prototypes were established on the VIDAS® automated immunoanalyser and analytical validation was carried out using post-DRE urine samples obtained from patients with PCa (n=23) or benign prostate hyperplasia (n=31). RESULTS: The assays had the same capture antibody (TGC44) but different detection antibodies (13A12 or 5C5), recognizing novel distinct epitopes. Both had a lower limit of quantification <1ng/mL and were highly specific for ANXA3, not cross-reacting with other annexins. Interassay imprecision was ≤11% and ≤15% for 13A12 and 5C5 assays, respectively. Surprisingly, a total lack of correlation was observed between ANXA3 levels measured by these two assays in post-DRE urines, indicating detection of distinct antigenic variants. Two freeze-thaw cycles did not affect analyte stability in either assay, whereas a lack of stability of antigenic variants was observed when samples were stored at -80°C for 1month. CONCLUSIONS: Two different antigenic variants of ANXA3 are present in post-DRE urines and their clinical significance for diagnosis of prostate cancer should be further investigated. These variants are not stable over time in samples preserved at -80°C. Until this issue is resolved, ANXA3 should only be measured in freshly collected samples.
Assuntos
Anexina A3/urina , Biomarcadores Tumorais/urina , Exame Retal Digital , Proteínas de Neoplasias/urina , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/urina , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Antineoplásicos/química , Criopreservação , Ensaio de Imunoadsorção Enzimática , Epitopos/urina , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estabilidade ProteicaRESUMO
The accumulation of oxidative damage in mitochondrial proteins, membranes and DNA during ageing is supposed to lead to mitochondrial inactivation, downstream molecular impairments and subsequent decline of biological systems. In a quantitative study investigating the age-related changes of mitochondrial proteins on the level of oxidative posttranslational modifications, we previously found a set of conserved biomarkers across ageing models in five species with consistent oxidative break-up of tryptophan residues and formation of N-formyl kynurenine. In an additional proteomic profiling of a long-living Drosophila mutant overexpressing mitochondrial Hsp22 and controls, we found age-related redundant isoforms of voltage-dependent anion channel 1 (VDAC-1). A re-examination of data from human umbilical vein endothelial cells (with normal and chemically accelerated in vitro ageing), revealed similar age-dependent alterations of voltage-dependent anion channel isoforms. Building on these results, we examined the expression of VDAC-1 in an in vitro model of excitotoxicity. We show that glutamate-induced calcium toxicity in neurons induces changes of voltage-dependent anion channel 1 related to downstream events of mitochondrial apoptosis like poly-ADP-ribosylation.
Assuntos
Envelhecimento/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Animais , Animais Geneticamente Modificados , Células Cultivadas , Senescência Celular/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Técnicas In Vitro , Masculino , Camundongos , Mitocôndrias/metabolismo , Mutação , Neurônios/citologia , Neurônios/metabolismo , Processamento de Proteína Pós-Traducional , ProteômicaRESUMO
OBJECTIVES: By differential quantitative protein expression, it has previously been shown that annexin A3 (ANXA3) expression is associated with prostate cancer. However little is known about the role and biology of ANXA3 in the human prostate. The aim of this study was to thoroughly analyze ANXA3 expression patterns and its potential as a prognostic marker in a large set of benign, preneoplastic, and neoplastic prostate tissue samples. METHODS: Immunohistochemistry-based ANXA3 protein expression was analyzed for 1589 prostate cancers as well as smaller subsets of benign epithelium and high-grade prostatic intraepithelial neoplasia (PIN) in a tissue microarray format. RESULTS: All samples of benign prostatic epithelium and PIN showed ANXA3 protein expression, with PIN lesions showing a decreased staining intensity compared with benign epithelium (p<0.0001). In cancer, ANXA3 protein expression was essentially reduced, resulting in a negative staining rate of 27.2%, which correlated with increasing pT stage and Gleason score (p<0.0001). ANXA3 status in cancer was shown to be an independent adverse prognostic factor and enabled substratification of the large group of intermediate-risk patients (n=969) into high- and low-risk subgroups. CONCLUSIONS: ANXA3 represents a promising candidate tissue marker, and when combined with the standard prognostic parameters, is suggested to provide a more precise prediction of prognosis in the individual patient, therefore harboring the potential to contribute to future patient management.
Assuntos
Anexina A3/biossíntese , Neoplasias da Próstata/metabolismo , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias da Próstata/patologiaRESUMO
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.
Assuntos
Amiloidose/tratamento farmacológico , Autofagia/efeitos dos fármacos , Azasteroides/farmacologia , Encefalopatias/tratamento farmacológico , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Peptídeos beta-Amiloides/farmacologia , Precursor de Proteína beta-Amiloide/genética , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Apoptose/efeitos dos fármacos , Encefalopatias/metabolismo , Encefalopatias/patologia , Modelos Animais de Doenças , Dutasterida , Inibidores Enzimáticos/farmacologia , Finasterida/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Translocases Mitocondriais de ADP e ATP/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , N-Metilaspartato/farmacologia , Neocórtex/efeitos dos fármacos , Neocórtex/metabolismo , Neocórtex/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Placa Amiloide/efeitos dos fármacos , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Cianeto de Potássio/farmacologia , Presenilinas/genética , Fatores Sexuais , Testosterona/farmacologiaRESUMO
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.
Assuntos
Anexina A3/metabolismo , Biomarcadores Tumorais , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismo , Radioisótopos , Adulto , Idoso , Diagnóstico Diferencial , Humanos , Masculino , Pessoa de Meia-Idade , Hiperplasia Prostática/diagnóstico , Neoplasias da Próstata/diagnósticoRESUMO
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.
Assuntos
Aconitato Hidratase/biossíntese , Aconitato Hidratase/química , Cinurenina/análogos & derivados , Mitocôndrias Cardíacas/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Aconitato Hidratase/genética , Animais , Biomarcadores/metabolismo , Bovinos , Perfilação da Expressão Gênica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinurenina/químicaRESUMO
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.
Assuntos
Neoplasias da Mama/química , Eletroforese em Gel Bidimensional/métodos , Lasers , Microdissecção/métodos , Proteínas de Neoplasias/análise , Proteômica/métodos , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Extratos Celulares/química , Criopreservação , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Radioisótopos do Iodo/análise , Mutação , Receptores de Estrogênio/genética , Receptores de Progesterona/genética , Tamoxifeno/uso terapêuticoRESUMO
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.