RESUMEN
Gastric cancer mortality is second only to lung cancer, and its prognosis is dismal. Using surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry, we previously identified a single best mass, which could separate gastric cancer from patients without cancer, with a sensitivity of 89.9% and a specificity of 90%. Using protein liquid chromatography systems with various chromatography media and MS/MS analysis, we were able to identify thrombin light chain A, a proteolytic fragment of prothrombin, as the single best mass for early detection of gastric cancer patients. These findings indicate that disturbances in the coagulation-system are early events in gastric cancer biology and that a decrease or loss of thrombin light chain A, which we termed negative serum protein profiling, may contribute to the diagnosis of cancer patients.
Asunto(s)
Dispepsia/diagnóstico , Proteoma , Neoplasias Gástricas/diagnóstico , Trombina , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Diagnóstico Diferencial , Dispepsia/metabolismo , Femenino , Humanos , Inmunoprecipitación , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Neoplasias Gástricas/metabolismo , Trombina/químicaRESUMEN
Using surface-enhanced laser desorption ionization mass spectrometry (SELDI/TOF-MS) and ProteinChip technology, coupled with a pattern-matching algorithm and serum samples, we screened for protein patterns to differentiate gastric cancer patients from noncancer patients. A classifier ensemble, consisting of 50 decision trees, correctly classified all gastric cancers and all controls of a training set (100% sensitivity and 100% specificity). Eight of 9 stage I gastric cancers (88.9% sensitivity for stage I) were correctly classified. In addition, 28 sera from gastric cancer patients taken in different hospitals were correctly classified (100% sensitivity). Furthermore, all 11 control sera obtained from patients without gastric cancer (100% specificity) were classified correctly and 29 of 30 healthy blood-donors were classified as noncancerous. ProteinChip technology in conjunction with bioinformatics allows the highly sensitive and specific recognition of gastric cancer patients.
Asunto(s)
Proteínas Sanguíneas/análisis , Proteínas de Neoplasias/sangre , Neoplasias Gástricas/diagnóstico , Algoritmos , Estudios de Casos y Controles , Humanos , Espectrometría de Masas , Análisis por Matrices de Proteínas , Sensibilidad y Especificidad , Neoplasias Gástricas/sangreRESUMEN
In this review article, we describe some of the studies that have been performed using the surface-enhanced laser desorption ionization (SELDI) time-of-flight mass spectrometry and ProteinChip technology over the past few years, and highlight both their findings as well as limitations. Proteomic applications, such as target or marker identification and target validation or toxicology, will be addressed. We will also provide an examination of SELDI technology and go into the question of where possible future research may lead us.
Asunto(s)
Análisis por Matrices de Proteínas/métodos , Proteínas/análisis , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Biomarcadores/análisis , Humanos , Análisis por Matrices de Proteínas/instrumentación , Proteínas/genéticaRESUMEN
Gastrointestinal cancers are usually diagnosed at advanced stages, making a curative treatment difficult. Biomarkers can help to overcome this problem by allowing earlier diagnosis, and thus better therapy. Proteomics tools are novel technologies to identify such biomarkers. This review summarizes advances in biomarker detection using two-dimensional gel electrophoresis (2D-PAGE), chromatography and mass spectrometry technologies. 2D-PAGE combined with mass spectrometry has led to the identification of several differentially expressed proteins in cancer tissue. However, for serum analysis, 2D-PAGE has severe limitations. For serum-based cancer diagnosis, surface-enhanced laser desorption-ionization time-of-flight (SELDI-TOF) mass spectrometry is a promising new technology. The potential of proteins identified with this technology as novel cancer biomarkers still needs to be confirmed in clinical trials.
Asunto(s)
Biomarcadores de Tumor , Neoplasias Gastrointestinales/diagnóstico , Proteínas de Neoplasias/análisis , Proteómica , Electroforesis en Gel Bidimensional , Neoplasias Gastrointestinales/genética , Humanos , Espectrometría de Masas/métodos , Análisis por Matrices de Proteínas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The purified membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri was studied with electron paramagnetic resonance (EPR) focusing on the properties of the iron-sulphur clusters. The EPR spectra showed signals from three different [4Fe-4S] clusters. Two of the clusters could be reduced under 101 kPa of H2, whereas the third cluster was only partially reduced. Magnetic interaction of one of the clusters with an unpaired electron localized on the Ni-Fe site indicated that this was the proximal cluster as found in all [NiFe]-hydrogenases. Hence, this cluster was assigned to be located in the EchC subunit. The other two clusters could therefore be assigned to be bound to the EchF subunit, which has two conserved four-Cys motifs for the binding of a [4Fe-4S] cluster. Redox titrations at different pH values demonstrated that the proximal cluster and one of the clusters in the EchF subunit had a pH-dependent midpoint potential. The possible relevance of these properties for the function of this proton-pumping [NiFe]-hydrogenase is discussed.
Asunto(s)
Proteínas Hierro-Azufre/química , Methanosarcina barkeri/enzimología , Oxidorreductasas/química , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Proteínas Hierro-Azufre/metabolismo , Modelos Biológicos , Modelos Químicos , Oxidación-Reducción , Unión Proteica , Protones , TemperaturaRESUMEN
Ech hydrogenase (Ech) from the methanogenic archaeon Methanosarcina barkeri catalyzes the reversible reduction of ferredoxin by H(2) and is a member of a distinct group of membrane-bound [NiFe] hydrogenases with sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I). To elucidate the physiological role(s) of Ech a mutant lacking this enzyme was constructed. The mutant was unable to grow on methanol/H(2)/CO(2), H(2)/CO(2), or acetate as carbon and energy sources but showed wild-type growth rates with methanol as sole substrate. Addition of pyruvate to the growth medium restored growth on methanol/H(2)/CO(2) but not on H(2)/CO(2) or acetate. Results obtained from growth experiments, cell suspension experiments, and enzyme activity measurements in cell extracts provide compelling evidence for essential functions of Ech and a 2[4Fe-4S] ferredoxin in the metabolism of M. barkeri. The following conclusions were made. (i) In acetoclastic methanogenesis, Ech catalyzes H(2) formation from reduced ferredoxin, generated by the oxidation of the carbonyl group of acetate to CO(2). (ii) Under autotrophic growth conditions, the enzyme catalyzes the energetically unfavorable reduction of ferredoxin by H(2), most probably driven by reversed electron transport, and the reduced ferredoxin thus generated functions as low potential electron donor for the synthesis of pyruvate in an anabolic pathway. (iii) Reduced ferredoxin in addition provides the reducing equivalents for the first step of methanogenesis from H(2)/CO(2), the reduction of CO(2) to formylmethanofuran. Thus, in vivo genetic analysis has led to the identification of the electron donor of this key initial step of methanogenesis.
