RESUMEN
This study was designed to quantify and identify differences in protein levels between tumor and adjacent normal breast tissue from the same breast in 18 women with stage I/II ER positive/Her2/neu negative invasive breast cancer. Eighteen separate difference gel electrophoresis (DIGE) gels were run (1 gel per patient). Relative quantification was based on DIGE analysis. After excision and tryptic digestion, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass mapping were used to identify protein spots. Two hundred and forty-three spots were differentially abundant between normal and cancer tissues. Fifty spots were identified: 41 were over abundant and nine were less abundant in cancers than in normal breast tissue. Western blotting provided independent confirmation for three of the most biologically and statistically interesting proteins. All 18 gels were replicated by another technician and 32% of the differentially abundant proteins were verified by the duplicate analysis. Follow-up studies are now examining these proteins as biomarkers in blood.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias de la Mama/química , Receptor ErbB-2/análisis , Receptores de Estrógenos/análisis , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Neoplasias de la Mama/patología , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional , Femenino , Humanos , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Variaciones Dependientes del Observador , Mapeo Peptídico , Proteómica/métodos , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The milk of the one-humped camel (Camelus dromedarius) reportedly offers medicinal benefits, perhaps because of its unique bioactive components. Milk proteins were determined by (1) two-dimensional gel electrophoresis and peptide mass mapping and (2) liquid chromatography-tandem mass spectrometry (LC-MS/MS) following one-dimensional polyacrylamide gel electrophoresis. Over 200 proteins were identified: some known camel proteins including heavy-chain immunoglobulins and others exhibiting regions of exact homology with proteins from other species. Indigenous peptides were also identified following isolation and concentration by two strategies: (1) gel-eluted liquid fraction entrapment electrophoresis and (2) small-scale electrophoretic separation. Extracts were analyzed by LC-MS/MS and peptides identified by matching strategies, by de novo sequencing and by applying a sequence tag tool requiring similarity to the proposed sequence, but not an exact match. A plethora of protein cleavage products including some novel peptides were characterized. These studies demonstrate that camel milk is a rich source of peptides, some of which may serve as nutraceuticals.
Asunto(s)
Camelus , Proteínas de la Leche/análisis , Fragmentos de Péptidos/análisis , Mapeo Peptídico/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Animales , Electroforesis en Gel Bidimensional , Femenino , Proteínas de la Leche/química , Proteínas de la Leche/clasificación , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/clasificaciónRESUMEN
INTRODUCTION: Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic autoimmune diseases with variable clinical outcomes. We investigated whether the synovial fluid (SF) proteome could distinguish a subset of patients in whom disease extends to affect a large number of joints. METHODS: SF samples from 57 patients were obtained around time of initial diagnosis of JIA, labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with expression verified by immunochemical methods. Protein glycosylation status was confirmed by hydrophilic interaction liquid chromatography. RESULTS: A truncated isoform of vitamin D binding protein (VDBP) is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p<0.05). Furthermore, sialylated forms of immunopurified synovial VDBP were significantly reduced in extended oligoarticular patients (p<0.005). CONCLUSION: Reduced conversion of VDBP to a macrophage activation factor may be used to stratify patients to determine risk of disease extension in JIA patients.