RESUMEN
Topology analysis of membrane proteins can be obtained by enzymatic shaving in combination with MS identification of peptides. Ideally, such analysis could provide quite detailed information about the membrane spanning regions. Here, we examine the ability of some shaving enzymes to provide large-scale analysis of membrane proteome topologies. To compare different shaving enzymes, we first analyzed the detected peptides from two over-expressed proteins. Second, we analyzed the peptides from non-over-expressed Escherichia coli membrane proteins with known structure to evaluate the shaving methods. Finally, the identified peptides were used to test the accuracy of a number of topology predictors. At the end we suggest that the usage of thermolysin, an enzyme working at the natural pH of the cell for membrane shaving, is superior because: (i) we detect a similar number of peptides and proteins using thermolysin and trypsin; (ii) thermolysin shaving can be run at a natural pH and (iii) the incubation time is quite short. (iv) Fewer detected peptides from thermolysin shaving originate from the transmembrane regions. Using thermolysin shaving we can also provide a clear separation between the best and the less accurate topology predictors, indicating that using data from shaving can provide valuable information when developing new topology predictors.
Asunto(s)
Proteínas de la Membrana/química , Proteómica/métodos , Termolisina/química , Endopeptidasa K/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Espectrometría de Masas , Proteínas de la Membrana/análisis , Pepsina A/química , Péptidos/análisis , Péptidos/química , Conformación Proteica , Estructura Terciaria de Proteína , Proteoma/análisis , Proteoma/química , Tripsina/químicaRESUMEN
Approximately 25% of eukaryotic proteins possessing homology to at least two transmembrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme α-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes.
Asunto(s)
Cerebelo/química , Proteínas de la Membrana/análisis , Neuronas/química , Proteínas PrPC/deficiencia , Proteoma/análisis , Secuencia de Aminoácidos , Animales , Células Cultivadas , Cerebelo/citología , Cerebelo/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteínas de la Membrana/clasificación , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Neuronas/metabolismo , Fragmentos de Péptidos/análisis , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteómica , Reproducibilidad de los Resultados , Espectrometría de Masas en TándemRESUMEN
Nontransient hypoxia is strongly associated with malignant lesions, resulting in aggressive behavior and resistance to treatment. We present an analysis of mRNA and protein expression changes in neuroblastoma cell lines occurring upon the transition from normoxia to hypoxia. The correlation between mRNA and protein level changes was poor, although some known hypoxia-driven genes and proteins correlated well. We present previously undescribed membrane proteins expressed under hypoxic conditions that are candidates for evaluation as biomarkers.
Asunto(s)
Biomarcadores/química , Membrana Celular/química , Hipoxia/metabolismo , Proteínas de la Membrana/química , Biomarcadores/metabolismo , Línea Celular Tumoral , Humanos , Espectrometría de Masas/métodos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Análisis por Micromatrices , Neoplasias/química , Neoplasias/metabolismo , Neoplasias/patología , ARN Mensajero/metabolismo , Electroforesis Bidimensional Diferencial en Gel/métodosRESUMEN
We present a mass spectrometry-based method for the identification and quantification of membrane proteins using the low-specificity protease Proteinase K, at very high pH, to digest proteins isolated by a modified SDS-PAGE protocol. The resulting peptides are modified with a fragmentation-directing isotope labeled tag. We apply the method to quantify differences in membrane protein expression of Bacillus subtilis grown in the presence or absence of glucose.