RESUMEN
An innovative sample preparation strategy is reported for protein identification in complex mixtures based on integration of affinity chromatographic selection and accelerated trypsin digestion using a continuous flow immobilized enzyme reactor (cf-IMER). Affinity selected glycoproteins were released to a cf-IMER column which converted native proteins to peptides in 5 min at elevated temperature. Digestion with the cf-IMER was compared to the traditional 16 h solution-based trypsin digestion of reduced and alkylated proteins. With immobilized antibody selection of Lewis x (Le(x)) glycan bearing glycoproteins from plasma, 66 proteins were identified in total with the two methods while approximately 1/3 of the total proteins and peptides were only observed with the cf-IMER. This suggests that proteomics based on protein identification by reduction and alkylation with solution-based trypsin digestion alone may not be identifying large numbers of proteins or peptides present at detectable levels in samples. Furthermore, except for proteins containing a high content of disulfide bonds, the majority of proteins did not require reduction and alkylation steps for their identification. The validity of the proposed proteolysis was evaluated in several ways by analyses of a model protein and yeast lysates where the reproducibility of quantification was essentially the same with both cf-IMER and solution-based proteolysis.
Asunto(s)
Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/genética , Proteómica/métodos , Secuencia de Aminoácidos , Enzimas Inmovilizadas/análisis , Enzimas Inmovilizadas/genética , Humanos , Datos de Secuencia Molecular , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
The intestinal epithelia form the first line of defense against harmful agents in the gut lumen of most monogastric vertebrates, including teleost fishes. Previous investigations into the effect of starvation on the intestinal epithelia of teleost fishes have focused primarily on changes in morphological characteristics and targeted molecular analysis of specific enzymes. The goal of this study was to use a comprehensive approach to help reveal how the intestinal epithelia of carnivorous teleost fishes acclimate to short-term nutrient deprivation. We utilized two-dimensional gel electrophoresis (2-DE) to conduct the proteomic analysis of the mucosal and epithelial layer of the anterior gut intestinal tract (GIT) from satiation fed vs. 4 week starved rainbow trout (Oncorhynchus mykiss). A total of 40 proteins were determined to be differentially expressed and were subsequently picked for in-gel trypsin digestion. Peptide mass fingerprint analysis was conducted using matrix assisted laser desorption time-of-flight/time-of-flight. Nine of the 11 positively identified proteins were directly related to innate immunity. The expression of α-1 proteinase inhibitor decreased in starved vs. fed fish. Also, the concentration of one leukocyte elastase inhibitor (LEI) isomer decreased in starved fish, though the concentration of another LEI isomer increased in due to starvation. In addition, starvation promoted an increased concentration of the important xenobiotic-transporter p-glycoprotein. Finally, starvation resulted in a significant increase in type II keratin E2. Overall, our results indicate that starvation promoted a reduced capacity to inhibit enzymatic stress but increased xenobiotic resistance and paracellular permeability of epithelial cells in the anterior intestine of rainbow trout.