A fast and accurate method to identify and quantify enzymes in brush-border membranes: In situ hydrolysis followed by nano LC-MS/MS
- Brun, Antonio
- Magallanes Alba, Melisa Eliana
- Martínez del Rio, Carlos
- Barrett Wilt, Gregory
- Karasov, William H.
- Caviedes Vidal, Enrique Juan Raul
DOIAbstract
A simple method for the identification of brush-border membrane α-glucosidases is described. The proteins were first solubilized and separated in a gel under native, non-denaturing, conditions. The gel was then incubated in substrate solutions (maltose or sucrose), and the product (glucose) exposed in situ by the oxidation of o-dianisidine, which yields a brown-orange color. Nano-liquid chromatography coupled to mass spectrometry analyses of proteins (nano LC-MS/MS) present in the colored bands excised from the gels, was used to confirm the presence of the enzymes. The stain is inexpensive and the procedure permits testing several substrates in the same gel. Once enzymes are identified, their abundance, relative to that of other proteins in...
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