A Fast and Accurate Method to Identify and Quantify Enzymes in Brush-Border Membranes: In Situ Hydrolysis Followed by Nano LC-MS/MS

Instituto Multidisciplinario de Investigaciones Biológicas de San Luis (IMIBIO-SL), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), 5700 San Luis, Argentina

Facultad de Ciencias de la Salud. Universidad Nacional de San Luis, 5700 San Luis, Argentina

Department of Zoology and Physiology, University of Wyoming, Laramie, WY 82071, USA

⁴

Mass Spectrometry and Proteomics Facility, Biotechnology Center, University of Wisconsin-Madison, WI 53706, USA

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Department of Forest and Wildlife Ecology, University of Wisconsin-Madison, Madison, WI 53706, USA

⁶

Departamento de Biología, Universidad Nacional de San Luis (UNSL), 5700 San Luis, Argentina

Author to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Methods Protoc. 2020, 3(1), 15; https://doi.org/10.3390/mps3010015

Received: 7 December 2019 / Revised: 2 February 2020 / Accepted: 3 February 2020 / Published: 10 February 2020

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Abstract

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 the brush border, can be semi-quantified using nano LC-MS/MS. View Full-Text

Keywords: α-glucosidase; polyacrylamide gels; detection; proteomics; activity

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Brun, A.; Magallanes, M.E.; Martínez del Rio, C.; Barrett-Wilt, G.A.; Karasov, W.H.; Caviedes-Vidal, E. A Fast and Accurate Method to Identify and Quantify Enzymes in Brush-Border Membranes: In Situ Hydrolysis Followed by Nano LC-MS/MS. Methods Protoc. 2020, 3, 15. https://doi.org/10.3390/mps3010015

AMA Style

Brun A, Magallanes ME, Martínez del Rio C, Barrett-Wilt GA, Karasov WH, Caviedes-Vidal E. A Fast and Accurate Method to Identify and Quantify Enzymes in Brush-Border Membranes: In Situ Hydrolysis Followed by Nano LC-MS/MS. Methods and Protocols. 2020; 3(1):15. https://doi.org/10.3390/mps3010015

Chicago/Turabian Style

Brun, Antonio; Magallanes, Melisa E.; Martínez del Rio, Carlos; Barrett-Wilt, Gregory A.; Karasov, William H.; Caviedes-Vidal, Enrique. 2020. "A Fast and Accurate Method to Identify and Quantify Enzymes in Brush-Border Membranes: In Situ Hydrolysis Followed by Nano LC-MS/MS" Methods Protoc. 3, no. 1: 15. https://doi.org/10.3390/mps3010015

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A Fast and Accurate Method to Identify and Quantify Enzymes in Brush-Border Membranes: In Situ Hydrolysis Followed by Nano LC-MS/MS

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