An optimized method for the isolation and identification of membrane proteins

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Abstract
The purpose of this study was to develop a protocol suitable for membrane protein extraction from limited starting material and to identify appropriate conditions for two‐dimensional (2‐D) gel electrophoresis. We used A549 cells, a human alveolar type II cell line, and evaluated three protein extraction methods based on different separation principles, namely protein solubility, detergent‐based and density‐based organelle separation. Detergent‐based extraction achieved the highest yield with 14.64% ± 2.35 membrane proteins but sequential extraction with 7.35% ± 0.78 yield and centrifugal extraction with 4.1% ± 0.54 yield produced the purest fractionation of membrane proteins. Only the sequential and the detergent‐based extraction proved suitable for small volumes of starting material. We identified annexin I + II, electron transfer flavoprotein β‐chain, H+‐transporting ATP synthase, mitofilin and protein disulfide isomerase A3 as membrane and cytokeratin 8 + 18, actin and others as soluble proteins using matrix assisted laser desorption/ionization‐time of flight (MALDI‐TOF) analysis and started to map the A549 cell proteome. Our data suggest that membrane proteins can be extracted efficiently from small samples using a simple sequential protein extraction method. They can be separated and identified successfully using optimized conditions in 2‐D gel electrophoresis. The presented methods will be useful for further investigations of membrane proteins of alveolar and bronchial carcinomas.

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