An approach for vacuole isolation and vacuolar protein identification by proteomics in apple

Apple, one of the most common fruits consumed worldwide, has a high economic value. Vacuoles are membrane-bound organelles in plant cells that perform many important functions. An increasing number of studies have reported a variety of proteins in a vacuole, with crucial functions in the transport, storage and metabolism of energy and substances. Here, we successfully extracted the vacuoles and vacuolar proteins from the ‘Orin’ apple fruit calli. We collected intact vacuoles with a density of 100/mm2 by mild osmotic lysis and Ficoll density gradient centrifugation. The result of SDS-PAGE showed that lots of vacuolar proteins were isolated and enriched from vacuole samples, and western blotting showed that the purity of the vacuolar protein was high. Furthermore, we showed GO enrichment and KEGG enrichment of isolated vacuolar proteins and selected 41 vacuolar proteins with known functions and 10 vacuolar proteins with unknown functions for functional annotation from the mass spectrometry analysis. This approach for vacuole isolation and vacuolar protein identification will provide a theoretical basis for further study of the function of vacuoles and vacuolar proteins.