Enrichment and identification of integral membrane proteins from barley aleurone layers by reversed-phase chromatography, SDS-PAGE and LC-MS/MS

Radovan Hynek, Birte Svensson, Ole Nørregaard Jensen, Vibeke Barkholt, Christine Finnie

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    The plasma membrane of the cereal aleurone layer is the site of perception of germination signals and release of enzymes to the starchy endosperm. Analysis of membrane proteins is challenging due to their hydrophobicity and low abundance, thus little is known about the membrane proteins involved in seed germination. A membrane fraction highly enriched for the plasma membrane H+-ATPase was prepared from barley aleurone layers by aqueous two-phase partitioning. Since detergent and salt washes did not efficiently remove soluble proteins from the membrane preparations, an alternative procedure was developed, comprising batch reversed-phase chromatography with stepwise elution of hydrophobic proteins by 2-propanol. Proteins in the most hydrophobic fraction were separated by SDS-PAGE and identified by LC-MS/MS and barley EST sequence database search. The method was efficient for enrichment of integral membrane proteins with relatively low levels of soluble contaminating proteins. Forty-six proteins associated with barley aleurone plasma membranes were identified, including proteins with more than 10 transmembrane domains. Among the identified proteins were two new isoforms of the plasma membrane H+-ATPase, two proteins possibly involved in ion-channel regulation and two proteins of unknown function. This represents the first analysis of membrane proteins involved in seed germination, using a proteomics approach.
    Original languageEnglish
    JournalJournal of Proteome Research
    Volume5
    Pages (from-to)3105-3113
    Publication statusPublished - 2006

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