Two‐dimensional gel electrophoresis pattern (pH 6–11) and identification of water‐soluble barley seed and malt proteins by mass spectrometry

Abstract

Abstract A protocol was established for two‐dimensional gel electrophoresis (2‐DE) of barley seed and malt proteins in the pH range of 6–11. Proteins extracted from flour in a low‐salt buffer were focused after cup‐loading onto IPG strips. Successful separation in the second dimension was achieved using gradient gels in a horizontal SDS‐PAGE system. Silver staining of gels visualized around 380 (seed) and 500 (malt) spots. Thirty‐seven different proteins from seeds were identified in 60 spots, among these 46 were visualized also in the malt 2‐D pattern. Proteins were identified by peptide mass fingerprinting and by tandem MS sequencing after in‐gel digestion by trypsin. In addition, the N ‐terminal sequence of 10 different proteins from 11 spots was determined after electroblotting to a polyvinylidene difluoride (PVDF) membrane. Five identified proteins (in 9 spots) are involved in glycolysis, 12 in defence against pathogens (21 spots), 4 in storage, folding, and synthesis of proteins, and in nitrogen metabolism (5 spots), 6 in carbohydrate metabolism (11 spots), and 4 in stress and detoxification (9 spots). Six proteins (7 spots) were not grouped in these categories, and 3 were not ascribed a function. The presented 2‐D patterns and identifications will be used to describe proteome differences between cultivars and changes during malting.