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

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.

A novel late embryogenesis abundant protein and peroxidase associated with black point in barley grains

“…This study presented a link between the expression of stressrelated proteins including peroxidases and black point symptoms. To date several studies have analysed the proteome of the barley grain during grain fill and during micromalting [16][17][18], however, none have examined black point related protein expression.…”

Integration of the barley genetic and seed proteome maps for chromosome 1H, 2H, 3H, 5H and 7H

“…Proteins in 26 of the spots were already identified as part of ongoing barley seed proteome analyses (Bak-Jensen et al 2004Finnie et al 2002Finnie et al , 2004Finnie et al , 2006Finnie and Svensson 2003;Laugesen et al 2007;Østergaard et al 2002. Proteins in an additional 22 varying spots were successfully identified by mass spectrometry (Supplementary Table S2) resulting in the characterisation of 48 out of the 69 variable spots.…”

“…Proteins in 100 or 200 μL extract (100 or 200 μg protein) for pI 4-7 or 6-11 gels, respectively, were precipitated with 4 volumes of acetone at −20°C for at least 24 h. Precipitated proteins were redissolved in the appropriate IPG rehydration buffer prior to the first dimension, as previously described (Østergaard et al 2002;Bak-Jensen et al 2004). For separation of proteins in the pI ranges 4-7 and 6-11, immobilised pH gradient 18 cm IPG strips were run on an IPGphor unit or a Multiphor II, respectively (GE-Healthcare), and the second dimension SDS-PAGE gels (12-14%, 18×24 cm, GE-Healthcare) were run on a Multiphor II as previously described (Østergaard et al 2002;Bak-Jensen et al 2004). Gels were stained with silver nitrate (Heukeshoven and Dernick 1985).…”

A Review: Biological and Technological Functions of Barley Seed Pathogenesis-Related Proteins (PRs)

“…The mature barley seed proteome is dominated by proteins involved in stress response including Pathogenesis-Related proteins (PRs) 16,247 . PRs have captured the attention of the scientific community for 40 years, since they were discovered 103,194 , being extensively studied mostly from an agricultural perspective.…”

“…To enhance the studies on malt quality, scientists embarked on a proteome analysis approach for barley seeds and malt 21,248 . The barley seed proteome was determined recently 16,247 . Differences in the protein composition between barley varieties, as well as changes in seed protein composition during development and germination, and under different environmental conditions have been surveyed using proteomics 15,24,[86][87][88][89][90][91][92]202,247,248 .…”