Proteome analysis of barley seeds: Identification of major proteins from two-dimensional gels (pI 4–7) Ole Østergaard 1 , Christine Finnie*, Sabrina Laugesen 2 , Peter Roepstorff 2 and Birte Svensson* 1 Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark 2 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark Germination of monocotyledonous plants involves activation and de novo synthesis of enzymes that degrade cell walls and starch and mobilize stored endosperm reserves for embryo growth. Two-dimensional (2-D) gel electrophoresis and mass spectrometry were applied to identify major water-soluble proteins in extracts of mature barley (Hordeum vulgare) seeds and to follow their fate during germination. About 1200 and 600 spots of pI 4–7 were detected on 2-D gels by silver staining and colloidal Coomassie Brilliant Blue staining, respec- tively. About 300 spots were selected for in-gel digestion followed by matrix-assisted laser desorption/ionization-mass spectrometry-peptide map fingerprint analysis. Database searches using measured peptide masses resulted in 198 identifications of 103 proteins in 177 spots. These include housekeeping enzymes, chaperones, defence proteins (including enzyme inhibitors), and proteins related to desiccation and oxidative stress. Sixty-four of the identifications were made using expressed sequence tags (ESTs). Numerous spots in the 2-D gel pattern changed during germination (micromalting) and an intensely stained area which contained large amounts of the serpin protein Z appeared centrally on the 2-D gel. Spots con- taining a-amylase also appeared. Identification of 22 spots after three days of germination represented 13 different database entries and 11 functions including hydrolytic enzymes, chaperones, housekeeping enzymes, and inhibitors. Keywords: Barley / Malt / Protein identification / Proteome analysis / Two-dimensional gel electrophoresis Received 9/12/03 Accepted 16/1/04 Proteomics 2004, 4, 2437–2447 2437 1 Introduction Cereal seed proteins are of importance in the brewing industry, for human and animal nutrition, plant breed- ing, and cultivar identification. Early studies of proteins extracted from seeds include starch gel electrophoresis to detect isozyme variations in barley, Hordeum vulgare [1], separation of barley hordeins on lactate polyacryl- amide gel electrophoresis [2], and analysis of hordeins and glycoproteins by SDS-PAGE or isoelectric focusing (IEF) for cultivar identification [3, 4]. Two-dimensional gel electrophoresis with IEF in the first- and SDS- PAGE in the second dimension allows improved resolu- tion of proteins in complex seed extracts with high re- producibility. This methodology was used for analysis of extracts from wheat, Triticum aestivum [5], maize, Zea mays [6], and barley [7, 8]. Today, proteomics, including high resolution 2-D gel electrophoresis, in-gel proteolytic digestion of protein spots and protein iden- tification by mass spectrometry and database searches, is increasingly used to address questions of development, physiology and quality in major crop plants Correspondence: Dr. Birte Svensson, Biochemistry and Nutri- tion Group, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark E-mail: bis@biocentrum.dtu.dk Fax: 145-4588-6307 Abbreviations: BMAI, barley monomeric a-amylase inhibitor; cCBB, colloidal Coomassie Brilliant Blue; CM-proteins, chloro- form-methanol-soluble proteins; DMAS, deoxymugineic acid synthase; LEA protein, late embryogenesis abundant protein; PDI, protein disulfide isomerase; sHSP , small heat shock protein; SOD, superoxide dismutase; TC sequence, tentative consensus sequence Supporting information for this article (Table 1) is available on the WWW under www.proteomics-journal.de or from the author. * Current address: Biochemistry and Nutrition Group, BioCentrum- DTU, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de DOI 10.1002/pmic.200300753