Proteomic Profiling of Barley during Various Stages of Malting.

Proteins play a vital role in determining the quality of barley in malting and brewing end-uses. Only a few well characterized enzymes involved in carbohydrate metabolism are currently used as markers for testing malting quality. Proteomic analysis during the various malting stages would be a useful resource for identifying novel protein markers associated with barley malting quality. In this study we used shotgun proteomics to analyze the changes in the proteome of the two-rowed barley cultivar ÔConradÕ during the process of malting. Five different stages during malting were examined - end of steeping, 1, 3, 5 days after germination, and kilning. Three biological replicates of these six samples were analyzed. Precipitated proteins were reduced, alkylated and digested with trypsin. Trypsin-digested peptides were desalted by solid phase extraction using the C18 cartridges. Digested peptides were analyzed using HPLC coupled to tandem mass spectrometry on a hybrid linear ion trap mass spectrometer equipped with a nanoelectrospray ion source. MS spectra of tryptic digests were collected over an m/z range of 380-2400. Raw MS/MS data files from Xcalibur software were submitted to Proteome Discoverer software with the Mascot search engine for peptide/ protein identification. Scaffold software was used to validate MS/MS based peptide and protein identification. This analysis led to the identification of more than 1400 barley proteins during these five malting stages. At the end of the kilning process, the tiny roots (referred to as chits) are removed from the seeds before they get into mashing process. Proteomic analysis of the chits collected from germinating seeds and kilned samples led to identification of more than 1700 proteins. A detailed bioinformatic analysis of this time-course series proteomics data from various stages of malting and a comparative analysis of the seed versus the chit proteomes will be presented.