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The protein composition of barley grains has been studied systematically for over a century, and barley grain proteins have been studied by two-dimensional gel electrophoresis for more than two decades. Mass spectrometry enables identification of proteins in complex mixtures and identification and characterization of their post-translational modifications, making it the cornerstone of proteome analysis. Recently, the experimental system based on isolated barley aleurone layers was exploited to gain more insight into the protein secretory system responsible for secretion of hydrolytic enzymes into the starchy endosperm during malting. Using proteomics, secretory hydrolases can be identified both within the aleurone layers and after their release to the incubation medium. Many proteins are N-glycosylated in the secretory pathway, but little is yet known about the post-translational modifications of grain enzymes. A glycopeptide-enrichment strategy enabled identification of 73 glycosylation sites in 65 proteins, a significant improvement in characterization of the barley glycoproteome. The proteins in mature cereal grains are major factors influencing quality. However grains are also colonized by complex microbial communities that actively interact with the plant via e.g. secretion of enzymes for breakdown of cell wall components. These may influence grain quality and be a source of enzymes for exploitation in grain processing. Analysis of surface-associated proteomes of barley grains enabled identification of 53 proteins of bacterial or fungal origin including cell-wall degrading enzymes such as xylanases, in agreement with microbial xylanase activity isolated from the grain surface. Ongoing efforts to characterise grain proteomes and post-translational modifications will provide new targets for functional analysis and improvement of grain quality.