Shotgun proteomics of the barley seed proteome

Abstract

BackgroundBarley seed proteins are of prime importance to the brewing industry, human and animal nutrition and in plant breeding for cultivar identification. To obtain comprehensive proteomic data from seeds, total protein from a two-rowed (Conrad) and a six-rowed (Lacey) barley cultivar were precipitated in acetone, digested in-solution, and the resulting peptides were analyzed by nano-liquid chromatography coupled with tandem mass spectrometry.ResultsThe raw mass spectra data searched against Uniprot’s Barley database using in-house Mascot search engine identified 1168 unique proteins. Gene Ontology (GO) analysis indicated that the majority of the seed proteins were cytosolic, with catalytic activity and associated with carbohydrate metabolism. Spectral counting analysis showed that there are 20 differentially abundant seed proteins between the two-rowed Conrad and six-rowed Lacey cultivars.ConclusionThis study paves the way for the use of a top-down gel-free proteomics strategy in barley for investigating more complex traits such as malting quality. Differential abundance of hordoindoline proteins impact the seed hardness trait of barley cultivars.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3408-5) contains supplementary material, which is available to authorized users.

Temporal Analyses of Barley Malting Stages Using Shotgun Proteomics

“…Previously we have reported on the barley seed proteome using the same Conrad cultivar . Of the 1168 proteins identified in the seeds, more than 75% were also identified in the Steep sample.…”

“…A careful analysis of the published proteomic studies using 2DE in barley and other cereals did not report on transport proteins . We speculate that the protein isolation procedures that were employed in this analysis were amenable for extracting membrane proteins and further supported by identification of a large number of transmembrane proteins in the barley seeds using the same strategy . Given that protein synthesis is rampant during malting, enrichment of transporters is important to ensure that the synthesized proteins reach their appropriate destinations to carry out their biological functions.…”

“…Approximately one gram of barley seeds (12–15 seeds) from each stage were placed in a precooled mortar and ground to a fine powder with pestle. Protein isolations, enzymatic “in Liquid” digestion and NanoLC‐MS/MS were conducted as described earlier . The protein pellet was solubilized in a urea buffer pH 8.5 (8 mol L −1 urea in 50 mmol L −1 NH 4 HCO 3 ) using 100 μL of buffer mg −1 weight of pellet.…”

“…Enzymatic “In Liquid” Digestion : Extracted seed protein (200 μg) was TCA/acetone precipitated (9% TCA, 28% acetone final concentration) and the pellet resolubilized and denatured in 30 μL of 8 m urea / 50 m m NH 4 HCO 3 (pH 8.5) / 1 m m Tris–HCl for 5 min. Subsequent reduction, alkylation, and tryptic digestion were conducted as described earlier . Fifty micrograms of digested proteins (1/4th digestion volume) were cleaned up using OMIX C18 SPE cartridges (Agilent, Palo Alto, CA) per manufacturer protocol and eluted in 20 μL of 60/40/0.1% ACN/H 2 O/TFA, dried to completion in the speedVac and finally reconstituted in 50 μL of 0.1% formic acid.…”

“…Recent advances in gel‐free proteomics have not been exploited for understanding barley malting. We have recently reported on a shotgun proteomics analysis that led to the identification of more than 1100 barley seed proteins . In this study, we have applied this strategy to examine changes in the proteome during five different malting stages of a two‐rowed spring barley with good malting quality traits.…”