The hordeins are the major barley seed storage proteins and are elicitors of coeliac disease - a condition that affects ~1% of the world population. There are four multi-genic hordein families in barley: B-hordeins representing 70-80% of total gluten; C-hordeins (10-20%); D-hordein (<5%); and, γ-hordeins (1-2%). Using conventional breeding a series of null lines were developed in which different classes or combinations of classes of hordeins are absent.
Data-independent acquisition (DIA) mass spectrometry (MS) was used to measure proteome-wide abundance differences between wild-type and selectively-bred hordein double-null barley lines. Data were acquired in information-dependent acquisition (IDA) and sequential window acquisition of all theoretical fragment-ion spectra-mass spectrometry (SWATH-MS) on a TripleTOF 6600 MS (SCIEX, USA). Statistical and functional analyses were performed on the proteins perturbed between wild-type and hordein double-null barley lines.
A total of 6,138 peptides mapping to 1,907 proteins were quantified at a 1% false discovery rate. Pairwise comparisons revealed proteome-wide alterations for BC-, BD- and CD-null lines in the order of ~16%, ~10% and ~14%, respectively. As an example, the comparison between wild-type and the BC-null line identified 151 up-regulated proteins (7.9%), while 145 (7.6%) proteins were down-regulated in the BC-null line. Contextualization within a protein-protein interaction network reveals the up-regulation of proteins associated with primary metabolism, transcription and enzymatic biosynthesis processes while down-regulation of heat shock proteins can be found in the BC-null line. Gene Ontology (GO) analysis (molecular function) revealed that enzymatic activities were up-regulated, whilst nutrient reservoir activities were down-regulated in the BC-null line.
Overall, the GO-based analysis provides an overview of the functional classes that are perturbed during the breeding process. Grain proteome profiling delivers an informative molecular portrait of the hordein double-null lines and the underlying storage protein biosynthesis mechanisms thereby shedding light on mechanism of proteome compensation.