Proteomic characterization of pilot scale hot-water extracts from the industrial carrageenan red seaweed Eucheuma denticulatum

Abstract

Seaweeds have a long history as a resource for polysaccharides/hydrocolloids extraction for use in the food industry due to their functionality as stabilizing agents. In addition to the carbohydrate content, seaweeds also contains a significant amount of protein, which may find application in food and feed. Here, we present a novel combination of transcriptomics, proteomics, and bioinformatics to determine the protein composition in two pilot-scale extracts from Eucheuma denticilatum (Spinosum) obtained via hot-water extraction. Although the quality of extracted protein appeared quite poor based on SDS-PAGE analysis, extracts were characterized by qualitative and quantitative proteomics using LC-MS/MS and a de-novo transcriptome assembly for construction of a suitable protein database. A novel concept of length-normalization for relative quantification of sub-optimal protein extracts with partial, non-specific digestion is introduced and validated against conventional methods for relative quantification of proteins. Despite a limited number of protein identifications due to poor protein quality, our data suggest that the majority of quantified protein in the extracts (>75%) is constituted by merely three previously uncharacterized proteins. Putative subcellular localization for the quantified proteins was determined by bioinformatic prediction using several predictors, and by correlating with the expected copy number from the transcriptome analysis, we find that the extracts appear highly enriched in extracellular proteins. This implies that the extraction method used predominantly extracts extracellular proteins, and thus appear ineffective for cellular disruption and subsequent release of intracellular proteins. Nevertheless, the highly abundant proteins may be potential substrates for targeted hydrolysis and release of bioactive peptides. Ultimately, this study highlight the potential of quantitative proteomics for characterization of alternative protein sources intended for use in foods and evaluating protein extraction process efficiency through novel combinations with bioinformatic analysis.