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

Simon Gregersen,Margarita Pertseva,Paolo Marcatili,Susan Løvstad Holdt,Charlotte Jacobsen,Pedro J García-Moreno,Egon Bech Hansen,Michael Toft Overgaard

doi:10.1016/j.algal.2021.102619

Simon Gregersen, Margarita Pertseva + Show 6 more

Open Access

https://doi.org/10.1016/j.algal.2021.102619

Copy DOI

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.

Highlights

Seaweeds are known to contain numerous compounds of interest, such as polysaccharides, proteins and other compounds with health beneficial properties such as anti-inflammatory, anti-oxidant, and anticancer [1,2]
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 pro teins
The transcript expression level was calculated in terms of transcripts per kilo megabase (TPM) and removed proteins with TPM below 100, which resulted in 1628 proteins retained for the database

Summary

Introduction

Seaweeds are known to contain numerous compounds of interest, such as polysaccharides, proteins and other compounds with health beneficial properties such as anti-inflammatory, anti-oxidant, and anticancer [1,2]. Eucheuma denticulatum (commercially referred to as “spinosum”) is among the most cultivated and harvested red seaweed species for the carrageenan industry. The most common industrial method to obtain carra geenan from E. denticulatum uses hot water at high pH (>12). If other compounds, such as protein, could be isolated prior to or as part of the industrial hot water extraction without compromising the yield and quality of carrageenan, this could be of interest, as the amount of available biomass is substantial. Proteins from E. denticulatum were shown to constitute only 3.8% of dry biomass, but were of high quality with respect to their amino acid profile and nutritional quality [4]. The protein is comparable to beef in regards to content of branched chained amino acids (i.e. leucine, isoleucine, and valine), which are of specific interest due to their diverse metabolic and physiological func tions [5]

Objectives

Methods

Results

Conclusion