Fine-tuned method to extract high purified proteins from the seagrass Halophila stipulacea to be used for proteome analyses

Abstract

The non-indigenous to the Mediterranean tropical seagrass Halophila stipulacea has the possibility to become more prevalent in the Mediterranean basin, exacerbated by the rapid increase of water temperature. Molecular profiling appears a promising tool to study the traits that render H. stipulacea tolerant and resilient and facilitate its rapid and vast geographical spread. Taking advantage from recent seagrass genomes sequencing, proteomics specialty has been applied to several seagrasses giving new insight on the biology and physiology of this group of angiosperms. Thus, it could be of interest to apply proteomics to H. stipulacea that it could be considered as a possible plant model species to study marine biological invasion. The first step to achieve this goal is to obtain high quality proteins from plant tissue. Tissue fixation and protein extraction protocol are the most challenging steps in proteomics . Here we report a fine-tuned procedure obtained by comparing protein yield from H. stipulacea plants frozen in liquid nitrogen or preserved in RNAlater and processed following two different extraction protocols. Higher protein yield have been extracted from the procedure that use the RNAlater preserved plants, extracted with trichloroacetic acid in water followed by trichloroacetic acid in acetone, compared to those obtained from all other procedures. Protein purity of these samples have been tested by the separation in SDS-PAGE comfirming a better resolved profile of peptide bands suitable for a gel-based proteomics. Then, to assess the quality of proteins the mHPLC-ESI-MS/MS mass spectrometry analyses and bioinformatics have been performed.Hundreds proteins have been identified against several seagrass genomic resources available at UniProt, NCBI, SeagrassDB and transcriptomic datasets, which were merged to form the first customized dataset useful for H. stipulacea proteomic investigations.

Seagrass Omics: How to Short the Workflow for Protein Expression Analyses

“…Since the last decade, proteomic technologies have been successfully applied to marine plants to obtain insights into their adaptive capacity and gene expression plasticity against environmental threats [1][2][3][4][5][6][7]. In seagrasses several species-specific multistep workflows for free-label differential protein expression have been optimized to obtain high quality protein samples [8][9][10]; these protocols were routinely coupled with the tryptic in-gel digestion as this method offers the advantage to remove any further contaminants (e.g., detergents, salts) during electrophoresis. The generated peptides can be, then, readily analysed by LC-MS/ MS to obtain the peptide mass fingerprinting.…”