Fine-tuned Method to Extract High Purified Proteins from the Seagrass Halophila Stipulacea to Be Used for Proteome Analyses

We report a fine tuned procedure to obtain high proteins yield and quality from the marine plant Halophila stipulacea. Two tissue fixation methods have been applied; sampled plants were preserved in RNAlater or frozen in liquid nitrogen. Both fixed plants have been processed following three different protein extraction protocols; we used a protocol optimized in this work, reported as Procedure 1 in which very small amount of tissue were used for the extraction in trichloroacetic acid/water, followed by trichloroacetic acid/acetone. The second protocol, reported as Procedure 2, is the well-established protocol developed for P. oceanica; following this protocol, proteins have been extracted from fivefold tissue amount than Procedure 1, without the trichloroacetic acid/acetone step. In the Procedure 3 the reverse approach between sample fixation and extraction protocols used in the previous two procedures have been applied. The lowest yield of 2.49 +/- 0.18 mg/g of tissue is obtained from RNAlater preserved plants processed with the Procedure 2, while the highest protein yield of 5.88 +/- 0.22 mg/g is obtained from the RNAlater preserved plants processed with the Procedure 1. These last protein samples gave the best resolved profile of peptide bands in SDS-PAGE showing higher proteins purity than those in all the other samples. The gel-based proteomics approach by the uHPLC-mu ESI-MS/MS analyses and bioinformatics against a customized dataset of genomic and transcriptomic seagrass sequences have been performed. Hundreds of proteins were identified from the Procedure 1 samples; lesser protein identification was obtained from Procedure 2 samples and no significant identifications have been detected from Procedure 3 samples. Statistics of classes of proteins that were identified in each procedures were also reported.