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

Abstract

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-µESI-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.

Supplemental data for this article is available online at https://doi.org/10.1080/11263504.2021.2020355 .

Keywords:

• Halophila stipulacea
• tissue fixation
• protein extraction
• seagrass proteomics

Acknowledgments

This research was co-financed by Greece and the European Union (European Social Fund- ESF) through the Operational Programme ‘Human Resources Development, Education and Lifelong Learning 2014–2020’ in the context of the project ‘I-ADAPT’ (MIS 5006611). Authors sincerely thank Gaurav Sablok and Regan Hayward who provided sequences of seagrass transcriptomes stored at SeagrassDB. We thank Julius Glampedakis and Thanos Dailianis for helping during field work. The authors thank also the project PON “Sistema Integrato di Laboratori per l’Ambiente (S.I.L.A.) – PON a3_00341A for the availability of the mass spectrometer equipments.

Disclosure statement

No potential conflict of interest was reported by the authors.