Peptidomic strategy for purification and identification of potential ACE-inhibitory and antioxidant peptides in Tetradesmus obliquus microalgae

• Published in: Analytical and bioanalytical chemistry Vol. 410; no. 15; pp. 3573 - 3586
• Main Authors: Montone, Carmela Maria, Capriotti, Anna Laura, Cavaliere, Chiara, La Barbera, Giorgia, Piovesana, Susy, Zenezini Chiozzi, Riccardo, Laganà, Aldo
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.06.2018; Springer; Springer Nature B.V
• Subjects: Algae; algorithms; Amino Acid Sequence; Analytical Chemistry; Angiotensin; Angiotensin-Converting Enzyme Inhibitors - analysis; Angiotensin-Converting Enzyme Inhibitors - isolation & purification; Angiotensin-Converting Enzyme Inhibitors - pharmacology; Animals; Antihypertensives; Antioxidants; Antioxidants - analysis; Antioxidants - isolation & purification; Antioxidants - pharmacology; Beads; bioactive properties; biochemical pathways; Biochemistry; Biological activity; Biological research; Cell walls; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chlorophyta - chemistry; Chromatography, Reverse-Phase - methods; Discovery of Bioactive Compounds; Food Science; glass; Glass beads; Hydrolysates; Identification; Laboratory Medicine; Liquid chromatography; Marine environment; Marine organisms; Microalgae; Microalgae - chemistry; milling; Monitoring/Environmental Analysis; Offshore structures; Peptides; Peptides - analysis; Peptides - isolation & purification; Peptides - pharmacology; Peptidyl-dipeptidase A; Physiological aspects; probability; Protein hydrolysates; Protein purification; Proteins; Proteomics - methods; protocols; Rabbits; Research Paper; reversed-phase liquid chromatography; Separation; Subtilisin; Tandem Mass Spectrometry - methods; Tetradesmus; tissues; Off-line two-dimensional chromatography; ACE-inhibitory peptides; High resolution mass spectrometry; Protein extraction methods; Antioxidant peptides; Microalgae; Peptidomics
• ISSN: 1618-2642; 1618-2650; 1618-2650
• DOI: 10.1007/s00216-018-0925-x

Microalgae are unicellular marine organisms that have promoted complex biochemical pathways to survive in greatly competitive marine environments. They could contain significant amounts of high-quality proteins which, because of their structural diversity, contain a range of yet undiscovered novel bioactive peptides. In this work, a peptidomic platform was developed for the separation and identification of bioactive peptides in protein hydrolysates. In this work, a peptidomic platform was developed for the extraction, separation, and identification of bioactive peptides in protein hydrolysates. Indeed, extraction of proteins from recalcitrant tissues is still a challenge due to their strong cell walls and high levels of non-protein interfering compounds. Therefore, seven different protein extraction protocols, based on mechanical and chemical methods, were tested in order to produce high-quality protein extracts. Proteins obtained by means of the best protocol, consisting of milling the recalcitrant tissue with glass beads, were subjected to enzymatic digestion with Alcalase® and subsequently the hydrolysate was purified by two-dimensional semi-preparative reversed phase liquid chromatography. Fractions were assayed for antioxidant and antihypertensive activities and only the most active ones were finally analyzed by RP nanoHPLC-MS/MS. Around 500 peptide sequences were identified in these fractions. The identified peptides were subjected to an in silico analysis by PeptideRanker algorithm in order to assign a score of bioactivity probability. Twenty-five sequenced peptides were found with potential antioxidant and angiotensin-converting-enzyme-inhibitory activities. Four of these peptides, WPRGYFL, GPDRPKFLGPF, WYGPDRPKFL, SDWDRF, were selected for synthesis and in vitro tested for specific bioactivity, exhibiting good values of antioxidant and ACE-inhibitory activity. Graphical abstract Workflow showing the entire peptidomic approach developed for identification of bioactive peptides in microalgae