Two-dimensional profiling and peptide sequencing of angiotensin-I converting enzyme (ACE) inhibitory proteolysate from winged bean (Psophopcarpus tetragonolobus) seeds

Winged bean seed (WBS) is an underutilised tropical leguminous crop that represents a green, sustainable source of plant protein. The potential of WBS to produce biofunctional peptides, particularly ACE-inhibitory peptide, remains unexplored. In the current work, the seeds were enzymatically proteolysed using papain, and the proteolysate was separated via a two-step mechanism (reverse-phase high performance liquid chromatography and isoelectric focusing). A peptide search was conducted via sequencing using liquid chromatography with tandem mass spectrometry. Three subfractions from isoelectric focusing, namely F-12-12 (ACE inhibition rate = 87.8%; peptide content = 1.0 mM; isoelectric point, pI = 10.0), F-16-2 (ACE inhibition rate = 50.1%; peptide content = 0.4 mM; pI = 3.6) and F-16-6 (ACE inhibition rate = 77.3%; peptide content = 0.9 mM, pI = 6.8) were selected for sequencing. Based on the structural requirements for a peptide to exhibit ACE-inhibitory properties, as well as the confidence score obtained during sequencing, a total of eleven peptides (TSISSEDKTPPQPRN, ISSEDKTPPQPR, SEDKTPPQPR, SISSEDKTPPQPR, ADQLDTAR, DVKERAKDYG, TASSVEEAT, ERAKDYG, STNPIT, RGVFPCLK, TQLDLPTQ) and four peptides (LSSEDKTP, EPALVP, MRSVVT, DMKP) were successfully identified upon a database search and de novo sequencing, respectively. All peptides were oligopeptides, with low molecular weights of <2 kDa (specifically, 489.9-1656.7 Da) and contained 4-15 amino acid residues. An in-depth technical interpretation of the sequencing parameters was subsequently elucidated. In short, the current work demonstrates the potential of the sustainable plant protein source WBS to produce ACE-inhibitory peptides, which could ultimately aid in blood pressure regulation, and concurrently sheds light on the technical fundamentals of peptide separation and sequencing.