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Identification of novel angiotensin‐converting enzyme‐inhibitory peptides from ovine milk proteins by CE‐MS and chromatographic techniques
Author(s) -
GómezRuiz José Ángel,
Ramos Mercedes,
Recio Isidra
Publication year - 2007
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200700324
Subject(s) - chemistry , chromatography , hydrolysate , tripeptide , trypsin , pepsin , casein , peptide , chymotrypsin , enzyme , hydrolysis , fast protein liquid chromatography , biochemistry , beta lactoglobulin , proteolysis , high performance liquid chromatography , whey protein
In this report, we present the use of CE‐MS as complement to RP separation for the identification of novel angiotensin‐converting enzyme‐inhibitory (ACEI) peptides from a complex milk protein hydrolysate. As preliminary step, fast protein LC (FPLC) was used to isolate the different casein fractions from raw ovine milk. Enzymatic hydrolysis of these fractions was performed by using proteolytic enzymes of gastrointestinal origin. The most active hydrolysate, corresponding to κ‐casein hydrolyzed with pepsin, chymotrypsin, and trypsin, was fractionated by RP‐HPLC and the peptides contained in the active fractions were sequenced by CE coupled to IT‐MS (CE‐MS). The use of CE‐MS allowed the identification of short peptides with ACEI activity included in the scarcely retained fraction obtained by semipreparative RP‐HPLC. Among the identified peptides, those with hydrophobic or positively charged residues at the C ‐terminal tripeptide were chemically synthesized to determine their ACEI activity. This procedure allowed us to identify four novel potent ACEI peptides from κ‐casein with sequences IAK, YQQRPVA, WQVLPNAVPAK, and HPHPHLSF. These active sequences could be obtained by enzymatic hydrolysis either of the individual κ‐casein fraction or the total casein fraction from ovine milk.