Identification of novel angiotensin-converting enzyme-inhibitory peptides from ovine milk proteins by CE-MS and chromatographic techniques

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...

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Bibliographic Details
Published in:Electrophoresis 2007-11, Vol.28 (22), p.4202-4211
Main Authors: Gómez-Ruiz, José Ángel, Ramos, Mercedes, Recio, Isidra
Format: Article
Language:English
Subjects:
ACE-inhibitory peptides
Amino Acid Sequence
Angiotensin-Converting Enzyme Inhibitors - analysis
Angiotensin-Converting Enzyme Inhibitors - isolation & purification
Animals
Antihypertensive activity
Caseins
Chromatography
Electrophoresis, Capillary
HPLC
Hydrolysis
Milk Proteins - analysis
Milk Proteins - isolation & purification
Peptide Hydrolases - metabolism
Peptides - analysis
Peptides - isolation & purification
Sheep, Domestic
Tandem Mass Spectrometry
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Summary: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.
ISSN:0173-0835
1522-2683
DOI:10.1002/elps.200700324