Analysis of Bovine Kappa-Casein Glycomacropeptide by Liquid Chromatography-Tandem Mass Spectrometry.

Yunyao Qu,Jeewon Koh,David C. Dallas,Bum-Jin Kim

doi:10.3390/foods10092028

Abstract

Caseinomacropeptide (CMP) is released from bovine kappa-casein after rennet treatment and is one of the major peptides in whey protein isolate. CMP has in vitro anti-inflammatory and antibacterial activities. CMP has two major amino acid sequences with different modifications, including glycosylation, phosphorylation and oxidation. However, no previous work has provided a comprehensive profile of intact CMP. Full characterization of CMP composition and structure is essential to understand the bioactivity of CMP. In this study, we developed a top-down glycopeptidomics-based analytical method to profile CMP and CMP-derived peptides using Orbitrap mass spectrometry combined with nano-liquid chromatography with electron-transfer/higher-energy collision dissociation. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) spectra of CMPs were annotated to confirm peptide sequence, glycan composition and other post-translational modifications using automatic data processing. Fifty-one intact CMPs and 159 CMP-derived peptides were identified in four samples (one CMP standard, two commercial CMP products and one whey protein isolate). Overall, this novel approach provides comprehensive characterization of CMP and CMP-derived peptides and glycopeptides, and it can be applied in future studies of product quality, digestive survival and bioactivity.

Highlights

Caseinomacropeptide (CMP) is a 64-amino-acid C-terminal fragment of bovine kappacasein that is released after rennet treatment
The fraction of CMP with glycosylation is referred to as glycomacropeptide, whereas CMP without glycosylation is referred to as aglycosylated CMP
Our top-down liquid chromatography (LC)–mass spectrometry (MS)/MS approach enabled the identification of intact aglycosylated CMP (aCMP) and gCMP and fragments of aCMP and gCMP

Summary

Introduction

Caseinomacropeptide (CMP) is a 64-amino-acid C-terminal fragment of bovine kappacasein that is released after rennet (chymosin) treatment. It is the third most abundant protein/peptide in cheese whey after β-lactoglobulin and α-lactalbumin and accounts for 20–25% of whey protein [1]. CMP has numerous in vitro bioactivities, including neutralization of enterotoxin [2], inhibition of bacterial and viral adhesion to Caco-2 cells [3], promotion of bifidobacterial growth [4] and modulation of the immune system response [5]. O-linked glycosylation (glycans linked to the oxygen atom of amino acids Thr or Ser) is the most common PTM in CMP. The only methionine of CMP can be oxidized to methionine sulfoxide [8]

Objectives

Methods

Results

Conclusion