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Kappa-casein-derived caseinomacropeptide (CMP)—a 64-amino-acid peptide—is released from kappa-casein after rennet treatment and is one of the major peptides in whey protein isolate (WPI). CMP has anti-inflammatory and antibacterial activities. It also has two major amino acid sequences with different modifications, including glycosylation, phosphorylation, and oxidation. To understand the potential biological role of CMP within the human body, there is a need to examine the extent to which CMP and CMP-derived fragments survive across the digestive tract, where they can exert these functions. In this study, three solid-phase extraction (SPE) methods—porous graphitized carbon (PGC), hydrophilic interaction liquid chromatography (HILIC), and C18 chromatography—were evaluated to determine which SPE sorbent is the most efficient to extract intact CMP and CMP-derived peptides from WPI and intestinal digestive samples prior to LC-MS/MS acquisition. The C18 SPE sorbent was the most efficient in extracting intact CMP and CMP-derived peptides from WPI, whereas the PGC SPE sorbent was the most efficient in extracting CMP-derived peptides from intestinal digesta samples.

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.