Top-Down Glycopeptidomics Reveals Intact Glycomacropeptide Is Digested to a Wide Array of Peptides in Human Jejunum

Bovine milk κ-casein-derived caseinomacropeptide (CMP) is produced in large quantities during cheese-making and has various biological activities demonstrated via in vitro and in vivo experiments. Previous studies examined protein degradation and peptide release after casein or whey protein consumption. However, whether purified intact CMP that is partially glycosylated survives intact to its presumed site of bioactivity within the gut remains unknown.

The aim of this study was to determine the extent to which purified intact CMP (including glycosylated forms) is digested into peptide fragments within the jejunum of healthy human adults after consumption.

Jejunal fluids were collected from 3 adult participants (2 men and 1 woman, age: 27 ± 7 y; BMI: 23 ± 1 kg/m2) for 3 h after consuming 37.5 g of purified intact CMP. CMP and CMP-derived peptides were isolated from the collected jejunal fluids by ethanol precipitation and solid-phase extraction and identified by MS-based top-down glycopeptidomics. Relative abundances of CMP and CMP-derived peptides were compared qualitatively between the feed and the jejunal fluids.

Intact CMP was dominant in feeding material, accounting for 90% of the total ion abundance of detected peptides, and in very low abundance (<2%) in the jejunal fluids. CMP-derived fragment peptides ranging from 11 to 20 amino acids in length were predominant (accounting for 68-88% of the total peptide ion abundance) in jejunal fluids during 1-3 h post consumption.

This study demonstrates that intact CMP (including glycosylated forms) is mostly digested in the human jejunum, releasing a wide array of CMP-derived peptide fragments. Some of the CMP-derived peptides with high homology to known bioactive peptides consistently survived across 3 h of digestion. Therefore, future research should examine the biological effects of the partially digested form-the CMP-derived fragments-rather than those of intact CMP.