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

Background Bovine milk κ-casein-derived caseinomacropeptide (CMP) is produced in large quantities during cheesemaking 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 which is partially glycosylated survives intact to its presumed site of bioactivity within the gut remains unknown. Objective 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. Methods Jejunal fluids were collected from three adult participants (two men and one woman, age: 27 ± 7, BMI: 23 ± 1) for three hours 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 mass spectrometry-based top-down glycopeptidomics. Relative abundances of CMP and CMP-derived peptides were compared qualitatively between the feed and the jejunal fluids. Results Intact CMP was dominant in feeding material, accounting for 90% of the total ion abundance of detected peptides, and in very low abundance (less than 2%) in the jejunal fluids. CMP-derived fragment peptides ranging from 11-20 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. Conclusions 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 that of intact CMP.