Short communication: Application of proteomics for characterization of caseinomacropeptide isoforms before and after desialidation

摘要

Caseinomacropeptide (CMP) is an important polypeptidefound in cheese whey that has various biologicalactivities and functional properties. Because sialylationsplay an important role in the functional properties ofCMP, the aim of the present work was to characterizeCMP isoform heterogeneity in a commercial glycosylatedCMP (gCMP) isolate using liquid chromatography–and gel-based proteomics before and afterdesialidation. Using 2-dimensional gel electrophoresis(2-DGE), we observed a shift in isoelectric point (pI)after enzymatic desialidation, indicating that sialylatedgCMP were located at pI 3.0 to 3.1, but desialylatedgCMP had repositioned to pI 3.7 to 3.9. We used liquidchromatography/mass spectroscopy (LC-ESI/MS) forfurther analysis of the glycan chains of gCMP. In total,we identified 19 CMP isoforms, of which 13 were glycosylatedand 6 were nonglycosylated. We also detected3 genetic variants (A, B, and E), with up to 3 glycosylationsattached per gCMP. Further, we identified up to4 isomers, reflecting different sites of glycosylation inthe peptide backbone of these isoforms. The dominatinggCMP isoform of genetic variant E appeared tocontain 4 N-acetyl-neuraminic acid (NeuAc) residues,whereas the dominating gCMP isoforms of variants Aand B appeared to contain 2 NeuAc. The identificationrevealed conversions of isoforms containing differentcombinations of genetic variants and degrees of glycosylation,sialylation, and phosphorylation to variousdesialylated counter-isoforms. The content of sialylatedgCMP relative to the total CMP content was 37% (wt/wt), which decreased to 1.5% after sialidase treatment,indicating 96% effectivity of the desialidation. This approachcan be valuable for future functionality studiesspecifically addressing the importance of NeuAc.