Stability of milk fat globule membrane proteins toward human enzymatic gastrointestinal digestion.

The milk fat globule membrane (MFGM) fraction refers to the thin film of polar lipids and membrane proteins that surrounds fat globules in milk. It is its unique biochemical composition that renders MFGM with some beneficial biological activities, such as anti-adhesive effects toward pathogens. However, a prerequisite for the putative bioactivity of MFGM is its stability during gastrointestinal digestion. We, therefore, subjected MFGM material, isolated from raw milk, to an in vitro enzymatic gastrointestinal digestion. Sodium dodecyl sulfate PAGE, in combination with 2 staining methods, Coomassie Blue and periodic acid Schiff staining, was used to evaluate polypeptide patterns of the digest, whereas mass spectrometry was used to confirm the presence of specific MFGM proteins. Generally, it was observed that glycoproteins showed higher resistance to endogenous proteases compared with non-glycosylated proteins. Mucin 1 displayed the highest resistance to digestion and a considerable part of this protein was still detected at its original molecular weight after gastric and small intestine digestion. Cluster of differentiation 36 was also quite resistant to pepsin. A significant part of periodic acid Schiff 6/7 survived the gastric digestion, provided that the lipid moiety was not removed from the MFGM material. Overall, MFGM glycoproteins are generally more resistant to gastrointestinal digestion than serum milk proteins and the presence of lipids, besides glycosylation, may protect MFGM glycoproteins from gastrointestinal digestion. This gastrointestinal stability makes MFGM glycoproteins amenable to further studies in which their putative health-promoting effects can be explored.

A new chemical approach to differentiate carboxy terminal peptide fragments in cyanogen bromide digests of proteins.

We present a novel approach to perform C-terminal sequence analysis by discriminating the C-terminal peptide in a mass spectral analysis of a CNBr digest. During CNBr cleavage, all Met-Xxx peptide bonds are cleaved and the generated internal peptides all end with a homoserine lactone (hsl)-derivative. The partial opening of the hsl-derivatives, by using a slightly basic buffer solution, results in the formation of m/z doublets (Deltam=18 Da) for all internal peptides and allows to identify the C-terminal peptide which appears as a singlet in the mass spectra. Using two model proteins we demonstrate that this approach can be applied to study proteins purified in gel or in solution. The chemical opening of the hsl-derivative does not require any sample clean-up and therefore, the sensitivity of the C-terminal sequencing approach is increased significantly. Finally, the new protocol was applied to characterize the C-terminal sequence of two recombinant proteins. Tandem mass spectrometry by MALDI-TOF/TOF allowed to identify the sequence of the C-terminal peptides. This novel approach will allow to perform a proteome-wide study of C-terminal proteolytic processing events in a high-throughput fashion.