CRYPTOMICS Platform

Generation of novel activities by protein fragmentation is part of normal biological function (1) and there is increasing evidence that proteolytic cleavage gives rise to ‘hidden’ peptides with bioactivities that are often unpredicted and totally distinct to the parent protein (2). HealthLinx has developed a ‘Cryptomic’ compound discovery platform that can significantly accelerate the discovery of novel proteins for nutraceutical and therapeutic use.

The Cryptomics platform entails a suite of technologies involving protein fragmentation predominantly by enzymes, high-performance liquid chromatography for separation of fragments, cell and non-cell based bioassays to monitor biological activity of protein fragments, and mass spectrometry for protein fragment identification.

The Cryptomics approach has isolated a novel anti-coagulant cryptein fragment from human plasma and a potential novel anti-angiogenic and osteoclast peptide from bovine milk protein. Thus, proteolytic liberation of crypteins with novel activities represents an important mechanism for increasing diversity of protein function and potentially offers new opportunities for protein-based therapeutics.

The Cryptomics approach has potentially liberated novel milk crypteins that was previously unpredicted and has provided a platform to the dairy industry to further mine the milk proteome for novel therapeutics and nutraceuticals.

Platform information

The protein fragmentation pattern clearly differs as fragmentation time increases with the decrease of native milk protein at time point T-1 to smaller peptide fragments at timepoint T-6. Time points T-4 and T-6 were assigned partial and near complete fragmentation based on their fragmentation patterns. Both unfractionated T-4 and T-6 and there respective protein controls, BSA-4 and BSA-6, were screened in cell proliferation assays (Fig. 2).

The partial and near complete fragmented milk proteins at T-4 and T-6, respectively, show a marked difference in activity where T-6 contains a potential anti-angiogenic activity. While the protein controls BSA-4 and BSA-6 showed no anti-angiogenic activity. The T-6 time point sample contains a fragment/s that has been generated due to increased fragmentation time that was not present in T-4 and would otherwise have be missed if only studying partial fragmentation. The active anti-angiogenic sample, T-6, was analysed by mass spectrometry (data not shown) revealing a complex heterogeneous mixture and both time points T-4 and T-6 were fractionated by RP-HPLC (Fig. 3).

The RP-HPLC chromatograms confirm in more detail the SDS-PAGE results with protein fragmentation increasing as fragmentation time increases. Monitoring the decrease of the native milk protein (T-1) at retention time 38 min to near complete fragmentation by time point T-6 was also confirmed by mass spectrometry (data not shown). The differences in chromatographic profiles from T-4 to T-6 show an increase in the number of peptide fragments and can explain the differences in anti-angiogenic activity. Time point T-6 has 86% sequence coverage when compared to theoretical fragmentation as determined by mass spectrometry, thus enabling peptide profiling and accurate hit identification. The fractionated T-6 library has to be re-assayed to confirm anti-angiogenic activity and further fractionated if necessary until activity is assigned to one peptide fragment.