Plasmodium falciparum parasite infects human red blood cells causing the most severe and life-threatening form of malaria in humans. In the human red blood cells, the parasite absolutely requires host haemoglobin digestion to supply amino acids for de novo protein synthesis. Digestion of haemoglobin occurs via a semi-ordered proteolytic cascade that is mediated by different proteases, and the exact interplay between the different classes of proteases and haemoglobin digestion is not clear. This limitation prompted us to develop an advanced metabolomics and peptidomics-based strategy to identify the ‘signature peptide’ libraries that are generated as a result of specific inhibition of each protease class involved in haemoglobin digestion.
The combined metabolomics and peptidomics analysis revealed that inhibition of specific haemoglobin digesting proteases resulted in unique changes in the abundance of haemoglobin derived endogenous peptides. Furthermore, metabolomics analysis of parasites cultured in media with labelled amino acids confirmed that the short chain peptides associated with protease inhibition were derived from haemoglobin. The results obtained could distinguish between different clans of the same class of protease. For example, inhibition of M1 aminopeptidase resulted in accumulation of peptides containing basic residues, while specific inhibition of M17 aminopeptidase resulted in accumulation of hydrophobic peptides.
In conclusion, this multi-platform approach provided an extensive coverage of endogenous peptides liberated during haemoglobin digestion within the parasite, and identified specific peptide signatures associated with inhibition of different classes of proteases involved in haemoglobin digestion.