Development of Chemical Proteomics for the Folateome and Analysis of the Kinetoplastid Folateome

The folate pathway has been extensively
studied in a number of organisms, with its essentiality exploited
by a number of drugs. However, there has been little success in developing
drugs that target folate metabolism in the kinetoplastids. Despite
compounds being identified which show significant inhibition of the
parasite enzymes, this activity does not translate well into cellular
and animal models of disease. Understanding to which enzymes antifolates
bind under physiological conditions and how this corresponds to the
phenotypic response could provide insight on how to target the folate
pathway in these organisms. To facilitate this, we have adopted a
chemical proteomics approach to study binding of compounds to enzymes
of folate metabolism. Clinical and literature antifolate compounds
were immobilized onto resins to allow for “pull down”
of the proteins in the “folateome”. Using competition
studies, proteins, which bind the beads specifically and nonspecifically,
were identified in parasite lysate (Trypanosoma brucei and Leishmania major) for each antifolate compound.
Proteins were identified through tryptic digest, tandem mass tag (TMT)
labeling of peptides followed by LC-MS/MS. This approach was further
exploited by creating a combined folate resin (folate beads). The
resin could pull down up to 9 proteins from the folateome. This information
could be exploited in gaining a better understanding of folate metabolism
in kinetoplastids and other organisms.