Development of chemical proteomics for the folateome and analysis of the kinetoplastid folateome

TY - JOUR

T1 - Development of chemical proteomics for the folateome and analysis of the kinetoplastid folateome

AU - Webster, Lauren

AU - Thomas, Michael

AU - Urbaniak, Michael

AU - Wyllie, Susan

AU - Ong, Han

AU - Tinti, Michele

AU - Fairlamb, Alan

AU - Boesche, Markus

AU - Ghidelli-Disse, Sonja

AU - Drewes, Gerard

AU - Gilbert, Ian

N1 - Extended thanks go to SULSA (Scottish Universities Life Sciences Alliance) for a studentship (LAW). The Wellcome Trust is also acknowledged for support: IHG and AHF WT100476; AHF WT079838. MT was supported by Wellcome Investigator Award 101842 to Michael AJ Ferguson.

PY - 2018/10/12

Y1 - 2018/10/12

N2 - 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.

AB - 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.

KW - Leishmania

KW - Trypanosoma brucei

KW - chemical proteomics

KW - folate

KW - kinetoplastid

KW - pull down

UR - https://www.scopus.com/pages/publications/85054292405

U2 - 10.1021/acsinfecdis.8b00097

DO - 10.1021/acsinfecdis.8b00097

M3 - Article

C2 - 30264983

SN - 2373-8227

VL - 4

SP - 1475

EP - 1486

JO - ACS Infectious Diseases

JF - ACS Infectious Diseases

IS - 10

ER -