Sugar nucleotide quantification by liquid chromatography tandem mass spectrometry reveals a distinct profile in Plasmodium falciparum sexual stage parasites

摘要

The obligate intracellular lifestyle of Plasmodium falciparum and the difficulties in obtaining sufficient amounts of biological material have hampered the study of specific metabolic pathways in the malaria parasite. Thus, for example, the pools of sugar nucleotides required to fuel glycosylation reactions have never been studied in-depth in well-synchronized asexual parasites or in other stages of its life cycle. These metabolites are of critical importance, especially considering the renewed interest in the presence of N -, O -, and other glycans in key parasite proteins. In this work, we adapted a liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on the use of porous graphitic carbon (PGC) columns and MS-friendly solvents to quantify sugar nucleotides in the malaria parasite. We report the thorough quantification of the pools of these metabolites throughout the intraerythrocytic cycle of P. falciparum . The sensitivity of the method enabled, for the first time, the targeted analysis of these glycosylation precursors in gametocytes, the parasite sexual stages that are transmissible to the mosquito vector.* ESI, : electrospray ionization; GDP, Guanosine diphosphate; GDP-Fuc, : GDP-fucose; GDP-Glc, : GDP-glucose; GDP-Man, : GDP-mannose; GIPL, : glycoinositol phospholipid; GPI, : glycosylphosphatidylinositol; hpi, hours post invasion; LC-MS/MS, : liquid chromatography tandem mass spectrometry; MACS, Magnetic-Activated Cell Sorting; MRM, : multiple reaction monitoring; MSP-1, : merozoite surface protein 1; MSP-2, : merozoite surface protein 2; PGC, : porous graphitic carbon; RP, : reversed phase; RPMI, Roswell Park Memorial Institute; SPE, : solid phase enrichment; TSR, : thrombospondin type 1 repeat; UDP, Uridine diphosphate; UDP-Gal, : UDP-galactose; UDP-Glc, : UDP-glucose; UDP-GlcNAc, : UDP-N-acetylglucosamine; uRBC, : uninfected red blood cell.