A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation

Abstract

Adaptation to starvation is integral to the Leishmania life cycle. The parasite can survive prolonged periods of nutrient deprivation both in vitro and in vivo and starvation plays a crucial role in the differentiation of the parasite from the non-infective promastigote form to infective metacyclics. The identification of parasite proteins synthesised during starvation is key to unravelling the molecular mechanisms facilitating adaptation to these conditions and the associated lifecycle differentiation. Additionally, as stress adaptation mechanisms in Leishmania are linked to virulence as well as infectivity, profiling of the complete repertoire of Newly Synthesized Proteins (NSPs) under starvation is important for drug target discovery. However, differential identification and quantitation of low abundance, starvation-specific NSPs from the larger background of the pre-existing parasite proteome has proven difficult, as this demands a highly selective and sensitive methodology. Herein we introduce an integrated chemical proteomics method in L. mexicana promastigotes that involves a powerful combination of the BONCAT technique and iTRAQ 4-plex quantitative proteomics Mass Spectrometry (MS), which enabled temporally resolved quantitative profiling of de novo protein synthesis in the starving parasite. Uniquely, this approach integrates the high specificity of the BONCAT technique for the NSPs, with the high sensitivity and multiplexed quantitation capability of the iTRAQ proteomics MS. Proof-of-concept experiments identified a total of 166 NSPs in the parasite and quantified the relative changes in abundance of these proteins as a function of duration of starvation. Our results show a starvation time-dependent differential expression of important translation regulators. GO analysis of the identified NSPs for Biological Process revealed translation (enrichment P value 6.93e-67) and peptide biosynthetic process (enrichment P value 1.85e-66) as extremely significantly enriched terms indicating the high specificity of the NSP towards regulation of protein synthesis. We believe that this approach will find wide-spread use in the study of the developmental stages of Leishmania species and in the broader field of protozoan biology.\n\nAuthor SummaryPeriodic nutrient scarcity plays crucial roles in the life cycle of the protozoan parasite Leishmania spp. Starvation triggers differentiation of the parasite from a non-infective form to an infective form. Although adaptation to nutrient stress has a pivotal role in Leishmania biology, the underlying mechanisms remain poorly understood. In a period of nutrient starvation, the parasite responds by decreasing its protein production to conserve nutrient resources and to prevent formation of toxic proteins. However, even during severe starvation, the parasite generates certain essential quality control and rescue proteins. Differential identification of the complete repertoire of these proteins synthesised during starvation from the pre-existing proteins in the parasite holds the key to understanding the starvation adaptation mechanisms. This has been challenging to accomplish due to technical limitations. Using a combination of chemical labelling techniques and protein mass-spectrometry, we selectively identified and measured the proteins generated in the starving Leishmania parasite. Our results show a starvation time-dependent differential expression of important protein synthesis regulators in the parasite. This will serve as an important dataset for a holistic understanding of the starvation adaptation mechanisms in Leishmania. We also believe that this method will find wide-spread applications in the field of protozoa and other parasites causing Neglected Tropical Diseases.