RT Journal Article
SR Electronic
T1 Methods matter: Influential purification and analysis parameters for intracellular parasite metabolomics
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 190421
DO 10.1101/190421
A1 Maureen A. Carey
A1 Vincent Covelli
A1 Audrey Brown
A1 Jessica G. Cooper
A1 Jennifer L. Guler
YR 2017
UL http://biorxiv.org/content/early/2017/09/18/190421.abstract
AB Due to improved instrument sensitivity and access, the use of metabolomics is gaining traction for the study of many organisms and pathogens. For the intracellular malaria parasite, Plasmodium falciparum, both targeted and untargeted metabolite detection has improved our understanding of pathogenesis, host-parasite interactions, parasite response to antimalarials, and impacts of resistance. However, protocols for purification are not optimized for investigations of intracellular pathogens and noise-limiting analysis parameters are not well defined. To explore influential parameters, we purified a diverse set of in vitro grown intra-erythrocytic P. falciparum parasites for untargeted metabolomics studies. Following metabolite identification, data processing included normalization to double stranded DNA, total protein, or parasite number to correct for different sample sizes and stage differences. We found that parasite-derived variables were most appropriate for normalization as they separate sample groups and reduce noise within the data set. However, these post-analysis steps did not remove the contribution from the host erythrocyte, in the form of membrane rich ‘ghosts’, and levels of technical sample variation persisted. In fact, we found that host contamination is as influential on the metabolome as sample treatment. This analysis also identified metabolites with potential to be used as markers to quantify host contamination levels. In conclusion, purification methods and normalization choices during the collection and analysis of untargeted metabolomics heavily affect the interpretation of results. Our findings provide a basis for development of improved experimental and analytical methods for future metabolomics studies of P. falciparum and other intracellular organisms.Importance Molecular characterization of pathogens, such as the malaria parasite, can lead to effective treatment strategies and improved understanding of pathogen biology. However, the distinctive biology of the Plasmodium parasite, such as its repetitive genome and requirement of growth within a host cell, hinders progress towards this goal. Untargeted metabolomics is one promising approach to learn about pathogen biology and how it responds to different treatments. By measuring many small molecules in the parasite at once, we gain a better understanding of important pathways that contribute to this response. Although increasingly popular, protocols for parasite isolation from the host cell and various analysis options are not well explored. The findings presented in this study emphasize the critical need for improvements in these areas to limit misinterpretation due to host metabolites and correct for variations between samples. This will aid both basic biological investigations and clinical efforts to understand important pathogens.