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Analysis of Membrane-Enriched and High Molecular Weight Proteins in Leishmania infantum Promastigotes and Axenic Amastigotes
journal contribution
posted on 2012-08-03, 00:00 authored by Marie-Christine Brotherton, Gina Racine, Amin Ahmed Ouameur, Philippe Leprohon, Barbara Papadopoulou, Marc OuelletteMembrane and high molecular weight (HMW) proteins tend
to be underrepresented
in proteome analyses. Here, we optimized a protocol designed for the
extraction and purification of membranes from the protozoan parasite Leishmania using a combination of serial centrifugation
and free-flow zone electrophoresis (ZE-FFE). We also enriched for Leishmania HMW proteins from total extracts using the Gelfree
8100 fractionation system. This allowed the study of expression of
both membrane-enriched and HMW proteins in Leishmania infantum promastigotes and amastigotes.
We identified 194 proteins with at least one transmembrane domain
(TMD) and 171 HMW proteins (≥100 kDa) in the invertebrate promastigote
stage and 66 proteins with at least one TMD and 154 HMW proteins in
the mammalian amastigote stage. Several of the proteins identified
in one of the stages are part of pathways consistent with the known
biology of the parasite, with many proteins involved in lipid synthesis,
numerous dynein heavy chains, and some surface antigen proteins 2
detected in the promastigote stage. Notably, some proteins involved
in transport and proteolysis were detected either in promastigote
or amastigote. The present study is using improved proteomic methods
for studying membrane-enriched and HMW proteins helping to achieve
a better understanding of the parasite life cycle.