Analysis of Membrane-Enriched and High Molecular Weight Proteins in <i>Leishmania infantum</i> Promastigotes and Axenic Amastigotes

Membrane 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 <i>Leishmania</i> using a combination of serial centrifugation and free-flow zone electrophoresis (ZE-FFE). We also enriched for <i>Leishmania</i> 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 <i>Leishmania infantum</i> 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.