Analysis of membrane-enriched and high molecular weight proteins in Leishmania infantum 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 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.
Centre de Recherche en Infectiologie, Département de Microbiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, Canada.
Marie-Christine
MC
Brotherton
Gina
G
Racine
Amin Ahmed
AA
Ouameur
Philippe
P
Leprohon
Barbara
B
Papadopoulou
Marc
M
Ouellette
D061245:Axenic Culture; D004591:Electrophoresis, Polyacrylamide Gel; D018314:Leishmania infantum / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism*; D015800:Protozoan Proteins / Q000302:isolation & purification / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D008565:Membrane Proteins; D020543:Proteome; D015800:Protozoan Proteins