Proteome coverage is limited by the dynamic range of proteins present in a sample and often is confined to the analysis of abundant proteins. We have developed a protein prefractionation protocol, based on the differential solubilization of membranes using digitonin, that has allowed an increase in the resolution and depth of comparative proteomic studies. This prefractionation protocol can also be used to infer the subcellular localization of hypothetical proteins as tested experimentally using green fluorescent fusion proteins. The abundant tubulins and associated proteins of the cytoskeleton were removed from the sample using digitonin extraction, hence facilitating the visualization of lower abundance proteins. The digitonin prefractionation protocol was applied for a comparative proteomic analysis of the promastigote and amastigote life cycle stages of Leishmania infantum and has allowed the identification of novel proteins expressed in a stage-specific manner.
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