Leishmania species are responsible for a broad spectrum of diseases, denominated Leishmaniasis, affecting over 12 million people worldwide. During the last decade, there have been impressive efforts for sequencing the genome of most of the pathogenic Leishmania spp. as well as hundreds of strains, but large-scale proteomics analyses did not follow these achievements and the Leishmania proteome remained mostly uncharacterized. Here, we report a comprehensive comparative study of the proteomes of strains representing L. braziliensis, L. panamensis and L. guyanensis species. Proteins extracted by SDS-mediated lysis were processed following the multi-enzyme digestion-filter aided sample preparation (FASP) procedure and analysed by high accuracy mass spectrometry. "Total Protein Approach" and "Proteomic Ruler" were applied for absolute quantification of proteins. Principal component analysis demonstrated very high reproducibility among biological replicates and a very clear differentiation of the three species. Our dataset comprises near 7000 proteins, representing the most complete Leishmania proteome yet known, and provides a comprehensive quantitative picture of the proteomes of the three species in terms of protein concentration and copy numbers. Analysis of the abundance of proteins from the major energy metabolic processes allow us to highlight remarkably differences among the species and suggest that these parasites depend on distinct energy substrates to obtain ATP. Whereas L. braziliensis relies the more on glycolysis, L. panamensis and L. guyanensis seem to depend mainly on mitochondrial respiration. These results were confirmed by biochemical assays showing opposite profiles for glucose uptake and O 2 consumption in these species. In addition, we provide quantitative data about different membrane proteins, transporters, and lipids, all of which contribute for significant species-specific differences and PLOS NEGLECTED TROPICAL DISEASES

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