In this proteomics study we have performed two levels of gelbased proteomic analysis using classical two-dimensional gel In classical 2DE analysis, patients suffering their first episode of malaria as well as few patients with a past history of malaria (relapse or recurrent) and higher level of parasitemia (.5000 infected RBCs/mL blood) were included since it was difficult to get bigger cohort of malaria patients with similar parameters. In gel-based proteomic analysis samples were studied individually (n = 63 for classical 2DE and n = 30 for 2D-DIGE) rather than sample pooling to achieve better insights about biological variability from individual samples. In proteomic analysis two major high-abundance serum proteins; albumin and IgG were removed using Albumin & IgG Depletion SpinTrap (GE Healthcare) to reduce the dynamic range of the serum proteome. Depletion of these top two highabundance proteins removes more than 60% of the total protein content in human plasma or serum allowing detection of more proteins by increasing the effective concentration of the lowabundance proteins. Depletion of albumin and IgG effectively increased the overall spot number in 2D gels ( Figure S1A ). The efficiency of albumin and IgG depletion from human serum was evaluated by densitometric analysis of SDS-PAGE gels containing resolved serum proteins before and after depletion ( Figure S1B ). The densitometric analysis revealed around 85% and 80% depletion of albumin and IgG, respectively ( Figure S1C ). Serum proteome analysis of FM patients and healthy controls by 2DE identified 22 statistically significant (p,0.05) differentially expressed (with changes from 24.28 to +78.73-fold) protein spots (Table S1 .1). After staining with GelCode Blue Safe Protein Stain, over 700 protein spots were detected reproducibly in each gel by IMP7 software. Representative 2DE images of serum proteome profile of FM subjects and healthy individuals, and bar-diagrammatic representation of the fold change and 3D views of few selected spots are illustrated in Figure 1A and B. In MS analysis 12 different proteins were identified from the 22 differentially expressed protein spots, since in few cases MS analysis revealed similar identity for multiple protein spots appearing as different entities in 2D gels. The similar identity of multiple spots indicates the possibility of presence of various isoforms of those particular proteins probably due to the complex combinations of posttranslational modifications. Among the 12 identified proteins; 7 proteins were up-regulated (serum amyloid A, hemopexin precursor, apolipoprotein E, a-1-antitrypsin precursor, leucinerich a-2-glycoprotein, a-1-BN glycoprotein and a-1-antichymotrypsin precursor) and 5 proteins were down-regulated (haptoglobin, ficolin 3 precursor, apolipoprotein A-I, clusterin precursor and serum albumin) ( Figure S2 ; Table 2 and S2.1). Interestingly, serum amyloid A (spot U13 and U14) was found to be highly over expressed (.25-fold) in all the FM patients.