Figure 3.

Mass spectrometry analysis of origin of extracellular histones in CM cases. (A) Schematic representation of the methodology used for isolation, purification, and mass spectrometry analysis. Using Skyline software and by aligning trypsin fragments to reference amino acid sequences, we were able to identify specific histone H2A and H4 peptides that were present in purified P falciparum (malarial) preparations, that were not present in purified human histones (H1, H2A, H2B, H3, and H4) and vice versa. Typical peptides are presented from malarial (B) and human (C) database searches. Using Skyline software, we were able to identify histone H4 peptides for each species that demonstrated different mass/charge ratios with distinct human and P falciparum peptides and also distinct H2A human and P falciparum peptides (data not shown). This enabled us to identify, with high specificity, P falciparum (D) and human (E) species-specific peptides derived from histones spiked into PBS (left side of histogram, Histones) or serum (right side of histogram, Serum + Histones) Control is serum alone. Data shown are for H4. (F) In Ret⁺CM patient serum (n = 10) we were able to identify P falciparum histones H2A and H4 in the samples as well as human H2A and H4 (data not shown). (G) We combined the contribution of these 2 components to estimate the variable proportions of circulating human and P falciparum in the patient serum, demonstrating a significant contribution of P falciparum histones to the total pool.