Figure 4.

(A) Stable isotope labeling by amino acids in cell culture. P. falciparum infected erythrocytes are maintained under standard culturing conditions in RPMI containing either ¹²C₆¹⁴N₁ isoleucine (light) or ¹³C₆¹⁵N₁ isoleucine (heavy) for 72 hours. After proteome labeling (>95%), the light culture was untreated and the heavy culture was treated with 1 μM of tricostatin A (TSA) for three hours. Treated (heavy) and untreated (light) cultures were saponin-permeabilized, washed, combined and extracted. Following trypsin digestion and SCX fractionation, samples were analyzed both directly (unenriched) and subsequent to enrichment by anti-acetyl lysine immunoprecipitation via high resolution nano-UPLC-MS and MS/MS. Relative ratios of the isoleucine bearing heavy (+7 Da) peptides to their light counterparts were measured to determine the ratios of acetylation in the original experiments. (B) Proteins containing the acetyl-lysine sites that were quantified as undergoing a >2-fold increase (heavy/light ratio >2) when treated with TSA. Proteins are represented as blue circles, individual acetyl-lysine sites as orange circles (with the residue position defined alongside/within). The size of each acetyl-lysine site is proportional to the increase in acetylation following TSA treatment (quantified as the treated/untreated heavy/light ratio). Network analysis was performed on the entire dataset to represent functionally-related proteins. Direct (physical) or indirect (functional) interactions were defined with experimental, databases and text-mining information sources using STRING 9.1 with a medium confidence cutoff (0.4) and are represented by a pink line between proteins. (C) A subset of acetyl-lysine sites that decreased following TSA treatment (<0.5 heavy/light ratio). Percent variability is a measure of the heavy/light ratio variation between repeated measurements, where a value of 0 represents repeated measurement at the extreme minimum quantification range. The Ac-K site indicates the lysine residue position that underwent decreased acetylation. Supplemental Table 5 and 6 contain the complete list of altered acetylation sites under TSA treatment.