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. 2017 Jan 31;16(4):567–580. doi: 10.1074/mcp.M117.066951

Fig. 1.

Fig. 1.

Affinity Purification of MMA peptides and data sets. A, Diagram of affinity purification strategy. T. gondii tachyzoites were harvested either from infected cells (top) or as free floating extracellular parasites (bottom) and were filtered to remove host cell debris. Parasites were lysed and digested with trypsin to release peptides (green lines). MMA-modified peptides (red dots) were enriched by immunoaffinity purification using a mixture of two monoclonal antibodies raised against arginine monomethylation at R* and R*GG motifs (* = site of arginine methylation). Purified peptides were identified by LC-MS/MS and database search using the parameters described in the materials and methods. B, Diagram of data sets. “All data” represents all 370 MMA proteins that were identified within this study. Six biological samples were analyzed in two technical replicates (merged data for both replicates are presented). Three data sets are composed of proteins detected in wild type intracellular tachyzoites: two biological replicates from wild type (WT) intracellular RHΔhxgprt tachyzoites (WT1, WT2), one data set from intracellular RHΔhxgprtΔku80 tachyzoites (WT3). Three data sets consist of proteins detected in: (1) Extracellular wild-type parasites (EXTRA); (2) TgPRMT1 knockout (PRMT1KO) parasites; and (3) TgPRMT1 knockout parasites genetically complemented with PRMT1mRFP (PRMT1COMP) (29). All proteins present in any of the three biological replicates of intracellular wild-type parasites are included in “Intracellular Union”. The other data sets shown are derived from comparative analysis of the initial data sets and manual filtering of the protein lists. The candidate “TgPRMT1 substrates” data set represents the proteins present in the WT Intracellular Union data set but not in the PRMT1KO data set. The “high confidence TgPRMT1 substrate” data set consists of those proteins in the TgPRMT1 substrate data set (i.e. not present in the PRMT1KO) for which methylation was restored in PRMT1COMP. The 'PRMT1KO exclusive' data set consists of the proteins present in the PRMT1KO and not present in the WT Intracellular Union data set.