Stones were separated by SDS-PAGE using 12 bis-tris precast gel (Bio-Rad, 345-0118) and MES buffer (Invitrogen, NP0002). The proteins were then transferred overnight at 4uC on to nitrocellulose membrane of pore size 0.20 mm in presence of 20 methanol in Nupage transfer buffer (Invitrogen, NP0006). The membrane was then blocked with 3 BSA in TBS buffer containing 0.05 Tween20 and 100 mM NaF. Commercial antibodies purchased from Abcam: anti-H3core (ab1791) [1:2500 dilution], anti-H3S10ph (ab5176) [1:1000 dilution], anti-H3S28ph (ab5169) [1:1000 dilution], and antiH3T11ph (ab5168) [1:1000 dilution] were then used to probe the membrane. After probing with appropriate secondary antibodies conjugated to horseradish peroxidase (GE Healthcare LifeHistone Phosphorylation in P. falciparumFigure 1. Schematic of strategies for phosphoprotein analysis of P. falciparum histones. Histones were purified from asynchronous culture of intra-erythrocytic malaria parasite using two improved protocols for acid GR79236 site extraction and high-salt extraction. Both acid and high-salt extracted histone samples were digested in-solution with trypsin. The acid extracted sample was analyzed via LC-MS/MS in two ways: either with or without phosphopeptide enrichment. A high-salt extracted sample was analyzed only after phosphopeptide enrichment. When the samples were enriched for phosphopeptides, we were able to identify 14 phosphomarks that were manually validated and are presented in Table 1. All phospho-marks detected in context with other PTMs at this step are presented in Table S1. doi:10.1371/journal.pone.0053179.gSciences), the membranes were developed with Super Signal West FEMTO Chemiluminescent Substrate (Thermo Scientific) following the manufacturer’s recommendations. Western blot analysis was performed on cytoplasmic and nuclear fractions of unsynchronized 3D7 parasites prepared as described in 16574785 [3,27]. Briefly, cytoplasmic and nuclear extracts were separated and transferred onto nitrocellulose membrane by standard SDS-PAGE protocol. The membranes were then blocked overnight at 4uC using blocking buffer containing 5 milk in TBS containing 0.25 Tween-20. The membranes were subsequently incubated with anti-14-3-3I antibody diluted in blocking buffer [1:1000 dilution]. Detection with the secondary antibody was as described above.Immunofluorescence Assay (IFA)IFA was performed on synchronized stages (Ring, Trophozoite, and Schizont) of the wild type 3D7 strains as described in [28]. The fixed cells were incubated with primary antibody anti-14-3-3I [1:100 dilution]. After incubating with appropriate secondary antibodies, the cells were examined with a Nikon microscope.Mass spectrometryHistones purified from unsynchronised 3D7 parasites were digested and analysed by liquid chromatography-tandem mass spectrometric (LC-MS/MS) for the detection and site-localization of phosphorylation.Sample preparation and tryptic digestion. Prior to insolution buy Gepotidacin digestion, the samples containing 5?0 mg protein were concentrated with Amicon 3 kDa MWCO filters (Millipore) and the buffer was simultaneously exchanged for 50 mM Ammonium bicarbonate for subsequent trypsin digestion. Proteins were reduced with DTT and alkylated with iodacedamide. Incubation with trypsin (1 mg enzyme/50 mg protein) was carried out overnight at 37uC. The reaction was stopped by adding 5 ul of 1 formic acid. Phos-TiO phosphopeptide enrichment kit (GL Sciences) was used in order to enrich for phosphopeptides acco.Stones were separated by SDS-PAGE using 12 bis-tris precast gel (Bio-Rad, 345-0118) and MES buffer (Invitrogen, NP0002). The proteins were then transferred overnight at 4uC on to nitrocellulose membrane of pore size 0.20 mm in presence of 20 methanol in Nupage transfer buffer (Invitrogen, NP0006). The membrane was then blocked with 3 BSA in TBS buffer containing 0.05 Tween20 and 100 mM NaF. Commercial antibodies purchased from Abcam: anti-H3core (ab1791) [1:2500 dilution], anti-H3S10ph (ab5176) [1:1000 dilution], anti-H3S28ph (ab5169) [1:1000 dilution], and antiH3T11ph (ab5168) [1:1000 dilution] were then used to probe the membrane. After probing with appropriate secondary antibodies conjugated to horseradish peroxidase (GE Healthcare LifeHistone Phosphorylation in P. falciparumFigure 1. Schematic of strategies for phosphoprotein analysis of P. falciparum histones. Histones were purified from asynchronous culture of intra-erythrocytic malaria parasite using two improved protocols for acid extraction and high-salt extraction. Both acid and high-salt extracted histone samples were digested in-solution with trypsin. The acid extracted sample was analyzed via LC-MS/MS in two ways: either with or without phosphopeptide enrichment. A high-salt extracted sample was analyzed only after phosphopeptide enrichment. When the samples were enriched for phosphopeptides, we were able to identify 14 phosphomarks that were manually validated and are presented in Table 1. All phospho-marks detected in context with other PTMs at this step are presented in Table S1. doi:10.1371/journal.pone.0053179.gSciences), the membranes were developed with Super Signal West FEMTO Chemiluminescent Substrate (Thermo Scientific) following the manufacturer’s recommendations. Western blot analysis was performed on cytoplasmic and nuclear fractions of unsynchronized 3D7 parasites prepared as described in 16574785 [3,27]. Briefly, cytoplasmic and nuclear extracts were separated and transferred onto nitrocellulose membrane by standard SDS-PAGE protocol. The membranes were then blocked overnight at 4uC using blocking buffer containing 5 milk in TBS containing 0.25 Tween-20. The membranes were subsequently incubated with anti-14-3-3I antibody diluted in blocking buffer [1:1000 dilution]. Detection with the secondary antibody was as described above.Immunofluorescence Assay (IFA)IFA was performed on synchronized stages (Ring, Trophozoite, and Schizont) of the wild type 3D7 strains as described in [28]. The fixed cells were incubated with primary antibody anti-14-3-3I [1:100 dilution]. After incubating with appropriate secondary antibodies, the cells were examined with a Nikon microscope.Mass spectrometryHistones purified from unsynchronised 3D7 parasites were digested and analysed by liquid chromatography-tandem mass spectrometric (LC-MS/MS) for the detection and site-localization of phosphorylation.Sample preparation and tryptic digestion. Prior to insolution digestion, the samples containing 5?0 mg protein were concentrated with Amicon 3 kDa MWCO filters (Millipore) and the buffer was simultaneously exchanged for 50 mM Ammonium bicarbonate for subsequent trypsin digestion. Proteins were reduced with DTT and alkylated with iodacedamide. Incubation with trypsin (1 mg enzyme/50 mg protein) was carried out overnight at 37uC. The reaction was stopped by adding 5 ul of 1 formic acid. Phos-TiO phosphopeptide enrichment kit (GL Sciences) was used in order to enrich for phosphopeptides acco.
