Literature DB >> 27762281

Widespread occurrence of lysine methylation in Plasmodium falciparum proteins at asexual blood stages.

Inderjeet Kaur1, Mohammad Zeeshan1,2, Ekta Saini1, Abhinav Kaushik2, Asif Mohmmed3, Dinesh Gupta2, Pawan Malhotra1.   

Abstract

Post-transcriptional and post-translational modifications play a major role in Plasmodium life cycle regulation. Lysine methylation of histone proteins is well documented in several organisms, however in recent years lysine methylation of proteins outside histone code is emerging out as an important post-translational modification (PTM). In the present study we have performed global analysis of lysine methylation of proteins in asexual blood stages of Plasmodium falciparum development. We immunoprecipitated stage specific Plasmodium lysates using anti-methyl lysine specific antibodies that immunostained the asexual blood stage parasites. Using liquid chromatography and tandem mass spectrometry analysis, 570 lysine methylated proteins at three different blood stages were identified. Analysis of the peptide sequences identified 605 methylated sites within 422 proteins. Functional classification of the methylated proteins revealed that the proteins are mainly involved in nucleotide metabolic processes, chromatin organization, transport, homeostatic processes and protein folding. The motif analysis of the methylated lysine peptides reveals novel motifs. Many of the identified lysine methylated proteins are also interacting partners/substrates of PfSET domain proteins as revealed by STRING database analysis. Our findings suggest that the protein methylation at lysine residues is widespread in Plasmodium and plays an important regulatory role in diverse set of the parasite pathways.

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Year:  2016        PMID： 27762281      PMCID： PMC5071865          DOI： 10.1038/srep35432

Source DB:  PubMed          Journal:  Sci Rep        ISSN： 2045-2322            Impact factor:   4.379


Plasmodium falciparum, a protozoan parasite responsible for the severe form of human malaria, has a complex life cycle in two hosts, namely Anopheles mosquito and human. Parasites in these two hosts invade different cell types and propagate in distinct microenvironments. Although transcriptional regulation plays an important role in helping the parasite to adapt to distinct environments, however relatively few regulatory motifs and transcriptional regulators have been reported in Plasmodium so far123. Evidences are emerging to suggest that post-translational modifications (PTMs) play an important role in regulation of fundamental processes of Plasmodium growth and host invasion- including cell signaling and epigenetic control of gene regulation. Protein trafficking and interactions between various PTMs are the two very important processes that fine-tune the functions of several Plasmodium proteins4. Although several PTMs- such as phosphorylation, acetylation, palmotylation, ubiquitylation and lipidation have been identified in Plasmodium, however, only phosphorylation/dephosphorylation have been studied extensively567891011. In the recent years, methylation of proteins has been ranked as the fourth common post-translational modification12 and is of common occurrence in human, Saccharomyces cerevisiae and Trypanosomes13141516. Protein methylation is mainly found on lysine and arginine residues, although there are reports of methylation of histidine and glutamic acid too17. Methylation, particularly lysine methylation is a well-studied phenomenon in histones, which involves addition of one to three methyl groups on the amino acid’s amine group to form mono, di or tri-methyllysine18. Histone lysine methylation is involved in transcriptional activation and silencing. The process is regulated by histone lysine methyltransferases (HKMTs) and histone lysine demethylases19. Recent proteome-wide lysine methylation studies indicate that the modifications also occur in non-histone proteins such as proteins linked to RNA processing, ribosome assembly, trafficking and signaling2021. Among the apicomplexan parasites, Plasmodium and Toxoplasma have orthologs of several chromatin remodeling proteins and enzymes responsible for protein methylation and acetylation2223. In P. falciparum, the histone posttranslational modifications, mainly acetylation and methylation have been shown to play significant role(s) in red blood cell invasion and in virulence gene regulation2425. Ten SET domain containing histone lysine methyltransferases (HKMTs), three histone-demethylase orthologs of lysine-specific demethylases (LSD1) and jumonji-C histone demethylases (jHDM) families have been described in Plasmodium. These proteins are the targets for novel drug development as the proteins show low sequence similarity to corresponding human counterparts2226. To understand the extent of lysine methylation in blood stage forms of Plasmodium falciparum, we analyzed the reactivity of anti-mono/dimethyl lysine and anti-trimethyl lysine antibodies with intact asexual blood stage Plasmodium parasites and further immunoprecipitated the Plasmodium lysates from the three blood stages, using these antibodies. Intriguingly, the LC-MS/MS analysis of the immunoprecipitates identified several non-histone methylated Plasmodium proteins linked with diverse functions such as transport, hemostatic processes and chromosome organization. These results suggest an important role of protein lysine methylation in regulation of various P. falciparum biological processes.

Materials and Methods

Plasmodium falciparum culture

Plasmodium falciparum 3D7 was cultured in complete RPMI (1640 (Invitrogen Corporation, USA), 50 mg/L hypoxanthine (Sigma Aldrich Co., USA), 0.5 g/L Albumax I (Gibco, Thermofisher Scientific Inc., USA) and 2 g/L sodium bicarbonate (Sigma Aldrich Co., USA) using O+ human erythrocytes (4% haematocrit) under mixed gas (5% O2, 5% CO2 and 90% N2). Cultures were synchronized with 5% sorbitol for at least two successive cycles and harvested using saponin treatment.

Immunofluorescence assay

Thin smears were prepared from Plasmodium falciparum culture at ring, trophozoite and schizont stages. The glass slides were air dried and fixed with pre-chilled absolute methanol for 30 minutes. The smears were incubated with 4% BSA in PBS for 2 h at room temperature (RT) to block the non-specific binding. After washing with PBS, the smears were probed with anti-mono/dimethyl lysine polyclonal antibody (abcam; ab23366)(1:20) for overnight at 4 °C, followed by Alexa-Fluor 488 conjugated goat anti-rabbit IgG antibody (A11008; Thermofisher Scientific Inc. USA) (1:200) for 1 h at RT. The slides were washed and mounted with 4′,6-diamidino-2-phenylindoledihydrochloride (DAPI, Molecular Probes, USA) antifade solution (Molecular Probes, USA). Images were captured using a Nikon A1-R confocal microscope.

Preparation of parasite lysate, Immunoprecipitation and Western blot analysis

Immuno-precipitation was performed using Pierce® Crosslink Immunoprecipitation Kit (Thermofisher Scientific Inc., USA) according to manufacturer’s protocol. Briefly, parasite cell lysates from synchronized cultures of three asexual stages (ring, trophozoite and schizont) were prepared using IP lysis buffer. The stage specific cell lysates were incubated overnight at 4 °C with anti-mono/dimethyl (abcam; ab23366) or anti-trimethyl lysine (Immunechem Pharmaceutical Inc.; ICP0601) antibodies, cross-linked with Protein A/G PlusAgarose by DSS cross-linker. The antibody cross-linked resin was washed with TBS followed by two washes with lysis buffer. Finally, the resin was washed with conditioning buffer and antibody bound proteins were eluted with elution buffer. Also, the peptides obtained after trypsin digestion of trophozoite stage parasite lysates were lyophilized, desalted and incubated overnight at 4 °C with anti-mono/dimethyl and anti-trimethyl lysine antibodies cross-linked with Protein A/G Plus agarose. The eluted peptides were lyophilized and separated into 12 fractions using hydrophilic liquid interaction chromatography (HILIC) over one hour. Each fraction was separately analyzed on LC-MS/MS. For western blot analysis, equal amount (50 μg) of total protein from three stages were resolved on 10% SDS-PAGE and transferred onto PVDF membrane (Merck Millipore, Merck KGaA, USA) pre-activated with methanol. The membrane was blocked with 4% BSA and incubated with polyclonal anti-methyllysine antibodies (ImmuneChem Pharmaceuticals Inc, Canada, ICP0601; 1:50) overnight at 4 °C. After three washings with PBST, the membrane was incubated with HRP conjugated anti-rabbit IgG secondary antibody (Sigma Aldrich Pvt. Ltd., USA) for 1 h at RT. The membrane was developed and visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermofisher Scientific Incorporation, USA) as per manufacturer’s instructions. For confirmation of proteins in the IP experiments, a fraction of IP eluates pulled using anti-methyllysine antibodies were boiled in 4X sample loading buffer, separated on 10% SDS-PAGE gels and transferred to a nitro-cellulose membrane. The membrane was blocked with blocking buffer (ODYSSEY infrared imaging systems; LI-COR) and probed with protein specific antisera (anti-PfP12; a 6-cysteine protein and anti-PfTSN; Tudor Staphylococcal Nuclease) using anti-mice IRDye 800CW secondary antibodies (LI-COR). Protein bands were imaged in ODYSSEY Infrared Imager (LI-COR).

Tryptic digestion and LC-MS/MS analysis

Eluted proteins from immunoprecipitation were subjected to in-solution digestion. Samples were subjected to subsequent reduction and alkylation of disulfide bonds with 10 mM dithiothreitol (DTT) and 40 mM iodoacetamide for 1 hr. at room temperature. Digestion was performed using trypsin (1:50, enzyme: total protein) (Promega corporation, USA) for overnight at 37 °C and stopped by adding 0.1% trifluoroacetic acid. The samples were cleared by centrifugation at 10000 rpm for 10 min. The digested proteins were concentrated using Speed Vac (Thermofisher Scientific Inc., USA) and analyzed on Orbitrap Velos Pro mass spectrometer coupled with nano-LC 1000 (Thermofisher Scientific Inc., USA). The peptide mixtures were loaded on to a reverse phase C-18 pre-column (Acclaim PepMap, 75 μm × 2 cm, 3 μm, 100 A°, Thermofisher Scientific Inc., USA), in line with an analytical column (Acclaim PepMap, 50 μm × 15 cm, 2 μm, 100 A°). The peptides were separated using a gradient of 5% to 50% of solvent B (0.1% formic acid in 95/5 acetonitrile/water) in 180 min for immunoprecipitates and 120 min for HILIC fractions. The peptides were analyzed in data dependent mode where the precursors were acquired (MS) in Orbitrap at a resolution of 60000 and a minimum of 1000 counts were needed to trigger the MS/MS. Top 20 precursors were allowed to fragment using CID (collision induced dissociation) in Ion trap with collision energy of 35 for the immunoprecipitates. For HILIC fractions, precursors were fragmented using high-energy collision dissociation (HCD) and detected in Orbitrap at a resolution of 7500. Charge state screening of precursors and monoisotopic precursor selection was enabled. Unassigned and singly charged ions were rejected. The parent ions once fragmented were excluded for next 50 s with exclusion mass width of ±10 ppm. The lock mass (m/z 445.120024) enabled the accurate measurement in MS for immunoprecipitated samples and in both MS as well as MS/MS for HILIC fractions. The acquired spectra were analyzed using SEQUEST algorithm in Proteome Discoverer (PD; version 1.4) software, with a precursor tolerance of 20 ppm and tolerance of 0.6 Da for MS/MS for CID and 0.1 Da for HCD against P. falciparum database version 10 downloaded from PlasmoDB27. Carbamidomethyl (C), Deamidation (NQ) and mono-methyl, di-methyl and trimethyl (K) were set as variable modifications and five missed cleavages were allowed. The resultant identified peptides were validated using Percolator at 5% False Discovery Rate (FDR) (q value < 0.05), which uses PEP (Posterior Error Probability) and q value for validations.

Lysine methylation motif analysis

The motif search included six amino acid residues N-terminal and C-terminal to each methylation sites. Putative lysine methylation sites were analyzed, based on previously known methyl lysine associated motifs in other organisms, for example LK and MK. To determine the sequence motifs, we developed a PERL script to fetch six amino acids upstream as well as downstream of a central lysine. The frequency of amino acids near lysine residue was also analyzed using WebLogo28. To compare neighboring residues in the methylated and non methylated lysines in the proteomics identified proteins, we used Two Sample Logo29.

Gene Ontology (GO) analysis

GO enrichment was performed for functional classification and cellular localization of the methylated proteins using PlasmoDB (version 13.0).

Interaction analysis using STRING

We downloaded the STRING30 Protein-Protein Interaction (PPI) network for P. falciparum and converted it into R object using igraph package31. Next, we removed the Edges with no experimental evidence of interaction (Experimental < 0). However, the network contained redundant edges and loops which were removed too, using simplify() function from igraph package. SET domain containing proteins along with its top 10 nearest neighbors were retrieved and the network was visualized using Cytoscape32.

Results

Anti-methyl lysine specific antibodies recognize Plasmodium proteins at asexual blood stages

Plasmodium genome sequencing analysis has revealed existence of a number of methyltransferases, particularly SET-domain-containing proteins and demethylases that are responsible for lysine methylation in the genome2233. To assess the extent of lysine methylation in Plasmodium proteins in asexual blood stages, we tested the reactivity of commercially available anti-mono/di methyl lysine and anti-trimethyl lysine antibodies with the parasite lysate and with intact blood stage P. falciparum parasites by western blot and immunofluorescence analysis respectively. As shown in Fig. 1A, anti-methyl lysine specific antibodies recognized specific bands at ring, trophozoite and schizont stages, although the extent of reactivity appeared more at the schizont stage. The results were further confirmed by immunofluorescence assay where a considerable staining was observed at all the three parasite blood stages, thereby indicating the extensive methylation of Plasmodium proteins at lysine residues. Staining was mainly observed in the periphery of nucleus as blue DAPI stain overlapped with the anti-lysine antibody immunostains, however some staining was also seen in parasite cytoplasm (Fig. 1B).
Figure 1

Extensive lysine methylation occurs at asexual blood stages of P. falciparum.

Anti-methyl-lysine specific antibodies recognize the proteins from three different blood stages of P. falciparum. (A) Representative western blot showing extent of lysine methylation at ring, trophozoite and schizont stages when the parasite lysates were probed with anti-methyl-lysine specific antibody. (B) The same antibodies also immuno-stained the asexual blood stages of the parasite as examined through immuno-fluorescence assay. The representative confocal microscopy images are shown above. DIC- bright field, DAPI-stained nucleus (blue), immunofluorescent cells labeled with anti-Methyl lysine antibody (green), and merged images.

Identification of P. falciparum lysine methylated proteins

Next, we carried-out global proteome analysis of lysine methylated Plasmodium proteins at asexual blood stages by immunoprecipitating the proteins from Plasmodium lysates prepared from the three asexual blood stages; ring, trophozoite and schizont using either anti-mono/di methyl lysine and anti-trimethyl lysine antibodies. Mass spectroscopic analysis of immunoprecipitated proteins identified a total of 570 putative lysine methylated proteins in P. falciparum asexual blood stages (Fig. 2). The two antibodies pulled-down approximately the same number of proteins at each stage, with considerable overlap. The one hundred and thirty two proteins identified in the proteome analysis were common to all the three stages (Fig. 2, Supplementary Table 1). To determine whether these putative methylated proteins have methylated lysine residues, we analyzed the spectra of the peptides generated in mass spectrometric analysis. As shown in Table 1, we could identify 364 K-methylated sites on 266 peptides corresponding to Plasmodium proteins. The representative spectra of few of the methylated peptides corresponding to Plasmodium proteins are shown in Supplementary Fig. 1.
Figure 2

Lysine methylated proteins of Plasmodiun falciparum.

Anti-methyl-lysine antibodies were used to immunoprecipitate stage specific lysine methylated proteins from P. falciparum lysates. N = number of proteins, MK, K = Proteins immunoprecipitated by anti-monomethyl lysine antibody, TMK, TK = Proteins immunoprecipitated by anti-trimethyl lysine antibody.

Table 1

List of the peptides with confirmed methylated lysine residues.

Peptide SequenceAccession no.ProteinModificationMethod
KVnGEkGSGEnNDQIITIRPF3D7_0100100erythrocyte membrane protein 1, PfEMP1 (VAR)K6(Dimethyl)IP
IDIKFGLSDEYGTPIAGPPNIEIPqKISGLVEQADQAAVEVAkDTSQSVAAkPF3D7_0101600rifin (RIF)K43(Dimethyl); K52(Dimethyl)IP
VLCkITIDYIYMFNPPIKPF3D7_0103100vacuolar protein sorting-associated protein 51, putative (VPS51)K4(Dimethyl)IP
ITYDLLGYGEMDDVSAYSLSYALNDVETIkPF3D7_0104200StAR-related lipid transfer proteinK30(Dimethyl)HILIC
HNNNDNNNDNDNIcNSSNNNMVAVDINEENNkPF3D7_0105700asparagine-rich antigen Pfa35-2K32(Dimethyl)HILIC
QTkEDIVTSLPHTSnkPF3D7_0106500conserved Plasmodium protein, unknown functionK3(Methyl); K16(Methyl)IP
KGEDSIIGILAQSSFVFYNWNIRTFIDPYNnFTDDEIVHALkLNGINLGKPF3D7_0112200multidrug resistance-associated protein 1 (MRP1)K42(Dimethyl)IP
QNEkIEQNEEDKPF3D7_0113400Plasmodium exported protein, unknown functionK4(Dimethyl)IP
kWFEKCITETqNGEIAkKPF3D7_0115000surface-associated interspersed protein 1.3 (SURFIN 1.3) (SURF1.3)K1(Trimethyl); K17(Methyl)IP
DETADknAANNGEQVMSRPF3D7_0202000knob-associated histidine-rich protein (KAHRP)K6(Trimethyl)HILIC
EALAIkDkLPGGLDEYQNQLYGICNETCTTcGPAAIDYVPADAPNGYAYGGSAHDGSHGNLrPF3D7_0202000knob-associated histidine-rich protein (KAHRP)K6(Trimethyl); K8(Trimethyl)IP
LNLcSSkFIHRPF3D7_02039005′-3′ exonuclease, N-terminal resolvase-like domain, putativeK7(Dimethyl)HILIC
ENNVSnIEEEnIILDSkPF3D7_0205100conserved Plasmodium protein, unknown functionK17(Trimethyl)HILIC
cDAIASDcFLSGnINVEkPF3D7_0207800serine repeat antigen 3 (SERA3)C-Term(Methyl)IP
AISESGFEHPSEVQqETIPAAITGTDILcQAkPF3D7_0209800ATP-dependent RNA helicase UAP56 (UAP56)K32(Trimethyl)HILIC
DLVEEEGHAQINnLIINDkPF3D7_0214800conserved Plasmodium membrane protein, unknown functionC-Term(Methyl)IP
NDINNNNNNNNININNNNINNSCSNnYGLkPF3D7_0218200conserved Plasmodium protein, unknown functionK30(Dimethyl)HILIC
EnILEESQVNDDIFNSLVkPF3D7_0220000liver stage antigen 3 (LSA3)K19(Dimethyl)HILIC
IFAGTYGITnYELIGnkPF3D7_0301300alpha/beta hydrolase, putativeC-Term(Methyl)IP
GVVSFIDSFEHkPF3D7_0302100serine/threonine protein kinase (SRPK1)K12(Dimethyl)HILIC
KkkkWICNHFHIYSINNDMINYTTTSTHYSILISYNkPF3D7_0304800conserved Plasmodium membrane protein, unknown functionK2(Trimethyl); K3(Trimethyl); K4(Dimethyl); K37(Dimethyl)IP
ELNNTLNkPF3D7_0305200conserved Plasmodium protein, unknown functionK8(Dimethyl)IP
ESNTEDDNIDSEQNSSInMnNSNDDSQNSSNSESNNNDDNDSNNSNEkPF3D7_0305800P-loop containing nucleoside triphosphate hydrolase, putativeK48(Trimethyl)HILIC
HNNNNNNNNNNNNNNNNNNNNNNcCTFkPF3D7_0309000dual specificity protein phosphatase (YVH1)K28(Dimethyl)HILIC
SCSDCAVIPnLFkPF3D7_0309500asparagine synthetase, putativeK13(Dimethyl)IP
CDSSIDNNGGDNGnGTEGnVnPDTPSCNDNDDDDkPF3D7_0315200circumsporozoite- and TRAP-related protein (CTRP)K35(Dimethyl)HILIC
NnIHnkPnGYNLKPF3D7_0317200cdc2-related protein kinase 4 (CRK4)K6(Trimethyl)HILIC
VLDELIVVkALADIPYFDLTEIEGFDFKPF3D7_0317400conserved Plasmodium protein, unknown functionK9(Trimethyl)IP
SSTFSNNSDYSDNSNNSNnSDYSDYSDYSYGInNSYEYVPENSNLkPF3D7_0317700CPSF (cleavage and polyadenylation specific factor), subunit A, putativeK46(Methyl)HILIC
SLCHPGECVGALAAqSIGEPATqMTLNTFHFAGVGSkNVTLGVPRLKELINIVkPF3D7_0318200DNA-directed RNA polymerase II subunit RPB1 (RPB1)K37(Methyl); K54(Trimethyl)IP
NNMMINNNNNIkPF3D7_0319400kinesin-8, putativeK12(Dimethyl)HILIC
IILTFknIELTNVELKYIINLLMLIVNLkPF3D7_0319700ABC transporter I family member 1, putative (ABCI3)K6(Trimethyl); C-Term(Methyl)IP
NCNDTnNYDNNSNNNNNNDNHNDNHSNNSGINSSFNNNNNVHNkPF3D7_0319700ABC transporter I family member 1, putative (ABCI3)K44(Methyl)HILIC
LYSFPNLSTVTSNNFNYHFLIENGIAYIAVFPVTYPkkLAFLFLnDIcKPF3D7_0320100protein transport protein SEC22 (SEC22)K37(Dimethyl); K38(Trimethyl)IP
TNkPnIVQGQKPF3D7_0321300conserved Plasmodium protein, unknown functionK3(Methyl)HILIC
NNCHGNHNNVcDGNHNNVCDGNHNNICNGNNNNNNNCDSHILTkPF3D7_0321500peptidase, putativeK44(Dimethyl)HILIC
EIcDMEIKKkPF3D7_0321600ATP-dependent RNA helicase, putativeC-Term(Methyl)IP
QIDYFNGPSPkDQGIIMLNNIILkDELkNDLGSLGEKGIVMCDESTNCDYKPF3D7_0321800WD repeat-containing protein, putativeK11(Trimethyl); K24(Trimethyl); K28(Methyl)IP
HDQPGLLSMANAGPnTnSSQFFITLVPcPWLDGkPF3D7_0322000peptidyl-prolyl cis-trans isomerase (CYP19A)K34(Methyl)HILIC
KPFFFIQLnTDSSQHEkPF3D7_0402300reticulocyte binding protein homologue 1 (RH1)K17(Trimethyl)HILIC
NSTNQQITLQELKqVqENVEkPF3D7_0402300reticulocyte binding protein homologue 1 (RH1)K21(Dimethyl)IP
NTQGNSNHDNNnnnNNNNCNNSNSNSTCSSHSkPF3D7_0403800alpha/beta hydrolase, putativeK33(Trimethyl)HILIC
NVDGINNVGDINNAGDTNNAGDINNVGDINnSVDIYnVEHIDEAEkKPnLDNPKPF3D7_04053006-cysteine protein (LISP2)K46(Dimethyl)IP
INTLFqkPF3D7_0405400pre-mRNA-processing-splicing factor 8, putative (PRPF8)K7(Dimethyl)HILIC
InknDGnYYYHNNFSNNSKPF3D7_0405700lysine decarboxylase, putativeK3(Dimethyl);HILIC
IIQKETMNSNANNIYFNNNNNINNINNNNINNNNVYNSHVENIINMKPLIYYGKEkPF3D7_0407600conserved Plasmodium protein, unknown functionC-Term(Methyl)IP
GNINENIFSMnnTYnNMkPF3D7_0409400heat shock protein 40 (DnaJ)K18(Trimethyl)IP
IHANENGDTNkPF3D7_0409400heat shock protein 40 (DnaJ)K11(Dimethyl)IP
QVNIYIENSDLnkPF3D7_0409800zinc finger protein, putativeC-Term(Methyl)HILIC
QTPPAPAPAAPPSPPRPLPkPkPPkPDLPPALkPF3D7_0412900erythrocyte membrane protein 1, PfEMP1 (VAR)K20(Trimethyl); K22(Trimethyl); K25(Trimethyl); K33(Dimethyl)IP
IESESNNQSISSFQnnILSLDIPLDSSLLnGDEEKLkPF3D7_0413900ubiquitin carboxyl-terminal hydrolase 13, putative (USP13)K37(Dimethyl)IP
DTLESYFSTFGEIDVVQIVLDSSGrSrCFGFVVFSNENSVAkVLkPF3D7_0414500RNA-binding protein, putativeK42(Trimethyl); K45(Trimethyl)IP
NNDNNDNNDNNDNNDnnDnEETLIIQIETkPF3D7_0415700conserved Plasmodium protein, unknown functionK30(Dimethyl)HILIC
MDDDkNDYnPEEEVVTGNWNTPKPF3D7_0419600ran binding protein 1, putativeK5(Trimethyl)HILIC
IqqILPqNGDkPGKPF3D7_0421300erythrocyte membrane protein 1, PfEMP1 (VAR)K11(Methyl)HILIC
NNNNNNNNNNNNNnNNDNGVkPF3D7_0423600conserved Plasmodium protein, unknown functionC-Term(Methyl)HILIC
QVEEGIkEnDTEGNDKPF3D7_0500800mature parasite-infected erythrocyte surface antigen (MESA)K7(Dimethyl)HILIC
qVEEGIkENDTESKDKPF3D7_0500800mature parasite-infected erythrocyte surface antigen (MESA)K7(Trimethyl)HILIC
DIYkPIqSYANNFSKPF3D7_0501000Plasmodium exported protein, unknown functionK4(Trimethyl)HILIC
NGkEEFFGTPDDLISSFFSDMKPF3D7_0501500rhoptry-associated protein 3 (RAP3)K3(Trimethyl)HILIC
SSSLALVGTnnNDPIFAYCEkDNkSEYYGTPDDLITSFFSIIKPF3D7_0501600rhoptry-associated protein 2 (RAP2)K21(Dimethyl); K24(Dimethyl)IP
nTSnINNkSICIKPF3D7_0504700centrosomal protein CEP120, putative (CEP120)K8(Dimethyl)IP
MnIMnKEkTKnKPF3D7_0504800conserved Plasmodium protein, unknown functionK8(Trimethyl)IP
DGLDERGHLIEEEEnDkRDEkPF3D7_0505100trafficking protein particle complex subunit 8, putative (TRS85)K17(Dimethyl); K21(Dimethyl)IP
DIEqVDDnkEDkPF3D7_0505700conserved Plasmodium membrane protein, unknown functionK9(Trimethyl); K12(Trimethyl)IP
DkDNDIIDEnInNkINYYEKDNIKPF3D7_0505700conserved Plasmodium membrane protein, unknown functionK2(Dimethyl); K14(Dimethyl)IP
QMNkPInkPINkPINKPINKPINKPINKPINKPINKPF3D7_0508900conserved Plasmodium protein, unknown functionK4(Dimethyl); K8(Dimethyl); K12(Trimethyl)HILIC
QMNkPINkPINkPINkPINKPINKPINKPINKPINKPF3D7_0508900conserved Plasmodium protein, unknown functionK4(Dimethyl); K8(Trimethyl); K12(Trimethyl); K16(Trimethyl)IP
MIDLILNNGNkCYQKPF3D7_0510100conserved Plasmodium protein, unknown functionK11(Dimethyl)IP
LPVEISVHFINAGFDTVDTLCTLSnnSLDDVEkPF3D7_0510300stripes inner membrane complex protein, putative (SIP)K33(Dimethyl)HILIC
MMnAGnDLnSLMYkPF3D7_0511500RNA pseudouridylate synthase, putativeC-Term(Methyl)IP
NNLNGYkPF3D7_0512500conserved Plasmodium protein, unknown functionK7(Dimethyl)IP
ETNNDNINNNDDGNNNnNDDDGNVFITEPLSYNNLKNkPF3D7_0514300aspartate–tRNA ligase, putativeC-Term(Methyl)IP
LFSKDGVLNQGIqIckPF3D7_0515300phosphatidylinositol 3-kinase (PI3K)K16(Trimethyl)IP
LILYNNFLDIIYEkSNELMNAkNIcLWIYPILSLcKPF3D7_0516000RAP protein, putativeK14(Dimethyl); K22(Dimethyl)IP
ANAIITnLYLDGTLIIEnkPF3D7_0517500UTP–glucose-1-phosphate uridylyltransferase, putativeK19(Dimethyl)HILIC
ANAIITNLYLDGTLIIENkPF3D7_0517500UTP–glucose-1-phosphate uridylyltransferase, putativeC-Term(Methyl)HILIC
VQSINSTkHNNNIKPF3D7_0518100RAP protein, putativeK8(Methyl)HILIC
nETLEEYWWcVESALTWGDGDDNGPDMIVDDGGDATLLVHkPF3D7_0520900S-adenosyl-L-homocysteine hydrolase (SAHH)K41(Methyl)HILIC
EDkENLNDMYSVNVTNHnDEkPF3D7_0521300conserved Plasmodium protein, unknown functionK3(Trimethyl); C-Term(Methyl)IP
QNLNASNSSGQENkQnESDGKPF3D7_052460050S ribosomal protein L12, apicoplast, putativeK14(Dimethyl)IP
VPQGALNNIFSICYTSGTTGYPkPF3D7_0525100acyl-CoA synthetase (ACS10)K23(Methyl)HILIC
DITkYWcAQTNkNNNINNNKIIIPIKPF3D7_0525500WD repeat-containing protein, putativeK4(Dimethyl); K12(Dimethyl)IP
TVEkIVEVPVYVNRPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K4(Trimethyl)HILIC
EVYLEkIVEVPQIKPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K6(Trimethyl)IP
IIYQEkIVEVPQIkPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K6(Trimethyl); K14(Methyl)IP
IIYQEkIVEVPQIKPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K6(Dimethyl)IP
IIYqEkIVEVPQIkTVEKPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K6(Dimethyl); K14(Trimethyl)IP
IIYqEkIVEVPQIKTVEkPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K6(Dimethyl); K18(Trimethyl)IP
IIYQEkIVEVPQIkTVEkIVEVPVYVNRPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K18(Dimethyl)IP
IVEIPKEVYLEkIVEVPQIKPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K12(Trimethyl)IP
IVYVEkVKPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K6(Trimethyl)IP
NVDKIIYQEkIVEVPQIkPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K10(Trimethyl); C-Term(Methyl)IP
NVDKIIYQEkIVEVPqIKPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K10(Dimethyl)IP
NVDKIIYQEkIVEVPQIKPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K10(Trimethyl)IP
NVDkIIYQEKIVEVPQIKPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K4(Dimethyl)IP
TVEkIVEVPVYVNRPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K4(Trimethyl)IP
TVEkIVEVPVYVNRPF3D7_0525800inner membrane complex protein 1g, putative (IMC1g)K4(Dimethyl)IP
DITSIELSNEVLTTnVMDIHNCSLDISkPF3D7_0526600conserved Plasmodium protein, unknown functionK28(Methyl)HILIC
VGLLDYLnkPF3D7_0529400.2conserved Plasmodium protein, unknown functionK9(Methyl)HILIC
NISSFSLDQLVSLCnAYSkPF3D7_0529700conserved Plasmodium protein, unknown functionK19(Dimethyl)HILIC
EIDELNINNSDNIVIIEkLKEELqrPF3D7_0529800conserved Plasmodium protein, unknown functionK18(Dimethyl)IP
DYDkPCPNDYNYLGSVHTDDDEIcAPSSTYEGPCSGEELNIkPF3D7_0530800CPW-WPC family proteinK4(Dimethyl); K42(Methyl)HILIC
VYMGkPFKDVYnEKkPF3D7_0532300Plasmodium exported protein (PHISTb), unknown functionK5(Methyl)IP
CAqnLGGIVAPSSGVLVGIAEGALYAWKPTAITAAkPF3D7_0600700rifin (RIF)K36(Methyl)IP
LSQEEIEkPF3D7_0602600conserved Plasmodium protein, unknown functionK8(Dimethyl)IP
MkLSqEEIEKPF3D7_0602600conserved Plasmodium protein, unknown functionK2(Dimethyl)IP
EnSTLQnkLSnEIKPF3D7_0603400trophozoite exported protein 1 (TEX1)K8(Trimethyl)HILIC
ISSIILnSkPF3D7_0604800RAP protein, putativeC-Term(Methyl)HILIC
EYDVYENGLNSCSCIAENSIEISQLInVISKNkIINNNIMLIYIkLIDkPF3D7_0604800RAP protein, putativeK33(Methyl); K45(Trimethyl)IP
QQIREALDLEkGIkPF3D7_0605500cyclin dependent kinase binding protein, putativeK11(Dimethyl); K14(Trimethyl)IP
QQIREALDLEkGIkPF3D7_0605500cyclin dependent kinase binding protein, putativeK11(Trimethyl); K14(Dimethyl)IP
MNInIENILLTNSNkPcINQLIWKLkPF3D7_0609500conserved Plasmodium protein, unknown functionK15(Dimethyl); K26(Methyl)IP
YGYCIIDLSKAcLTnYVLLQkPF3D7_0609600hypothetical proteinC-Term(Methyl)IP
NTNKNNNIQSDTLSVNINnSNDFNkPF3D7_0611600conserved Plasmodium protein, unknown functionK25(Trimethyl)IP
SLNINLNNITkSVADMKPF3D7_0612200leucine-rich repeat protein (LRR6)K11(Dimethyl)HILIC
TITINGSNGNPSSkPF3D7_06127006-cysteine protein (P12)K14(Methyl)HILIC
nPFQnIknINNKPF3D7_0613300rhoptry protein ROP14 (ROP14)K7(Methyl)HILIC
NnTMDnnNNDENNGHSGDEKSGHHNDDQkPF3D7_0615600conserved Plasmodium protein, unknown functionC-Term(Methyl)IP
TGYFDMLkVENGcMIkPF3D7_0616900conserved Plasmodium protein, unknown functionK8(Dimethyl); K16(Methyl)IP
KVQQIkYASFVLVnLALITLISSIFSINrEYNIPLcVQNDGSSDICMDGKPF3D7_0618000conserved Plasmodium membrane protein, unknown functionK6(Trimethyl)IP
kSSSNSMDSSYNPnMKPF3D7_0622900transcription factor with AP2 domain(s), putative (ApiAP2)K1(Methyl)IP
NITTNNNnnnNSNNNNSNNNNSNNNNSNNDCVNNEETNFkPF3D7_0623100nuclear polyadenylated RNA-binding protein NAB2, putative (NAB2)K40(Methyl)HILIC
NLITFDYkQMELFVMAYLSFDEQLLKLLnYSDVFIETAKVLFnTNDVTNELrPF3D7_0625300DNA polymerase 1, putativeK8(Dimethyl)IP
VDLPIISEkDknDILnFAIPMGCNFIAASFIQSADDVRPF3D7_0626800pyruvate kinase (PyrK)K9(Dimethyl); K11(Dimethyl)IP
DLSSnEEIInISNEKKENDkPF3D7_0627600conserved Plasmodium protein, unknown functionK20(Dimethyl)IP
KNSSNNNnnnTIVDISDGDYTNDEEGTNkPKPF3D7_0629500amino acid transporter, putativeK29(Dimethyl)IP
nNMSNMNNMNNMNNMNNMNNMNNMNNMNNMDSINNVISYSCTNPNMkPF3D7_0629700SET domain protein, putative (SET1)K47(Methyl)HILIC
NFKcDNCIkCDnCLLHFDSSLKPF3D7_0629700SET domain protein, putative (SET1)K9(Dimethyl)IP
VnnHTLLTDVCLAAkFEAESLKPF3D7_0632800erythrocyte membrane protein 1, PfEMP1 (VAR)K15(Dimethyl)HILIC
SINLYkPINPDnGTKPF3D7_0700100erythrocyte membrane protein 1, PfEMP1 (VAR)K6(Methyl)HILIC
YYDCEINNIKSIIQNEISERIkPF3D7_0703600conserved Plasmodium protein, unknown functionK22(Trimethyl)IP
INqNnILkPF3D7_0704100conserved Plasmodium membrane protein, unknown functionK8(Dimethyl)HILIC
YNITTcDALDNAMDTQNGNIKNDNIKNDNIkPF3D7_0704200tRNA m5C-methyltransferase, putativeK31(Dimethyl)IP
SNLSKLGkLWSIIEPDMIGEIkVFSYnnDLTSNFVETYKPF3D7_0704400phosphoinositide-binding protein, putativeK8(Trimethyl); K22(Trimethyl)IP
GDGECDGEGEGEDDDEDDDDnnnNNkPF3D7_0704600E3 ubiquitin-protein ligase (UT)K26(Trimethyl)HILIC
LNIITGPnMGGkPF3D7_0706700DNA mismatch repair protein MSH2, putative (MSH2-2)K12(Dimethyl)HILIC
IIPDkTnnTLTIEDSGIGMTKPF3D7_0708400heat shock protein 90 (HSP90)K5(Trimethyl)HILIC
IIPDkTNnTLTIEDSGIGMTKPF3D7_0708400heat shock protein 90 (HSP90)K5(Trimethyl);HILIC
kPEEVTnEEYASFYKPF3D7_0708400heat shock protein 90 (HSP90)K1(Trimethyl)HILIC
NESDESSnEEGSSTSATSLSFLCEGYDSLDnnkPF3D7_0708500heat shock protein 86 family proteinK33(Trimethyl)HILIC
EDNINNINNVDNNISMVnnVNNVNNVNNDQYkPF3D7_0709600ribonucleases P/MRP protein subunit POP1, putative (POP1)K32(Trimethyl)IP
nEAEIILSSkQIIGYVSSGGHVLSkGYGYGVAHISFYLFLHNLLNHLFALKPF3D7_0709600ribonucleases P/MRP protein subunit POP1, putative (POP1)K10(Dimethyl); K25(Dimethyl)IP
LIqVnnYDkPF3D7_0710000conserved Plasmodium protein, unknown functionK9(Trimethyl)HILIC
LNEqINVTLEnkPF3D7_0710200conserved Plasmodium protein, unknown functionK12 Dimethyl)IP
LNEqINVTLEnkPF3D7_0710200conserved Plasmodium protein, unknown functionC-Term(Methyl)IP
SVSLSLSSNEKSSSSFFSTkPF3D7_0711400histone deacetylase complex subunit SAP18, putative (SAP18)K20(Dimethyl)HILIC
TnITNQkPF3D7_0713900conserved Plasmodium protein, unknown functionK7(Dimethyl)IP
NNnDIINNNISVNkPF3D7_0715800drug/metabolite exporter, drug/metabolite transporterC-Term(Methyl)IP
NIFENIDVNYILQNINkELLINRPF3D7_0717600conserved Plasmodium protein, unknown functionK17(Trimethyl)HILIC
NIGNVcYLkPF3D7_0717800conserved Plasmodium protein, unknown functionK9(Trimethyl)HILIC
IFIISNLNEILTKIEEnLVLnQNLLIINIYEIYHIDIINNQNMMINANAIINTLkPF3D7_0718000dynein heavy chain, putativeK55(Dimethyl)IP
VESLLNNQPIGGkkrPF3D7_0720100small subunit rRNA processing protein, putativeK13(Dimethyl); K14(Dimethyl)IP
NNNNNNkkPF3D7_0721000conserved Plasmodium membrane protein, unknown functionK7(Trimethyl); K8(Dimethyl)IP
ENSPLNIHNNDDnDDnDDnDENNGDNNNNNDDNNNNNDDNNNkPF3D7_0721300DEAD/DEAH box ATP-dependent RNA helicase, putativeK43(Dimethyl)HILIC
DGLLnFEIDNLNDNkNNDNIGSNkPF3D7_0723800conserved Plasmodium protein, unknown functionK15(Methyl); K24(Trimethyl)IP
NVVTqSnnkPF3D7_0724000Rab GTPase activator and protein kinase, putativeK9(Methyl)IP
ESnnNNTNNVNNNEDINFSNIDETQkPF3D7_0726300DNA mismatch repair protein PMS1, putative (PMS1)K26(Dimethyl)HILIC
EYIEHIqSVTNNKPSYYkPF3D7_0726400conserved Plasmodium membrane protein, unknown functionC-Term(Methyl)IP
KEDLYIEDENYPSYNIDANSNTLSkVLYkKPF3D7_0726500ubiquitin carboxyl-terminal hydrolase, putativeK25(Trimethyl); K29(Trimethyl)IP
GNLNIDEkVDDVNFncnDFISKPF3D7_0727800cation transporting ATPase, putativeK8(Trimethyl)HILIC
NLLFISFInLFYHkPF3D7_0728100conserved Plasmodium membrane protein, unknown functionK14(Dimethyl)IP
YYIknnIINKPF3D7_0728700alpha/beta hydrolase, putativeK4(Methyl)HILIC
FFqNGDSqPLSGkPVTQSSDKPF3D7_0800300erythrocyte membrane protein 1, PfEMP1 (VAR)K13(Methyl)HILIC
IPkEHIPMnKPTYQPYINILNEKPF3D7_0801900conserved Plasmodium protein, unknown functionK3(Methyl)HILIC
MLknIFSEYLkSYDnKPF3D7_0802600adenylyl cyclase beta (ACbeta)K3(Dimethyl); K11(Dimethyl)IP
ENMLCTTLKDqSKkkPF3D7_0806100conserved Plasmodium protein, unknown functionK14(Trimethyl); K15(Dimethyl)IP
YVVAGTqTVVSkSSnAAAqATKPF3D7_0808800rifin (RIF)K12(Trimethyl)HILIC
EEEGETcTPASPAPAPAPSEDPPVPAPAGDqkPF3D7_0809100erythrocyte membrane protein 1, PfEMP1 (VAR)K32(Methyl)HILIC
KYNSNNNkNNNNNNNNNDNNDDNNCGNnHGCnnYSEIASSLKPF3D7_0809200asparagine-rich antigen Pfa55-14 (pfa55-14)K8(Trimethyl)IP
FSILNYDSkPF3D7_0811900RNA-binding protein, putativeC-Term(Methyl)HILIC
TqGTPFDVKVIAILkPF3D7_0811900RNA-binding protein, putativeK15(Dimethyl)IP
YICkIIYRFLEYnFSVTIMDYSCFPINEKMSNKEKPF3D7_0812100conserved Plasmodium protein, unknown functionK4(Dimethyl)HILIC
ESNISNEYDnnINNASINkWSSLKSLNDLNNNDYFENKcISSFNGIIVSILNNMGLIKPF3D7_0812100conserved Plasmodium protein, unknown functionK19(Dimethyl)IP
IYVQVEDGNGcYNIccLQkPF3D7_0813300conserved Plasmodium protein, unknown functionK19(Methyl)HILIC
NMNLNINSGENVLLLGKnGIGkPF3D7_0813700ABC transporter F family member 1, putative (ABCF1)K22(Trimethyl)IP
GLHQITrCGSTVITDQYVSGQDnSEHVVQEkTVSFIEILLSREQLDMkPF3D7_0814200DNA/RNA-binding protein Alba 1 (ALBA1)K31(Dimethyl); K48(Methyl)IP
NVDDEEGSSDnSEDnDSSDFDVDIDEENDDTINGNINNGIEkPF3D7_0816000ribosome assembly protein RRB1, putative (RRB1)K42(Dimethyl)HILIC
NSkEIEnnLkPF3D7_0817200conserved Plasmodium protein, unknown functionK3(Dimethyl); K10(Dimethyl)HILIC
TLVEQcVnnDkDELTVEERPF3D7_081820014-3-3 protein (14-3-3I)K11(Methyl)HILIC
TLVEQcVNnDkDELTVEERPF3D7_081820014-3-3 protein (14-3-3I)K11(Trimethyl)HILIC
LQPAEIETcMkPF3D7_0818900heat shock protein 70 (HSP70)K11(Methyl)HILIC
SGVDEkPMIEVTYqGEKPF3D7_0818900heat shock protein 70 (HSP70)K6(Trimethyl);HILIC
kSqIFTTYADnQPGVLIQVYEGERPF3D7_0818900heat shock protein 70 (HSP70)K1(Dimethyl)IP
VNGQnVSEQnDISnQkPF3D7_0820200phosphatidylglycerophosphate synthase (PGPS)K16(Trimethyl)HILIC
DEDGkEEPVLcFVDnQnQKPF3D7_0821900conserved Plasmodium protein, unknown functionK5(Dimethyl)IP
SCYcSEEFAEPcTSEDLVDINNINQIMDNAFCEYNnNDNNNNDNNkPF3D7_0822700conserved Plasmodium protein, unknown functionK46(Trimethyl)HILIC
kNISHSILGCInnLKPF3D7_0826200alpha/beta hydrolase, putativeK1(Methyl)HILIC
IDFVLIGSEIVTDnGGIInkPF3D7_0828500translation initiation factor EIF-2b alpha subunit, putativeK20(Dimethyl)HILIC
HPYkInEEIInIWLnRPF3D7_0831100surface-associated interspersed protein 8.1 (SURFIN 8.1) (SURF8.1)K4(Methyl)HILIC
ATSGDTHLGGEDFDNkPF3D7_0831700heat shock protein 70 (HSP70-x)K16(Dimethyl)HILIC
ATSGDTHLGGEDFDNkPF3D7_0831700heat shock protein 70 (HSP70-x)K16(Dimethyl)IP
IINEPTAAAIAYGLDkPF3D7_0831700 ;PF3D7_0917900heat shock protein 70 (HSP70-x)K16(Dimethyl)HILIC
DTNVDPEqEEEEEEDDSEEqDATVNGPEDGGGEATqQPQGPSEkPF3D7_0833500erythrocyte membrane protein 1, PfEMP1 (VAR)K44(Trimethyl)HILIC
CGLAVAkNSYHTcNFGkALkCTELCSQANCSNEATIADGVNKILSTAKPF3D7_0901100rifin (RIF)K7(Trimethyl); K17(Dimethyl); K20(Dimethyl)IP
LQYQVSCATDPLGETSVLCFYKTIGEPNATAAVVENAkIVVTDAIqEASEVAFkPF3D7_0901500rifin (RIF)K38(Dimethyl); K54(Dimethyl)IP
nLISPHILNVcTkPF3D7_0903300conserved Plasmodium membrane protein, unknown functionC-Term(Methyl)HILIC
nKINDNNNNNINcDNTkPF3D7_0903400DEAD/DEAH box helicase, putativeK17(Dimethyl)IP
TDLDkSFISDIDInCTnNFNEKPF3D7_0903800LCCL domain-containing protein (CCp4)K5(Dimethyl)HILIC
KPFFFDMISNMLqkPKPF3D7_0905100nucleoporin NUP100/NSP100, putative (NUP100)K14(Trimethyl)HILIC
EDNNnEkNEEIEkGKPF3D7_0906000exoribonuclease II (RNaseII)K7(Dimethyl); K13(Dimethyl)IP
LPNVNNAGILNNPkPF3D7_0909900helicase with Zn-finger motif, putativeK14(Trimethyl)HILIC
LPNVNNAGILNNPkPF3D7_0909900helicase with Zn-finger motif, putativeK14(Dimethyl)HILIC
IIYHSSFDTDMNKkHFVVCSnVcIEnISIITNTVIKLPNVNNAGILNNPKPF3D7_0909900helicase with Zn-finger motif, putativeK14(Methyl)IP
NSNNNNNNNNNLknKPF3D7_0911100conserved Plasmodium protein, unknown functionK13(Dimethyl);HILIC
NkGTNHYIIKNEINNEGESFQELFYKPF3D7_0912000conserved Plasmodium protein, unknown functionK2(Methyl)IP
LSqKMHTLYYNNNIFKNIIqnIPSkPF3D7_0913900arginine–tRNA ligase, putativeK25(Dimethyl)IP
KNNNNNNNnnNIISQNVErLNDENLIKkPF3D7_0914100conserved Plasmodium protein, unknown functionC-Term(Methyl)IP
TLQFLLTNkDDEIEQIKPF3D7_0914500conserved Plasmodium protein, unknown functionK9(Trimethyl)HILIC
DEKNICNNNNNDCNNNNDCNnnnDCNNSCIDkPF3D7_0916400conserved Plasmodium protein, unknown functionC-Term(Methyl)IP
qATkDAGTIAGLNIVRPF3D7_0917900heat shock protein 70 (HSP70-2)K4(Trimethyl)HILIC
VNALQHFAALPnVELTDVPSSGPMGnkPF3D7_0918000glideosome-associated protein 50 (GAP50)C-Term(Methyl)HILIC
nAQICINYAFAHFISSGDkTTNLVITAKPF3D7_0918400conserved Plasmodium protein, unknown functionK19(Methyl)HILIC
NPMSSICEQNNEcVLDPNNTCESPGkPF3D7_0918400conserved Plasmodium protein, unknown functionK26(Dimethyl)HILIC
NNLPkPEPLTSVYSFSTSGTYSFGPF3D7_0919200PPPDE peptidase, putativeK5(Trimethyl)HILIC
EFVkASnELIDnDVVFVYVDTISLAKTADNFEIkPF3D7_0919400protein disulfide isomerase (PDI9)K4(Dimethyl); C-Term(Methyl)IP
SIIEkSLIEnNNNNNNNNNNNNIPKPF3D7_0919800TLD domain-containing proteinK5(Trimethyl)IP
QVSETTLTSQVkNVENFLYLYnFEEIIVLMKPF3D7_0920400conserved Plasmodium protein, unknown functionK12(Methyl)HILIC
kSNSSYSALYTSnNEEEQEDEDEDENEDEDENEDEDENEDEDEEDVENEQNkkPF3D7_0922100ubiquitin-like protein, putativeK1(Dimethyl); K52(Trimethyl); K53(Trimethyl)IP
NNTTTTNNNNNNNNNNNnSGYnnNNSGYNNNNSGHYNIYEEEkPF3D7_0922100ubiquitin-like protein, putativeC-Term(Methyl)IP
LnSYEFSDGkPF3D7_0922800conserved Plasmodium protein, unknown functionK10(Dimethyl)HILIC
SEDSENSKcEEENTDDYMLnFEQIYNSYNNIETTSFFSkPF3D7_0922800conserved Plasmodium protein, unknown functionK39(Dimethyl)IP
kNNINKPQYNEKPF3D7_0923100OTU-like cysteine protease, putativeK1(Dimethyl)IP
nVkEnFGDIYYSFGKPF3D7_0924200conserved Plasmodium protein, unknown functionK3(Dimethyl)HILIC
ENnTILYGnNNNNNNNNNNNNNNNNNNNNNNNNNNNNGcDLLCNIkPF3D7_0925800conserved Plasmodium protein, unknown functionK46(Dimethyl)HILIC
NLENDILkPF3D7_0926100protein kinase, putativeK8(Dimethyl)HILIC
NSCDTAISTMNnqTIDGDTITIILGkKPF3D7_0929200RNA-binding protein, putativeK26(Dimethyl)IP
NSCDTAISTMnnqTIDGDTITIILGKKIIDkPF3D7_0929200RNA-binding protein, putativeC-Term(Methyl)IP
NEHLVSEDPnDDCFInYPLATINLDISDPYkEISEDLIkPF3D7_0929400high molecular weight rhoptry protein 2 (RhopH2)K31(Dimethyl); K39(Dimethyl)HILIC
NNnnEDEDHLNDLCYSPLLMEDIIkPF3D7_0934100TFIIH basal transcription factor complex helicase XPD subunit (XPD)K25(Dimethyl)HILIC
IPADQHLTSYSGPSPFEIFGkPF3D7_0934900conserved Plasmodium protein, unknown functionK21(Dimethyl)HILIC
KPAPTAGGEEDQTEkPF3D7_1000100erythrocyte membrane protein 1, PfEMP1 (VAR)C-Term(Methyl)HILIC
MAAQSSGGGGGCGEEDkPF3D7_1000100erythrocyte membrane protein 1, PfEMP1 (VAR)K17(Methyl)IP
FnESCMPRPPGSVPGPVIDRAFCDTVDTLVLPSGTGSQTSASTnAVIkPF3D7_1000200rifin (RIF)C-Term(Methyl)IP
EcLkCAqNLGGIVAPSTGVLGEIAALAVnAWKPF3D7_1000400rifin (RIF)K4(Dimethyl)IP
GLAAGNAHGMnIVIYHLkPF3D7_1000500rifin (RIF)K18(Trimethyl)HILIC
EYMTHHVDTKkNNnHnEHHINDNNNNNNIVIIPkDKPF3D7_1002600conserved Plasmodium protein, unknown functionK11(Dimethyl); K34(Trimethyl)IP
IVEVPQIkEVVRPF3D7_1003600inner membrane complex protein 1c, putative (IMC1c)K8(Trimethyl)HILIC
TVEVPIIkTVEKPF3D7_1003600inner membrane complex protein 1c, putative (IMC1c)K8(Trimethyl)HILIC
IEIVEkVVERPF3D7_1003600inner membrane complex protein 1c, putative (IMC1c)K6(Dimethyl)IP
IEIVEkVVERPF3D7_1003600inner membrane complex protein 1c, putative (IMC1c)K6(Trimethyl)IP
IIEkWHDKIVEVPQIkEVVRPF3D7_1003600inner membrane complex protein 1c, putative (IMC1c)K4(Trimethyl); K16(Dimethyl)IP
IIEkWHDkIVEVPQIkEVVrPF3D7_1003600inner membrane complex protein 1c, putative (IMC1c)K8(Methyl); K16(Methyl)IP
IVEVPQIkEVVRPF3D7_1003600inner membrane complex protein 1c, putative (IMC1c)K8(Dimethyl)IP
NVTHIVEkIVEVPEVKPF3D7_1003600inner membrane complex protein 1c, putative (IMC1c)K8(Trimethyl)IP
NVTHIVEkIVEVPEVKPF3D7_1003600inner membrane complex protein 1c, putative (IMC1c)K8(Dimethyl)IP
TIIQEkIIHVPKPF3D7_1003600inner membrane complex protein 1c, putative (IMC1c)K6(Trimethyl)IP
TIIQEkIIHVPKPF3D7_1003600inner membrane complex protein 1c, putative (IMC1c)K6(Dimethyl)IP
TVEVPIIkTVEKPF3D7_1003600inner membrane complex protein 1c, putative (IMC1c)K8(Trimethyl)IP
YIEkIVEVPHIHYKPF3D7_1003600inner membrane complex protein 1c, putative (IMC1c)K4(Trimethyl)IP
KNYncDDIInIISLIkNHWDIFIGKPF3D7_1003900conserved Plasmodium protein, unknown functionK16(Dimethyl)HILIC
KLnFVTnkPF3D7_1005100conserved protein, unknown functionK8(Dimethyl)IP
QAQMGNFSFPGFNSPVLPTNNNILTnTLDInNkSTLPNIPLYIPNTTSNINNINNINNLLQQPIGNNIIYPPkPF3D7_1009400zinc finger protein, putativeK33(Methyl); K73(Trimethyl)HILIC
HTNIKNHSYLFYIFSFVkNHLQNYPFHHQLIQHINkNANMLQRPF3D7_1009900conserved Plasmodium protein, unknown functionK18(Trimethyl); K36(Trimethyl)IP
MINNNLFVFFLLSFFLSkIcTCLYVTDGSSIILEnIGTkYKLFSTDMKPF3D7_1010700dolichyl-phosphate-mannose protein mannosyltransferase, putativeK18(Trimethyl); K39(Trimethyl)IP
SYcNDQSTGTLEIVSEDLScLkPF3D7_1012400hypoxanthine-guanine phosphoribosyltransferase (HGPRT)C-Term(Methyl)HILIC
SVSIcIDDSDYIWkEnSScIKPF3D7_1012700NLI interacting factor-like phosphatase, putative (NIF4)K14(Trimethyl)HILIC
SNILNNELLNTTNNDINKHEEEkDEEHAILNCNIVNNnLLDLSnERIkPF3D7_1013200conserved Plasmodium protein, unknown functionK23(Trimethyl); K48(Trimethyl)IP
NkcTSSSVSSLTNVSSISSNNTMNSDIkPF3D7_1014200male gamete fusion factor HAP2, putative (HAP2)K2(Dimethyl); K28(Trimethyl)IP
EVEnVKEVEnVKEGENVkPF3D7_1014300conserved protein, unknown functionK18(Dimethyl)IP
DkNCYITSIDYNNNNNNNNNSINNSNNEYTGNNYnnNNkPF3D7_1015400conserved Plasmodium protein, unknown functionK2(Trimethyl); C-Term(Methyl)IP
IAMDVAASEFYnSENkTYDLDFKPF3D7_1015900enolase (ENO)K16(Trimethyl)HILIC
NADNkEDLTSADPEGQIMRPF3D7_1016300glycophorin binding protein (GBP)K5(Trimethyl)HILIC
NDEKSEYNYSGNVVEDFnFYMDkPF3D7_1016600Plasmodium exported protein (PHISTc), unknown functionK23(Dimethyl)IP
EDLNNSSSVPTTNINELnkPF3D7_1018200serine/threonine protein phosphatase 8, putative (PPP8)C-Term(Methyl)IP
NSTWcNNDLMcLPAILTKPYECYEDSSLnLENkVQYPNVYYDSLKPF3D7_1018300conserved Plasmodium protein, unknown functionK33(Dimethyl)HILIC
TKkCIHIIkNNSRqNDKPF3D7_1018800conserved protein, unknown functionK3(Dimethyl); K9(Trimethyl)IP
ILQNIIPLLFSkPF3D7_1018900conserved Plasmodium protein, unknown functionK12(Dimethyl)IP
SLDQEKnKTEIEnTGSKSIPnDSNEGANNkPF3D7_1021900conserved Plasmodium protein (10b antigen), unknown functionK30(Dimethyl)IP
KVTPNSNSNSNSNSSSSSNSSSSNNNHFEkPF3D7_1022000RNA-binding protein, putativeK30(Trimethyl)IP
DVCGLIGYNDISIWKNENCLENIKCIKNTVESYLnTAQEITkGnEEILTkPF3D7_1023700conserved Plasmodium protein, unknown functionK42(Dimethyl); K50(Dimethyl)IP
GDEYDEEDEDEEEEEEDDEnEDDDDDDVEDEDDDEESGECDkPF3D7_1023900chromodomain-helicase-DNA-binding protein 1 homolog, putative (CHD1)K42(Methyl)HILIC
VHNDFTIPNEYEEkPF3D7_1024700conserved Plasmodium protein, unknown functionK14(Dimethyl)IP
IMnSHFFGLSnETAScCGIMAYMGNRDASkPF3D7_1025100glutamine–fructose-6-phosphate aminotransferase [isomerizing], putative (GFPT)K30(Dimethyl)IP
MMLQYGNINAQGNYMNGQTNNVMNGQGNNYMNGQTNNVMNGqGNNYMNGqkPF3D7_1025400conserved Plasmodium membrane protein, unknown functionK51(Dimethyl)HILIC
TDkNSASTDADITEKPF3D7_102780060S ribosomal protein L3 (RPL3)K3(Trimethyl)HILIC
MDELNkEEIVDNINNEQAKPF3D7_1028400nucleolar preribosomal assembly protein, putativeK6(Trimethyl)HILIC
LIVTSATLDAEkPF3D7_1030100pre-mRNA-splicing factor ATP-dependent RNA helicase PRP22, putative (PRP22)C-Term(Methyl)HILIC
SNSSDGSSSDGSSSDGSSSDGNSSDGSSSSSSNYkPF3D7_1032000ribosome maturation factor RimM, putative (RimM)K35(Dimethyl)HILIC
AYCNNGNMDnnTkSNSSDGSSSDGSSSDGSSSDGNSSDGSSSSSSNYkPF3D7_1032000ribosome maturation factor RimM, putative (RimM)K13(Methyl); K48(Trimethyl)HILIC
DTNNcVIkSYDIDTnDTFFKPF3D7_1034000Sec1 family protein, putativeK8(Methyl)HILIC
FLDLHNAYSRNIKGIIIESLYKLSNTYNTSFENLCKHYLnInIPSINkPF3D7_1037000DNA polymerase zeta catalytic subunit, putativeK48(Trimethyl)IP
IMNSYVEINkSqnMLkPF3D7_1037000DNA polymerase zeta catalytic subunit, putativeK10(Methyl)IP
nIEnIIkLSDGIMIARPF3D7_1037100pyruvate kinase 2 (PyKII)K7(Dimethyl)IP
INILNVqnAnDIIkPF3D7_1038300conserved Plasmodium protein, unknown functionK14(Trimethyl)HILIC
MILFNFLInTLLLPHYEnSQnkPF3D7_1040200stevorK22(Dimethyl)HILIC
nHCDGDGFDCSEIGPNENGSFAIFkCPSCAISCRPF3D7_1100200erythrocyte membrane protein 1, PfEMP1 (VAR)K25(Dimethyl)IP
MENSDSNkDLQDSKPF3D7_1103700casein kinase II beta chain (CK2beta1)K8(Trimethyl)HILIC
INPLINDASLVSSFNPPDLkPF3D7_1103800CCR4-NOT transcription complex subunit 1, putative (NOT1)K20(Dimethyl)HILIC
kNEVNEYLLENNNYEQENNNYGQEKQFVSINTVDIENEILTQkPF3D7_1104100syntaxin, Qa-SNARE family (SYN13)K1(Methyl); K43(Dimethyl)IP
LSNVFVIGDnTkPYISLPRPF3D7_110540040S ribosomal protein S4, putativeK12(Trimethyl)HILIC
LPLILNkPF3D7_1106800protein kinase, putativeK7(Methyl)HILIC
nLPLDVLSnnNSSANIkPF3D7_1106800protein kinase, putativeK17(Dimethyl)HILIC
NLPLDVLSNNNSSANIkPF3D7_1106800protein kinase, putativeC-Term(Methyl)HILIC
VnInNDITkPF3D7_1107300polyadenylate-binding protein-interacting protein 1, putative (PAIP1)K9(Dimethyl)IP
McDEEAEAEEnVEMDGEEDnDDGVNNGkPF3D7_1107800transcription factor with AP2 domain(s) (ApiAP2)K28(Trimethyl)HILIC
NCTCCkPF3D7_1107900mechanosensitive ion channel proteinK6(Dimethyl)HILIC
IcnkPNLINYLKPF3D7_1113800conserved Plasmodium membrane protein, unknown functionK4(Methyl)HILIC
NPIPSNESQPIISFPNEDDNHAQnEGSInAPSEGEHNNTDNkPF3D7_1116000rhoptry neck protein 4 (RON4)C-Term(Methyl)IP
NTTQTGnkDTnEMDLENYEDTLNSPKPF3D7_1116700dipeptidyl aminopeptidase 1 (DPAP1)K8(Methyl)HILIC
SNYnFEkPFLWLARPF3D7_1117700GTP-binding nuclear protein RAN/TC4 (RAN)K7(Trimethyl)HILIC
EVFIrELISNSSDAIEkPF3D7_1118200heat shock protein 90, putativeK17(Dimethyl)IP
CLnLkKIYIQLLnEDEKPF3D7_1118300insulinase, putativeK5(Dimethyl)IP
VDNnNNNDDNNNDNNNNNkPF3D7_1118600histone acetyltransferase (MYST)C-Term(Methyl)IP
QVNNDIETLkKPF3D7_1120700conserved Plasmodium protein, unknown functionK10(Dimethyl)IP
MNNHIcAFrDDTRSkEADEFVrPF3D7_1121000palmitoyltransferase, putative (DHHC3)K15(Trimethyl)IP
NkNAnEnSNEIETNKPF3D7_1122900dynein heavy chain, putativeK2(Dimethyl)IP
GFNFcISSNKnnLEIVKGNkPF3D7_1126700autophagy-related protein 23, putative (ATG23)C-Term(Methyl)IP
DVEIkNAVnDVFLLYnAIYKPF3D7_1128400geranylgeranyl pyrophosphate synthase, putative (GGPPS)K5(Dimethyl)HILIC
MEDNkEENcELNKPF3D7_112920026S proteasome regulatory subunit RPN7, putative (RPN7)K5(Trimethyl)HILIC
NkNNNSISINIFGYSLGcSVTLqLVLDIAKSLYNDFFEDIKKVCYEGKPF3D7_1129300conserved Plasmodium protein, unknown functionK2(Methyl)IP
NSqEIIDnkPF3D7_1129300 K9(Dimethyl)IP
FNMkPFSYGVDVRPF3D7_113020060S ribosomal protein P0 (PfP0)K4(Trimethyl)HILIC
FCNDSLQkLVSnKPF3D7_1130700structural maintenance of chromosome protein, putativeK8(Trimethyl)HILIC
kPLGLIIRPF3D7_1132700mitochondrial ribosomal protein L2 precursorK1(Dimethyl)HILIC
EEEEDDkNEEVEEQnEEVVEKPF3D7_1133200conserved Plasmodium protein, unknown functionK7(Trimethyl);HILIC
NLTDSknEAETLIYSSEKPF3D7_1134000heat shock protein 70 (HSP70-3)K6(Trimethyl);HILIC
TQTEINLPFITANqTGPkPF3D7_1134000heat shock protein 70 (HSP70-3)K18(Trimethyl)HILIC
MSqSkPnqEISSRPF3D7_1134600zinc finger protein, putativeK5(Dimethyl)IP
LPQIYLnHkPF3D7_1135000.3conserved Plasmodium protein, unknown functionK9(Dimethyl)HILIC
NPESkPFCDLIVSGYKPF3D7_1135800conserved Plasmodium protein, unknown functionK5(Methyl)HILIC
TPQSNSDGYIPqCSDDkGrPF3D7_1136300tudor staphylococcal nuclease (TSN)K17(Dimethyl)IP
SkNNIMDILNnKPF3D7_1137600conserved Plasmodium protein, unknown functionK2(Dimethyl)HILIC
IGLHYGSCVGGVIGSGrLRYDLWGIDVLTGnLMESnGIPGkPF3D7_1138400guanylyl cyclase (GCalpha)K41(Dimethyl)IP
FEEIPDDPNNSLDNTEnSEHMnTNNNSDQNEkPF3D7_1138500protein phosphatase 2C (PPM2)K32(Methyl)HILIC
TVFkTInLnNINNKPF3D7_1138800WD repeat-containing protein, putativeK4(Dimethyl)HILIC
TkNTSTqEIDGDTINKPF3D7_1141100conserved Plasmodium protein, unknown functionK2(Dimethyl);IP
KNkPVEYPFAISnKPF3D7_1142800conserved Plasmodium protein, unknown functionK3(Dimethyl)HILIC
DMLQkIEkNYDNNDINNDNNNNDNNNNNDNNNNNNNNNNNNNnNNNQKPF3D7_1144400conserved Plasmodium protein, unknown functionK5(Trimethyl); K8(Trimethyl)IP
NSEIVkILNAPNKPF3D7_1144800conserved Plasmodium protein, unknown functionK6(Dimethyl)IP
IMDEnkETEQTEEGnTEEFVQEKPF3D7_1149000antigen 332, DBL-like protein (Pf332)K6(Methyl)HILIC
FCLncGFGLGSGVLQSLGLFGGSGIYAWTIGAPAAAIAAAKEAGAAAGIKAGHAVGATkVVELVNSkPF3D7_1150000rifin (RIF)K59(Dimethyl); K67(Trimethyl)IP
nAESIWQGMLcGLSHAVSArDkPF3D7_1200400erythrocyte membrane protein 1, PfEMP1 (VAR)K22(Trimethyl)IP
GADDcDDNSNIECkPF3D7_1200600erythrocyte membrane protein 1, PfEMP1 (VAR2CSA)K14(Trimethyl)HILIC
kFIEDCkGGDGTAGSSWVkRPF3D7_1200600erythrocyte membrane protein 1, PfEMP1 (VAR2CSA)K1(Trimethyl); K7(Trimethyl); K19(Methyl)IP
MGDDGDDnDDDDDGDDDnNNNNkPF3D7_1202600conserved protein, unknown functionK23(Methyl)IP
nIVnIINCkPF3D7_1203300conserved Plasmodium protein, unknown functionK9(Dimethyl)IP
MIKNIRGTEIqTNNLSMATIENHIDNkPF3D7_1204900probable protein, unknown functionK27(Dimethyl)IP
FKNFGSSYNSYPISYIAFSCVVGIErPELENFVSkLDNAIDYFIkFFkPF3D7_1205100O-phosphoseryl-tRNA(Sec) selenium transferase, putative (SEPSECS)K35(Trimethyl); K45(Trimethyl)IP
cGNNNYSnSkPF3D7_1207200conserved Plasmodium protein, unknown functionK10(Dimethyl)IP
ISLTEISEPSVLIkPF3D7_1207700blood stage antigen 41-3 precursorK14(Dimethyl)HILIC
SDDESDnESDDESDnESDDESDDkPF3D7_1207800conserved Plasmodium protein, unknown functionK24(Dimethyl)HILIC
NKNVNVNDNDNGNNNNNSNNNNNNSNGNSATLNNNNNMCVMckPF3D7_1208200cysteine repeat modular protein 3 (CRMP3)K43(Dimethyl)HILIC
TIALQNICGLnLkPF3D7_1208700conserved protein, unknown functionK13(Dimethyl)HILIC
NVGqNDDTLNNNNNNNINSVNNNNNHVVGGLHqTQTCEGkPF3D7_1209400cytosolic iron-sulfur protein assembly protein 1, putative (CIA1)K40(Trimethyl)HILIC
IIEqAkLKHNqIYNKELHNLArPF3D7_1211300DNA helicase MCM8, putative (MCM8)K6(Dimethyl)IP
YYDHNNMCGDNNICDDNNIcGDNEIYGDNkPF3D7_1218200conserved Plasmodium protein, unknown functionK30(Trimethyl)HILIC
NNSkEnINFIKPF3D7_1218700conserved Plasmodium protein, unknown functionK4(Dimethyl)HILIC
NNTIINMYNQNIrHSNSNnnTINDMNNNNINkPF3D7_1220000conserved Plasmodium protein, unknown functionK32(Dimethyl)IP
WDHVDInEDEnNkPF3D7_1220300cell cycle associated protein, putativeK13(Dimethyl)HILIC
IDGNILDNNKIDGnILDnnkIDGNILDNNKPF3D7_1221000histone-lysine N-methyltransferase, H3 lysine-4 specific (SET10)K20(Trimethyl)IP
DMVnDPnYDSVkVEETDDPNKPF3D7_1222300endoplasmin, putative (GRP94)K12(Methyl)HILIC
DMVnDPNYDSVkVEETDDPNKKPF3D7_1222300endoplasmin, putative (GRP94)K12(Trimethyl)HILIC
ILLTDnYnkPF3D7_1223300DNA gyrase subunit A (GyrA)K9(Trimethyl)HILIC
FNDLqkGnEQEKPF3D7_1223300DNA gyrase subunit A (GyrA)K6(Trimethyl)IP
nTEDEnnTSSSYLFSLSFQNSGkPF3D7_1224400conserved Plasmodium protein, unknown functionK23(Dimethyl)HILIC
ENVNKInnNNNNNNkkPF3D7_1225900conserved Plasmodium protein, unknown functionK15(Dimethyl); K16(Trimethyl)IP
NIVnQLFNYISkPF3D7_1227200potassium channel (K1)K12(Trimethyl)HILIC
KNVIDNNIYkPF3D7_1227300conserved Plasmodium protein, unknown functionK10(Methyl)HILIC
NKYYkDNIYDGNnIcDGnNIYCNNNNICCNNNNICCNNNNIYCNNNNIYDNNTCDKPF3D7_1227400conserved Plasmodium protein, unknown functionK5(Methyl)HILIC
NKYYkDNIYDGNNICDGNnIYCNNNNICCNNNNICCNNNNIYCNNNNIYDNNTCDkPF3D7_1227400conserved Plasmodium protein, unknown functionK5(Dimethyl); K56(Dimethyl)HILIC
ELnTSYDnnSPTDSTYkPF3D7_1231600pre-mRNA-splicing factor ATP-dependent RNA helicase PRP2, putative (PRP2)C-Term(Methyl)IP
TVYLFDIFLNEqSkPF3D7_1232000phenylalanine–tRNA ligase (aFRS)C-Term(Methyl)HILIC
GMESDnINEMVSDNINEMASDNINEMVSDNINEMTSDNINkMANQMNYEQNTDGIIIkPF3D7_1233600asparagine and aspartate rich protein 1 (AARP1)K41(Dimethyl); C-Term(Methyl)IP
LNVqRDKTFnEEDnIkPF3D7_1234100bromodomain protein, putativeK16(Dimethyl)IP
NNNEPFSTLNLECNTkPF3D7_1235200V-type K+-independent H+-translocating inorganic pyrophosphatase (VP2)K16(Dimethyl)HILIC
GDDDDDDDDDDDDDDDDDDDnDDDDDDDDDDDDDDDGDNQITkPF3D7_1237200conserved Plasmodium protein, unknown functionK43(Trimethyl)HILIC
VNVDNICIPNkPF3D7_1237500conserved Plasmodium protein, unknown functionK11(Dimethyl)HILIC
TGDIITnDDSLTNNLCIFkPF3D7_1237500conserved Plasmodium protein, unknown functionK19(Methyl)IP
FLEnEDkVDLEIDkVDELLYFEEIKPF3D7_1238800acyl-CoA synthetase (ACS11)K7(Dimethyl); K14(Dimethyl)IP
AIGDAnSErHPcGIGkPF3D7_1240400erythrocyte membrane protein 1, PfEMP1 (VAR)K16(Trimethyl)IP
NDNNGDDYkEDNYDDDDDDDDDDDEkPF3D7_1245600kinesin, putativeK9(Methyl); K26(Methyl)IP
VnInGNVNNkPF3D7_1246900RAC-beta serine/threonine protein kinase (PKB)C-Term(Methyl)HILIC
IINQEkPRPF3D7_1247800dipeptidyl aminopeptidase 2 (DPAP2)K6(Dimethyl)HILIC
CNNDNITEEGENMNVQnLESLQnDGNGVIELGCGLGQISkPF3D7_1249900apicoplast dimethyladenosine synthase, putativeK40(Methyl)HILIC
AHLLqqNqLQGLTHkINFENNLGKPF3D7_1250100osmiophilic body protein (G377)K15(Trimethyl)IP
VAMATAEkVGIQLGIDAGNAAGIKPF3D7_1254200rifin (RIF)K8(Methyl)HILIC
DFYqQLQSGYGDVNAFLELLnkETTcKPF3D7_1300100erythrocyte membrane protein 1, PfEMP1 (VAR)K22(Trimethyl)IP
GILDINDPSVTNnVnEVHDASNTQGSVSNTSDITNGHSESSLNRTTNAQDIkPF3D7_1301600erythrocyte binding antigen-140 (EBA140)C-Term(Methyl)IP
DAVFSLPPTnEkPF3D7_130280040S ribosomal protein S7, putativeK12(Dimethyl)IP
NENIVDMVkPYDDFCkEIEYNYFIPIQILYKPF3D7_1306500MORN repeat protein, putativeK9(Trimethyl); K16(Dimethyl)HILIC
AVGYNNNYLNNNNNMnSAVnNNSSNGNNMkPF3D7_1312900eukaryotic translation initation factor 4 gamma (EIF4G)C-Term(Methyl)HILIC
NPLYYVSLLFNkPNSFPYLIKPF3D7_1313100conserved Plasmodium protein, unknown functionK12(Methyl)HILIC
LSLFYnknSInNIKPF3D7_1317200transcription factor with AP2 domain(s) (ApiAP2)K7(Trimethyl);HILIC
NELGnnECNNNNNNYISkPF3D7_1317200transcription factor with AP2 domain(s) (ApiAP2)K18(Dimethyl)HILIC
NVLkNIYNNNIINNNDnILnkPF3D7_1319600conserved Plasmodium protein, unknown functionK4(Dimethyl); K21(Trimethyl)IP
QNSYDISEVSINCYYDDVIkCYMDYTMHGMEDETnFYLCEFCEQnIFDMNNMIKPF3D7_1322100variant-silencing SET protein (SETvs)K20(Dimethyl)HILIC
QINDTINkPF3D7_1322100variant-silencing SET protein (SETvs)K8(Dimethyl)IP
GcDNADNDDDNNDDNNDGDNNNDDDNNNDDNNIDDNDGDnnNDEkPF3D7_1324000conserved Plasmodium protein, unknown functionK45(Methyl)HILIC
nDDTIVkNYMNNIENIkPF3D7_1324500DEAD box helicase, putativeK7(Dimethyl); C-Term(Methyl)IP
ALDTSHTNVMAYSNckPF3D7_1324900L-lactate dehydrogenase (LDH)K16(Methyl)HILIC
QMFNVHPYDSFDNDIGkGnTnIICkPF3D7_1326600conserved Plasmodium protein, unknown functionK17(Dimethyl); K25(Dimethyl)IP
KPSIFIkPLSnSPKPF3D7_1327300conserved Plasmodium protein, unknown functionK7(Trimethyl)HILIC
GILFLFILIGFVLkkPDEIkPLLkPF3D7_1328900conserved Plasmodium protein, unknown functionK14(Dimethyl); K15(Dimethyl); K20(Trimethyl); K24(Methyl)IP
NIFEnNDLSPLrkPF3D7_1334100conserved Plasmodium protein, unknown functionK13(Dimethyl)IP
LYNLGDVFNHVVDISnkkPF3D7_1335100merozoite surface protein 7 (MSP7)K17(Dimethyl); K18(Dimethyl)IP
NEQEISTQGQEVQkPAqGGESTFQkPF3D7_1335100merozoite surface protein 7 (MSP7)K14(Methyl); K25(Trimethyl)IP
NEQEISTQGQEVQkPAqGGESTFqkPF3D7_1335100merozoite surface protein 7 (MSP7)K14(Dimethyl); K25(Dimethyl)IP
EIYVLYnKLLnLnkPF3D7_1335800conserved Plasmodium protein, unknown functionC-Term(Methyl)IP
NILLNGGNDLSkPF3D7_1336000conserved Plasmodium protein, unknown functionK12(Dimethyl)IP
LPVLVTSHGSIFESNAVckPF3D7_1338300elongation factor 1-gamma, putativeK19(Trimethyl)HILIC
HINNNIIDIDInInEkPF3D7_1338500conserved Plasmodium protein, unknown functionK16(Dimethyl)HILIC
EkNNNIVHInINKPF3D7_1339700conserved Plasmodium protein, unknown functionK2(Trimethyl)HILIC
EkNNNIVHInINKPF3D7_1339700conserved Plasmodium protein, unknown functionK2(Trimethyl)HILIC
SANInITnHnINVQMNDSMNGHLVDEkPF3D7_1342900transcription factor with AP2 domain(s) (ApiAP2)K27(Dimethyl)HILIC
EnKLIDLWkPF3D7_1343300conserved Plasmodium protein, unknown functionK9(Trimethyl)IP
KENFLDAAnLInDDSGLNNLkPF3D7_1343700kelch protein K13 (K13)K21(Dimethyl)IP
NIPQLNENGNSNSNGNSnGNSNGNSNGNSNGNSNGNSNkPF3D7_1343800conserved Plasmodium protein, unknown functionK39(Methyl)HILIC
LTKNIkHEYIINPNYFEGYVEIYLSLIPTDHGERPFSRcFSSWGIqrPF3D7_1343800conserved Plasmodium protein, unknown functionK6(Methyl)IP
ILCnnnNkPF3D7_1343900U4/U6 small nuclear ribonucleoprotein PRP4, putative (PRPF4)K8(Dimethyl)IP
KnVDIAVSSSSkPIINAGNGTGEHPTQSLLDFYTIHNYFPFILDRNINKPF3D7_1344800aspartate carbamoyltransferase (ATCase)K12(Trimethyl)IP
NEEqKEnPDnkPF3D7_1345800conserved Plasmodium protein, unknown functionK11(Trimethyl)IP
AVNEnGEkPDEEVKPF3D7_1347500DNA/RNA-binding protein Alba 4 (ALBA4)K8(Trimethyl)HILIC
NDDDIYDCNNESTIDHSNNNNNNNNVYYNNTNIYNNQDLSkPF3D7_1348400conserved Plasmodium membrane protein, unknown functionK41(Trimethyl)HILIC
GNVNCNIEHIIMNEHMMEYIDFScLnYDEkPF3D7_1349500conserved Plasmodium protein, unknown functionC-Term(Methyl)HILIC
InNNNNNNkPF3D7_1350400ubiquitin-activating enzyme E1, putativeK9(Dimethyl)IP
nYITSEnFNDkPF3D7_1351000phosphatidylinositol transfer protein, putativeK11(Trimethyl)IP
SYnLLYQNEAkPF3D7_1352300conserved Plasmodium protein, unknown functionK11(Trimethyl)IP
nkDnISDINIIEKPF3D7_1353400Ran-binding protein, putativeK2(Methyl)HILIC
EISSEIYLVGLNFLGkKVDKPF3D7_1354300large subunit rRNA methyltransferase, putativeK16(Trimethyl)IP
RAMDFknEELMkIQKPF3D7_1354300large subunit rRNA methyltransferase, putativeK6(Trimethyl); K12(Trimethyl)IP
MEnVLVDHkDnAkPF3D7_1356800serine/threonine protein kinase, putative (ARK3)K9(Trimethyl); K13(Dimethyl)IP
SQAAGqAYLEAkPF3D7_1356800serine/threonine protein kinase, putative (ARK3)K12(Dimethyl)IP
NVSVkEIKPF3D7_1357100elongation factor 1-alphaK5(Trimethyl)IP
NVSVkEIKPF3D7_1357100elongation factor 1-alphaK5(Trimethyl)IP
TYHTLNNLSLNSnnNNkPF3D7_1359300exosome complex exonuclease RRP44 (DIS3)K17(Trimethyl)IP
ILEALLVCISILLLTFGVYYEkNknMIDICTHFCSNPYLSINNLDHMNISCLCkPF3D7_1360500guanylyl cyclase beta (GCbeta)K22(Dimethyl); K24(Dimethyl); C-Term(Methyl)IP
FTTkGIVGDAEVALkPrPF3D7_1361900proliferating cell nuclear antigen 1 (PCNA1)K4(Trimethyl); K15(Dimethyl)IP
ALGEDTPFTHISGSEVYSLEMSkPF3D7_1362200RuvB-like helicase 3 (RUVB3)K23(Trimethyl)HILIC
qEkNTKKAEIVYSIPF3D7_136330050S ribosomal protein L9, mitochondrial, putativeK3(Methyl)HILIC
FDIPLLGDNTATSIIGLNIKNEDkPF3D7_1364000conserved Plasmodium protein, unknown functionC-Term(Methyl)IP
nTLAnVTFEQkPF3D7_13641006-cysteine protein (P92)K11(Trimethyl)IP
EAQMDTTSDDFknVQEFISQMLALLkSYLHVVILIEDHIIKTnLENLLKPF3D7_1364400conserved Plasmodium protein, unknown functionK12(Methyl); K26(Methyl)IP
ENILEILDEEkPF3D7_1366400rhoptry protein RHOP148 (RHOP148)K11(Trimethyl)HILIC
ETnFYYEnGGQIYDTGIIQNDNMkFQVLNVQKPF3D7_1367700alanine–tRNA ligase (AlaRS)K24(Trimethyl)IP
YQqSNDTYkQLHELLEKPF3D7_1372500stevor, pseudogeneK9(Methyl)HILIC
SSLPGSIqkPF3D7_1373400rifin (RIF)K9(Dimethyl)IP
FcLTQNGSAGGGGSGnASGSSGDcGGGNSDSSLCEPWkPF3D7_1373500erythrocyte membrane protein 1, PfEMP1 (VAR)K38(Trimethyl)HILIC
IHIFLNnENMDELIkNNILELSFGLHFGWAIEGAIGSSYKPF3D7_1404600.2adenylyl cyclase alpha (ACalpha)K15(Trimethyl)IP
IDHIcYnSTNESEGkPF3D7_1407600conserved Plasmodium protein, unknown functionK15(Dimethyl)HILIC
MNINEkDKLAEQNLETLDVTKPF3D7_1410600eukaryotic translation initiation factor 2 gamma subunit, putativeK6(Trimethyl)IP
MNINEKDkLAEQNLETLDVTKPF3D7_1410600eukaryotic translation initiation factor 2 gamma subunit, putativeK8(Trimethyl)IP
MDMLNPqFEEIGkEFVNHYFQLFNSGRPF3D7_1412300nuclear transport factor 2, putative (NTF2)K13(Trimethyl)HILIC
cINNLCLnLkNDDIDYYINCkPF3D7_1412400conserved Plasmodium protein, unknown functionK10(Trimethyl); K21(Trimethyl)IP
AIGAGAGAGAGAGSASASGGQLMkPF3D7_1414100conserved Plasmodium protein, unknown functionK24(Methyl)HILIC
SNHGNLISWSEQGVFLLnTSLTVEENkPASHKNYGWETFTDTVINIINRQKPF3D7_1415000uracil-DNA glycosylase (UDG)K27(Methyl)IP
SNIIITTSNNAkPF3D7_1416200metacaspase-like protein (MCA3)K12(Trimethyl)HILIC
MLYIFFNNNLLNLFkPF3D7_1420500conserved Plasmodium membrane protein, unknown functionC-Term(Methyl)HILIC
NDDInnFnSkIIEGQVLFDKPF3D7_1420600pantothenate kinase, putative (PANK)K10(Dimethyl)HILIC
FSGCPAHFHNINYnLCDEEkPF3D7_1421100conserved Plasmodium protein, unknown functionC-Term(Methyl)IP
TLqNqEIIHNNDTNQkNNKVSSINKINTNKPF3D7_1422700conserved Plasmodium protein, unknown functionK16(Trimethyl)IP
NDPnIknILNKPF3D7_1426100basic transcription factor 3b, putativeK6(Methyl);HILIC
MEPTTETkTEEIDLEKPF3D7_1428300proliferation-associated protein 2g4, putativeK8(Trimethyl)HILIC
NkDITKYNNNNNDNNNNDNnnnDNNNNDNKNGCDNIKPF3D7_1429400rRNA (adenosine-2’-O-)-methyltransferase, putativeK2(Dimethyl)IP
ASIqNISSEqkPF3D7_1430800conserved Plasmodium protein, unknown functionC-Term(Methyl)IP
nETDHNLFWTCcSEENVEkPRPF3D7_1432200conserved Plasmodium protein, unknown functionK19(Dimethyl)HILIC
NSScNkqTHMNCVQENYMTQnSSFDILSTEKPF3D7_1433200conserved Plasmodium protein, unknown functionK6(Trimethyl)IP
EDKDDAGqDnqkWEDDIKPF3D7_1434500dynein-related AAA-type ATPase, putativeK12(Trimethyl)IP
nSVSAYnGPEFEDLDDSLQTSLDEWLANLGVDSELCDFIDSCSIDkPF3D7_1434800mitochondrial acidic protein MAM33, putativeK46(Trimethyl)HILIC
SDMTDDnYIDVSEkHVIILGGGKTAVDCISLAIrPF3D7_1435300NAD(P)H-dependent glutamate synthase, putativeK14(Dimethyl)IP
VSTISAcEkSDVnVTTNGISRcHEDKPF3D7_1435600conserved Plasmodium protein, unknown functionK9(Methyl)IP
IPNIGScYFVIIGHMkPIQLYVIINELQSTYnNLDEIIILTSLPIkPF3D7_1436100conserved Plasmodium membrane protein, unknown functionK16(Trimethyl); K46(Dimethyl)HILIC
ATFEGNVDDDDTVYDEnEEnYDCLkkPF3D7_1438100secretory complex protein 62 (SEC62)K25(Dimethyl); K26(Dimethyl)IP
FTENYNFALLPQDHSkPF3D7_1438400metacaspase-like protein (MCA2)C-Term(Methyl)HILIC
knVSGnINMPSFVKPF3D7_1438700DNA primase small subunitK1(Dimethyl)IP
FLVQNKcLEVVQENYVLNYYATPLGHIASMYYIkCETVYFFYTSIQAGkPF3D7_1439100DEAD/DEAH box helicase, putativeK34(Trimethyl); K49(Trimethyl)IP
LISnNDSkDELFSNLNRPF3D7_1440000conserved Plasmodium protein, unknown functionK8(Methyl)IP
MEknKDMENLKNADILKPF3D7_1440500allantoicase, putativeK3(Trimethyl)IP
EMINEYkTNkLnDSNIALMIEEKKPF3D7_1440600conserved Plasmodium protein, unknown functionK7(Dimethyl); K10(Trimethyl)IP
EEVTQIDIINnkEQNISSNSVIkPF3D7_1442700conserved Plasmodium protein, unknown functionK12(Dimethyl); K23(Trimethyl)IP
ISnLTDVNTNIkPF3D7_1444100conserved Plasmodium protein, unknown functionK12(Dimethyl)IP
INYDVMIEDFDEnnkPF3D7_1444100conserved Plasmodium protein, unknown functionC-Term(Methyl)IP
NPNFYICGNGYIAPLDIkPF3D7_1444200calmodulin-like proteinK18(Dimethyl)HILIC
ALQEnGVLLEGALLkPNMVTAGYEcTAKPF3D7_1444800fructose-bisphosphate aldolase (FBPA)K15(Trimethyl);HILIC
NEAGVPMVnLLHnENIIPGIkPF3D7_1444800fructose-bisphosphate aldolase (FBPA)K21(Methyl)HILIC
NNNIVAPTPIqIqGWPIALSGkPF3D7_1445900ATP-dependent RNA helicase DDX5, putative (DDX5)K22(Methyl)HILIC
EKnnKEEnVVkPF3D7_1446500conserved Plasmodium protein, unknown functionK11(Dimethyl)IP
TNIISNHLLDIQNVNSVQkLqSHVPHSYGNHISNGcSENTGLTYSkPF3D7_1450300NADPH–cytochrome P450 reductase, putative (CPR)K19(Methyl); K46(Dimethyl)IP
WNDInINLNMcDVMPCNkPF3D7_1450800conserved Plasmodium protein, unknown functionK18(Trimethyl)IP
ETVTEESTITcLGkPF3D7_1451100elongation factor 2 (eEF2)K14(Methyl)HILIC
GYVQPVSLTSSDYFHSCYTSSYESQMFDCnAGFSGnnQEkPF3D7_1451600LCCL domain-containing protein (LAP5)K40(Methyl)HILIC
VCPkINNDNVLKcEFEESSLYNPkPF3D7_1452000rhoptry neck protein 2 (RON2)K4(Trimethyl); C-Term(Methyl)IP
EIYDKqQINkPF3D7_1453800glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (GluPho)K10(Dimethyl)IP
YQEITDkSnTVDDASKPF3D7_1457000signal peptide peptidase (SPP)K7(Trimethyl)HILIC
NIAFYnLVIGNNNkPF3D7_1457400conserved Plasmodium protein, unknown functionK14(Trimethyl)HILIC
DHPTVHCFTNGDnFLYDTGkIIqDkPF3D7_1460400ubiquitin carboxyl-terminal hydrolase isozyme L3 (UCHL3)K20(Dimethyl); C-Term(Methyl)IP
NGICPNIDnTLMLVEDSLkPF3D7_1461800conserved Plasmodium protein, unknown functionC-Term(Methyl)HILIC
NMLPCIENTNTSLNrANQNHFDkNEVrPF3D7_1461800conserved Plasmodium protein, unknown functionK23(Trimethyl)IP
HNLNYINIFTLDGHINENGGkPF3D7_1461900valine–tRNA ligase, putativeC-Term(Methyl)IP
STILLPETYFKPFIkPLQHSSIINHNCLINInEQLFnYFLFLFFNkPF3D7_1462100conserved Plasmodium protein, unknown functionK15(Dimethyl); K46(Dimethyl)IP
KNDTNNQTnDLNSNkDETSGSQVTNRPF3D7_1462300conserved Plasmodium protein, unknown functionK15(Trimethyl)HILIC
VSVFAEkDPSqIPWGKPF3D7_1462800glyceraldehyde-3-phosphate dehydrogenase (GAPDH)K7(Trimethyl);HILIC
NSDTNSNIMSNTYEnMYEDFSSnYDDSAckPF3D7_1464500conserved Plasmodium membrane protein, unknown functionK30(Dimethyl)HILIC
kPnIAnKPF3D7_1466200conserved Plasmodium protein, unknown functionK1(Methyl)HILIC
IDELInIFkPF3D7_1467100DNA-3-methyladenine glycosylase, putativeK9(Dimethyl)HILIC
ELNENEIkGnHRPF3D7_1467900rab GTPase activator, putativeK8(Dimethyl);HILIC
EIWDQcTIAVYNnTLNAVESkPLLFLHGILNECrPF3D7_1471100exported protein 2 (EXP2)K21(Trimethyl)IP
ELNNFHFYLIVkINnLnIVPYLNLYMCrPF3D7_1471900conserved Plasmodium protein, unknown functionK12(Methyl); C-Term(Methyl)HILIC
ELVNIQMnDkInEKPF3D7_1474300DNA repair metallo-beta-lactamase protein, putativeK10(Trimethyl);HILIC
CLGTLINLcSDGSTGYqcNNCSkPF3D7_1475400cysteine repeat modular protein 4 (CRMP4)K23(Trimethyl)HILIC
cGFGLSGVAGSIGLFGAVAInIWkPAALKAAIAKAITEGTADIAAAGVkAGEVTGKPF3D7_1479400rifin (RIF)K24(Trimethyl); K49(Dimethyl)IP
GcLRcGSILGAAMPELGSVGGSLLYALNTWKPAAIIAAkEAALAEATDLATQAGIDTVVAQLkPF3D7_1479700rifin (RIF)K39(Trimethyl); K63(Trimethyl)IP

The sequences in bold represent two inner membrane complex proteins (IMC1c and IMC1g) for which a large number of lysine methylation sites were identified.

A number of reports have shown that the pre-fractionation of tryptic peptides either by SCX or HILIC provides a sort of enrichment of methylated peptides that can be better captured on LC-MS/MS for site identification. Between the two methods, HILIC has provided more number of methylation sites as compared to SCX in S. cerevasiae34. We performed HILIC chromatography analysis on trypsin digested parasite lysate generated from trophozoite stage. Twelve fractions were collected in two replicates and each of the fractions was separately analyzed on LC-MS/MS. As shown in Table 1, we could identify 247 sites in 236 peptides corresponding to Plasmodium proteins. Representative spectra of few of these K-methylated peptides are shown in the Supplementary Fig. 2. In total, 605 methylated lysine sites in 502 peptides were identified corresponding to 422 Plasmodium proteins. A couple of previous reports in human/mouse cell lines and Saccharomyces cerevisiae have shown that many of the lysine methylated sites are associated with EK, LK and MK motifs. We searched the motifs in 570 putative lysine methylated proteins identified by immunoprecipitation of parasite lysates. We could observe LK and EK motifs in many Plasmodium proteins (Fig. 3). The “Two Sample Logo” visualization of the residues surrounding methylated and non methylated lysines amongst the proteomics identified proteins reveal that the residues surrounding methylated lysines are different from those surrounding the non methylated lysines (Supplementary Fig. 4). Among the Plasmodium methylated peptides, we identified 152 monomethyl, 249 dimethylated and 210 trimethylated sites in the mass spectrometry analysis corresponding to these proteins (Fig. 3).
Figure 3

Analysis of identified lysine-methylated proteins for the presence of known motifs.

(A) Motif representation of methylated lysine sites along with a consensus sequence logo in P. falciparum. All the 605 confirmed sites were examined to know the presence of conserved motifs. (B) Motif representation of previously reported sites in other organisms.

To confirm the identity of non-histone lysine methylated proteins, we subjected the immunoprecipitated Plasmodium lysate to western blot analysis using anti-PfP12 or anti-PfTSN antisera. As shown in Fig. 4, PfP12 and PfTSN are detected in IP lysate, thus confirming the lysine methylation of the proteins. Altogether, several mass spectrometric analysis runs as well as western blot analysis indicate extensive lysine methylation of Plasmodium proteins at asexual blood stages.
Figure 4

Western blot confirms the presence of methylated lysine proteins in immuno precipitates.

Representative western blots (A) with anti-PfP12 and (B) anti-PfTSN antisera validating the LC-MS/MS results of immunoprecipitation experiments.

Classification of lysine methylated proteins

Mass spectrometry analysis of the anti-methyl lysine antibody immuno-precipitated proteins from the Plasmodium lysates suggested that over 10% of the proteins encoded in the P. falciparum genome are modified by lysine methylation. To get insight into the role of lysine methylation in the parasite growth and development, we utilized the fully annotated genome database (PlasmoDB) to describe correlations for lysine methylated proteins, their families, subcellular localization and biological function. The identified lysine methylated proteins were classified based on their subcellular localization with GO term analysis using PlasmoDB. As seen in Fig. 5A, we were able to obtain the GO assigned localization of only 78% of the lysine methylated proteins, since a large number of the query proteins were hypothetical. The percentage of proteins with known or predicted subcellular localization was highest for cytoplasmic proteins (Fig. 5A). Many of the identified lysine methylated proteins appeared to be part of protein-protein or protein-DNA complexes associated with chromosome or ribosome.
Figure 5

Classification of lysine methylated proteins in P. falciparum.

(A) based on cellular components and (B) based on function and (C) on the basis of conserved domains. The 570 identified lysine methylated proteins were categorized based on their known or likely functions and cellular localization. Proteins with no annotations in PlasmoDB were categorized as unknown proteins. A pie chart shows the distribution of the proteins based on domain super families.

The diverse localization profile of lysine-methylated proteins suggests that this modification regulates a wide range of cellular functions. To define the functional classes of lysine methylated proteins, we data mined the P. falciparum annotated genome database for all identified methylated proteins. Similar to the localization GO terms, we were able to predict functional classes for only 62% of the identified lysine methylated proteins (Fig. 5B). Many of these annotated proteins seem to be associated with the transport/trafficking processes, chromosomal organization and translation regulation (Fig. 5B). To get insights into the role(s) of lysine methylated Plasmodium proteins identified by mass spectrometry analysis, we sorted the proteins based on other conserved domains. As shown in Fig. 5C, several of these proteins belong to major super families such as protein kinase C (PKc), ATS, P-loop_ NTPase, PHD_SF and AdoMet_MTases_SF. The set also included 21 PfEMPs and 17 rifin proteins, indicating a role of lysine methylation in antigenic diversity in P. falciparum.

Lysine methyltransferase-substrate interactome networks and cross talks between PTMs

For further exploration of the breadth of Plasmodium protein lysine methylation and associated lysine methyltransferases (KMTs), we generated KMT-substrate networks using STRING database and published literature. These networks are depicted in Supplementary Fig. 3 and are described in Supplementary Table 2. All the Plasmodium SET domain proteins show a number of associated partners and many of them are non-histone proteins such as endoplasmin putative (GRP94), bromodomain protein 1 (BP1), proliferating cell nuclear antigen1, guanylyl cyclase (GCalpha). Importantly, some of the lysine-methylated proteins identified in our proteome analysis are also part of this KMTs-substrate network (Supplementary Table 2). For example endoplasmin putative (GRP94) and proliferating cell nuclear antigen 1 (PCNA1) proteins are identified in STRING as well as in our mass spectrometry analysis. We also observed associations among the set domain proteins. For example SET3 protein shows association with SET8 and SET4 proteins in the STRING analysis (Supplementary Table 2). Extensive cross talk has been reported and predicted between different PTMs in different organisms, including Plasmodium. We compared the methylated lysine containing proteins with the previously published studies for phosphorylation and acetylation in P. falciparum56835. This comparison showed that 209 lysine-methylated proteins were phosphorylated too. Further, 173 Plasmodium proteins were acetylated as well as lysine methylated and 113 proteins possess all three modifications (Supplementary Table 3). Several proteins from the STRING analysis were either acetylated or phosphorylated (Supplementary Table 2), thus indicating extensive cross talks between various PTMs.

Discussion

Processes related to intraerythrocytic development of malaria parasite that contributes to malaria associated morbidity and mortality are tightly regulated at transcription as well as translation levels363738. Genome sequencing has shown that basal transcription and translational machineries are conserved in Plasmodium parasite, although few recognizable transcription factors have been identified so far3940. Like in other eukaryotes, PTMs such as phosphorylation, ubiquitination, sumoylation, acetylation and methylation play an important role in regulating the functions of several Plasmodium proteins4. In recent times, methylation of proteins has emerged as one of the most prevalent post-translational modifications41, in the present study we have examined the lysine methylproteome of P. falciparum. Plasmodium genome encodes nine SET domain and two JmjC-domain genes, indicating the presence of protein lysine methylation in malaria parasites22. Our current understanding of protein lysine methylation in Plasmodium is mainly restricted to histones and its role(s) in regulation of var gene expression. Tri-methylated histone 3 lysine 9 (H3K9me3) has been linked to exclusive expression of certain var genes42, while H3K36me3 has been shown to be involved in the repression of these genes25. To know the extent of lysine methylation in Plasmodium parasites in particularly the non-histone lysine methylation, we performed immunoprecipiataion of Plasmodium asexual blood stage lysates with two Methyl lysine specific antibodies and identified lysine methylated proteins by LC-MS/MS analysis. We further validated many of these lysine methylated proteins and also identified additional lysine methylated proteins by HILIC fractionation followed by LC-MS/MS analysis at trophozoite stage. Similar approaches have been previously applied to identify Saccharomyces cerevisiae and human lysine methylated proteins4344. Based on spectral analysis, we could identify 364 sites in IP analysis and 247 sites in HILIC analysis. In total, we have identified 605 lysine methylated sites in 422 proteins. A number of previous studies in human and yeast have shown that methylated lysine amino acid residues are part of a motif that has either leucine or methionine at – 1 position4546. We could indeed identify leucine residue at -1 position in a number of Plasmodium specific methylated peptides identified in our study too. To get functional insights into the roles of methylated lysine containing proteins, especially the non-histone proteins, Gene Ontology (GO) analysis of these Plasmodium proteins was performed as described earlier4446. Besides histones, we identified a large number proteins involved in transport, protein folding, translational elongation and other important biological processes. For example, lysine-methylated peptides corresponding to a number of ribosome-associated proteins, translation elongation factors, HSPs and DNA mismatch repair proteins were identified in our study. We also identified lysine methylation in several helicases, hydrolases, histone deacetylase complex proteins, kinases and phosphatases. Many of these proteins are methylated in other organisms too4447. Remarkably, a number of Plasmodium specific proteins are also identified in the present study. These include many surface/secretory proteins; RON3, ROP14, 6-cysteine protein (p12), trophozoite excretory protein (TEX1) Rifin and PfEMP1. Interestingly, two inner membrane complex proteins; 1g and 1c (IMC 1g and 1c) are heavily methylated with 16 and 15 lysine methylation sites, respectively. Intriguingly, many of the lysine methylated proteins, especially the Plasmodium surface proteins and proteins involved in gliding motility of merozoites during invasion, have also been shown to be phosphorylated5. Summarily, the results suggest an important role of PTMs and their cross talks in the invasion of RBC by merozoites. It is important to mention here that in our analysis we could identify many methylated proteins belonging to PfEMP and Stevor family, whose transcription and antigenic variations have been linked with chromatin/epigenetic memory that includes methylation of histones254248. To validate our lysine methylome analysis, we used the STRING protein interaction database49 to understand the protein interaction networks for PfSET domain proteins and compared the results with the data generated in our methylome analysis. A few common non-histone proteins were identified in the two datasets. In addition, we also examined the proteins identified in STRING analysis for acetylation and phosphorylation. A number of Plasmodium proteins showed two or more PTMs, thereby suggesting cross talks among various PTMs. Such cross talks among PTMs such as between methylation and acetylation or between methylation and phosphorylation or between two methylation sites on the same proteins are reported in other organisms too4144. Such cross talks probably fine tune the function of individual proteins and elucidation of such cross talks between several PTMs may shed new lights on system biology of this human pathogen. Finally, the data presented here shows that protein lysine methylation is quite wide spread in P. falciparum which may be an important gene regulatory processes. However, in order to gain deeper understanding of the role of lysine methylation in Plasmodium development and sustenance, a conditional knockdown of each of the nine PfSET domain proteins followed by a quantitative lysine methylome analysis will be required. Additionally, it will be important to experimentally explore each of the nine PfSET domain proteins that may also be important for development of novel chemotherapeutics for malaria. Thus the parasite lysine methylome analysis performed by us is a step forward in elucidating the complex nature of the gene regulatory processes in Plasmodium, where only a few transcription factors have been functionally characterized.

Additional Information

How to cite this article: Kaur, I. et al. Widespread occurrence of lysine methylation in Plasmodium falciparum proteins at asexual blood stages. Sci. Rep. 6, 35432; doi: 10.1038/srep35432 (2016).
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