FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2770680654> ?p ?o ?g. }

• W2770680654 endingPage "60" @default.
• W2770680654 startingPage "43" @default.
• W2770680654 abstract "Despite recent efforts toward control and elimination, malaria remains a major public health problem worldwide. Plasmodium falciparum resistance against artemisinin, used in front line combination drugs, is on the rise, and the only approved vaccine shows limited efficacy. Combinations of novel and tailored drug and vaccine interventions are required to maintain the momentum of the current malaria elimination program. Current evidence suggests that strain-transcendent protection against malaria infection can be achieved using whole organism vaccination or with a polyvalent vaccine covering multiple antigens or epitopes. These approaches have been successfully applied to the human-infective sporozoite stage. Both systemic and tissue-specific pathology during infection with the human malaria parasite P. falciparum is caused by asexual blood stages. Tissue tropism and vascular sequestration are the result of specific binding interactions between antigens on the parasite-infected red blood cell (pRBC) surface and endothelial receptors. The major surface antigen and parasite ligand binding to endothelial receptors, PfEMP1 is encoded by about 60 variants per genome and shows high sequence diversity across strains. Apart from PfEMP1 and three additional variant surface antigen families RIFIN, STEVOR, and SURFIN, systematic analysis of the infected red blood cell surface is lacking. Here we present the most comprehensive proteomic investigation of the parasitized red blood cell surface so far. Apart from the known variant surface antigens, we identified a set of putative single copy surface antigens with low sequence diversity, several of which are validated in a series of complementary experiments. Further functional and immunological investigation is underway to test these novel P. falciparum blood stage proteins as possible vaccine candidates. Despite recent efforts toward control and elimination, malaria remains a major public health problem worldwide. Plasmodium falciparum resistance against artemisinin, used in front line combination drugs, is on the rise, and the only approved vaccine shows limited efficacy. Combinations of novel and tailored drug and vaccine interventions are required to maintain the momentum of the current malaria elimination program. Current evidence suggests that strain-transcendent protection against malaria infection can be achieved using whole organism vaccination or with a polyvalent vaccine covering multiple antigens or epitopes. These approaches have been successfully applied to the human-infective sporozoite stage. Both systemic and tissue-specific pathology during infection with the human malaria parasite P. falciparum is caused by asexual blood stages. Tissue tropism and vascular sequestration are the result of specific binding interactions between antigens on the parasite-infected red blood cell (pRBC) surface and endothelial receptors. The major surface antigen and parasite ligand binding to endothelial receptors, PfEMP1 is encoded by about 60 variants per genome and shows high sequence diversity across strains. Apart from PfEMP1 and three additional variant surface antigen families RIFIN, STEVOR, and SURFIN, systematic analysis of the infected red blood cell surface is lacking. Here we present the most comprehensive proteomic investigation of the parasitized red blood cell surface so far. Apart from the known variant surface antigens, we identified a set of putative single copy surface antigens with low sequence diversity, several of which are validated in a series of complementary experiments. Further functional and immunological investigation is underway to test these novel P. falciparum blood stage proteins as possible vaccine candidates. An estimated 3.2 billion people - nearly half the world's population - are at risk of contracting malaria (1.WHO. (2016) WHO Malaria Report 2016,Google Scholar). Although the mortality rate has decreased over the last decade, malaria remains an acute public health problem in many countries and regions. In 2015 alone, there was an estimated 214 million clinical cases of malaria leading to about 438,000 deaths, most of them children under the age of five (1.WHO. (2016) WHO Malaria Report 2016,Google Scholar). Severe and fatal malaria cases can largely be attributed to one parasite species, Plasmodium falciparum. After an initial and asymptomatic liver cycle, parasite propagation takes place in host red blood cells (RBC) 1The abbreviations used are: RBC, red blood cell; pRBC, parasite-infected red blood cell; PfEMP1, plasmodium falciparum erythrocyte membrane protein 1; Stevor, sub-telomeric variable open reading frame; Rifin, repetitive interspersed families of polypeptides; Surfin, surface-associated interspersed gene family; VSA, variant surface antigen; EPCR, endothelial protein C receptor; CD36, cluster of differentiation 36; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1; MCM, malaria Culture Medium; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; BSA, bovine serum albumin; PBS, phosphate buffered saline; RPMI, Roswell Park Memorial Institute; Hb, hemoglobin; MACS, magnet-activated cell sorting; LS-MS/MS, liquid chromatography tandem mass-spectrometry; DDA, data dependent acquisition; FDR, false discovery rate; iTRAQ, isobaric tag for relative and absolute quantification; DTT, dithiothreitol; ACN, acetonitrile; PIP, precursor ion purity; PEXEL, plasmodium export element; PSM, peptide spectrum match; RNA, ribonucleid acid; FKPM, fragments per kilobase per million; IMAC, immobilized metal affinity column; PEPP, protein expression and purification platform; FBS, fetal bovine serum; BF, bright field; DIC, differential interference contrast; CSA, chondroitin sulfate A; MAHRP1, membrane-associated histidine-rich protein 1; SBP1, skeleton binding protein 1; PHIST, plasmodium helical interspersed sub-telomeric family; PIESP2, plasmodium falciparum infected erythrocyte surface protein 1; PfMC-2TM, plasmodium falciparum Maurer's clefts 2 transmembrane domaine protein. 1The abbreviations used are: RBC, red blood cell; pRBC, parasite-infected red blood cell; PfEMP1, plasmodium falciparum erythrocyte membrane protein 1; Stevor, sub-telomeric variable open reading frame; Rifin, repetitive interspersed families of polypeptides; Surfin, surface-associated interspersed gene family; VSA, variant surface antigen; EPCR, endothelial protein C receptor; CD36, cluster of differentiation 36; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1; MCM, malaria Culture Medium; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; BSA, bovine serum albumin; PBS, phosphate buffered saline; RPMI, Roswell Park Memorial Institute; Hb, hemoglobin; MACS, magnet-activated cell sorting; LS-MS/MS, liquid chromatography tandem mass-spectrometry; DDA, data dependent acquisition; FDR, false discovery rate; iTRAQ, isobaric tag for relative and absolute quantification; DTT, dithiothreitol; ACN, acetonitrile; PIP, precursor ion purity; PEXEL, plasmodium export element; PSM, peptide spectrum match; RNA, ribonucleid acid; FKPM, fragments per kilobase per million; IMAC, immobilized metal affinity column; PEPP, protein expression and purification platform; FBS, fetal bovine serum; BF, bright field; DIC, differential interference contrast; CSA, chondroitin sulfate A; MAHRP1, membrane-associated histidine-rich protein 1; SBP1, skeleton binding protein 1; PHIST, plasmodium helical interspersed sub-telomeric family; PIESP2, plasmodium falciparum infected erythrocyte surface protein 1; PfMC-2TM, plasmodium falciparum Maurer's clefts 2 transmembrane domaine protein.. On RBC invasion, parasites start an active process of host cell remodeling. As mature RBCs lack organelles and intracellular membranes, parasite-derived trafficking machinery is introduced into the host cell cytosol to export parasite antigens to the RBC and its plasma membrane. Such antigens can establish new permeation pathways for nutrient uptake, induce adherence of parasitized RBCs (pRBC) to the vascular lining in the deep tissue (sequestration), bind unparasitized RBCs (rosetting), or evade immunity by antigenic variation (reviewed in (2.Maier A.G. Cooke B.M. Cowman A.F. Tilley L. Malaria parasite proteins that remodel the host erythrocyte.Nature Rev. Microbiology. 2009; 7: 341-354Crossref PubMed Scopus (314) Google Scholar)). Most of the known pRBC surface antigens are encoded by large multi-gene families, collectively named variant surface antigens (VSA). VSAs include P. falciparum erythrocyte membrane protein (PfEMP1) (3.Su X.Z. Heatwole V.M. Wertheimer S.P. Guinet F. Herrfeldt J.A. Peterson D.S. Ravetch J.A. Wellems T.E. The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes.Cell. 1995; 82: 89-100Abstract Full Text PDF PubMed Scopus (995) Google Scholar, 4.Smith J.D. Chitnis C.E. Craig A.G. Roberts D.J. Hudson-Taylor D.E. Peterson D.S. Pinches R. Newbold C.I. Miller L.H. Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes.Cell. 1995; 82: 101-110Abstract Full Text PDF PubMed Scopus (820) Google Scholar, 5.Baruch D.I. Pasloske B.L. Singh H.B. Bi X. Ma X.C. Feldman M. Taraschi T.F. Howard R.J. Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes.Cell. 1995; 82: 77-87Abstract Full Text PDF PubMed Scopus (885) Google Scholar), P. falciparum–encoded repetitive interspersed families of polypeptides (RIFIN)(6.Fernandez V. Hommel M. Chen Q. Hagblom P. Wahlgren M. Small, clonally variant antigens expressed on the surface of the Plasmodium falciparum-infected erythrocyte are encoded by the rif gene family and are the target of human immune responses.J. Exp. Med. 1999; 190: 1393-1404Crossref PubMed Scopus (170) Google Scholar, 7.Kyes S.A. Rowe J.A. Kriek N. Newbold C.I. Rifins: a second family of clonally variant proteins expressed on the surface of red cells infected with Plasmodium falciparum.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 9333-9338Crossref PubMed Scopus (270) Google Scholar), surface-associated interspersed gene family (SURFIN)(8.Winter G. Kawai S. Haeggstrom M. Kaneko O. von Euler A. Kawazu S. Palm D. Fernandez V. Wahlgren M. SURFIN is a polymorphic antigen expressed on Plasmodium falciparum merozoites and infected erythrocytes.J. Exp. Med. 2005; 201: 1853-1863Crossref PubMed Scopus (135) Google Scholar), and possibly others such as sub-telomeric variable open reading frame (STEVOR)(9.Niang M. Yan Yam X. Preiser P.R. The Plasmodium falciparum STEVOR multigene family mediates antigenic variation of the infected erythrocyte.PLoS Pathogens. 2009; 5: e1000307Crossref PubMed Scopus (93) Google Scholar, 10.Niang M. Bei A.K. Madnani K.G. Pelly S. Dankwa S. Kanjee U. Gunalan K. Amaladoss A. Yeo K.P. Bob N.S. Malleret B. Duraisingh M.T. Preiser P.R. STEVOR is a Plasmodium falciparum erythrocyte binding protein that mediates merozoite invasion and rosetting.Cell Host Microbe. 2014; 16: 81-93Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). PfEMP1s are the best characterized VSA, and multiple paralogs have been demonstrated to mediate adhesion of pRBCs to various endothelial receptors present in the microvasculature, including CD36, ICAM-1, VCAM-1 and endothelial protein C receptor (EPCR) (reviewed in (11.Rowe J.A. Claessens A. Corrigan R.A. Arman M. Adhesion of Plasmodium falciparum-infected erythrocytes to human cells: molecular mechanisms and therapeutic implications.Expert Rev. Mol. Med. 2009; 11: e16Crossref PubMed Scopus (250) Google Scholar)). Sequestration of pRBCs in the microvasculature is a key pathophysiological feature of malaria infection, and several VSAs including PfEMP1 induce strong antibody-mediated immune responses (12.Chan J.A. Fowkes F.J. Beeson J.G. Surface antigens of Plasmodium falciparum-infected erythrocytes as immune targets and malaria vaccine candidates.Cell Mol. Life Sci. 2014; 71: 3633-3657Crossref PubMed Scopus (106) Google Scholar). The RIFIN proteins belong to the largest VSA family, with roughly 150 paralogs. These are classified into the surface-exposed type A and type B that localize to Maurer's clefts (13.Petter M. Haeggstrom M. Khattab A. Fernandez V. Klinkert M.Q. Wahlgren M. Variant proteins of the Plasmodium falciparum RIFIN family show distinct subcellular localization and developmental expression patterns.Mol. Biochem. Parasitol. 2007; 156: 51-61Crossref PubMed Scopus (89) Google Scholar, 14.Joannin N. Abhiman S. Sonnhammer E.L. Wahlgren M. Sub-grouping and sub-functionalization of the RIFIN multi-copy protein family.BMC Genomics. 2008; 9: 19Crossref PubMed Scopus (64) Google Scholar) - parasite-derived membranous structures underneath the RBC plasma membrane (15.Mundwiler-Pachlatko E. Beck H.P. Maurer's clefts, the enigma of Plasmodium falciparum.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 19987-19994Crossref PubMed Scopus (72) Google Scholar). Like PfEMP1, RIFINs are immunogenic and have recently been shown to be involved in sequestration and rosetting of blood group A RBCs (16.Goel S. Palmkvist M. Moll K. Joannin N. Lara P. Akhouri R.R. Moradi N. Ojemalm K. Westman M. Angeletti D. Kjellin H. Lehtio J. Blixt O. Idestrom L. Gahmberg C.G. Storry J.R. Hult A.K. Olsson M.L. von Heijne G. Nilsson I. Wahlgren M. RIFINs are adhesins implicated in severe Plasmodium falciparum malaria.Nat. Med. 2015; 21: 314-317Crossref PubMed Scopus (139) Google Scholar). Recently the two paralogs of the P. falciparum Hyp8 family (17.Sargeant T.J. Marti M. Caler E. Carlton J.M. Simpson K. Speed T.P. Cowman A.F. Lineage-specific expansion of proteins exported to erythrocytes in malaria parasites.Genome Biol. 2006; 7: R12Crossref PubMed Scopus (330) Google Scholar) have also been localized at the pRBC surface (18.Hermand P. Ciceron L. Pionneau C. Vaquero C. Combadiere C. Deterre P. Plasmodium falciparum proteins involved in cytoadherence of infected erythrocytes to chemokine CX3CL1.Sci. Reports. 2016; 6: 33786Crossref PubMed Scopus (16) Google Scholar). The importance of VSAs such as PfEMP1 and RIFINs as immune targets strongly supports their development as vaccine candidates (12.Chan J.A. Fowkes F.J. Beeson J.G. Surface antigens of Plasmodium falciparum-infected erythrocytes as immune targets and malaria vaccine candidates.Cell Mol. Life Sci. 2014; 71: 3633-3657Crossref PubMed Scopus (106) Google Scholar). However, major challenges toward this goal are the significant sequence diversity and large genetic repertoires of VSAs, as well as variant expression patterns in the population. Hence, there is a strong rationale to identify conserved pRBC surface antigens (or at least epitopes) with minimal allelic variation that could induce strain-transcendent immunity. Two previous studies aiming at identifying parasite antigens at the pRBC surface have used surface biotinylation techniques followed by isolation of membranes and subsequent mass spectrometry (19.Sharling L. Sowa K.M. Thompson J. Kyriacou H.M. Arnot D.E. Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites.Malar J. 2007; 6: 66Crossref PubMed Scopus (6) Google Scholar, 20.Florens L. Liu X. Wang Y. Yang S. Schwartz O. Peglar M. Carucci D.J. Yates 3rd, J.R. Wub Y. Proteomics approach reveals novel proteins on the surface of malaria-infected erythrocytes.Mol. Biochem. Parasitol. 2004; 135: 1-11Crossref PubMed Scopus (147) Google Scholar). In a third study, parasite antigens were metabolically labeled before isolation of membranes (21.Vincensini L. Richert S. Blisnick T. Van Dorsselaer A. Leize-Wagner E. Rabilloud T. Braun Breton C. Proteomic analysis identifies novel proteins of the Maurer's clefts, a secretory compartment delivering Plasmodium falciparum proteins to the surface of its host cell.Mol. Cell. Proteomics. 2005; 4: 582-593Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). These pioneering studies identified several antigens, and several of them have subsequently been validated as markers of the Maurer's clefts. However, none of the putative pRBC surface antigens have since been confirmed. Limited information on parasite antigen composition at the pRBC surface is likely because of low antigen solubility (because these are usually membrane-anchored proteins), as well as low antigen expression. Here we present the most comprehensive proteomic investigation of the pRBC membrane so far. For this purpose, we used two complementary approaches: (1) surface shaving and proteomic analysis of shaved and unshaved pRBC membranes in combination with profiling of immunogenic surface antigens by protein array (here termed membrane proteomics), and (2) surface shaving and proteomic analysis of released protein ectodomains in the supernatant (here termed supernatant proteomics). Our data demonstrate the identification and validation of a series of nonvariant pRBC surface antigens, and provide a rational basis for novel vaccine approaches targeting blood stage malaria parasites. P. falciparum Pf2004 genomic DNA was sequenced with an Illumina HiSeq platform, using 101 base pair (bp) paired end reads from a small-insert (200 bp) library. The raw data have been deposited in the NCBI Sequence Read Archive under accession number SAMN02630800. Variants were called using GATK Unified Genotyper (22.DePristo M.A. Banks E. Poplin R. Garimella K.V. Maguire J.R. Hartl C. Philippakis A.A. del Angel G. Rivas M.A. Hanna M. McKenna A. Fennell T.J. Kernytsky A.M. Sivachenko A.Y. Cibulskis K. Gabriel S.B. Altshuler D. Daly M.J. A framework for variation discovery and genotyping using next-generation DNA sequencing data.Nat. Genet. 2011; 43: 491-498Crossref PubMed Scopus (7098) Google Scholar). Plasmodium falciparum strain Pf2004–164/TdTomato (23.Brancucci N.M. Goldowitz I. Buchholz K. Werling K. Marti M. An assay to probe Plasmodium falciparum growth, transmission stage formation and early gametocyte development.Nat. Protocols. 2015; 10: 1131-1142Crossref PubMed Scopus (39) Google Scholar) was kept in continuous culture according to standard procedures (24.Trager W. Jensen J.B. Human malaria parasites in continuous culture.Science. 1976; 193: 673-675Crossref PubMed Scopus (6161) Google Scholar) with O+ red blood cells (RBCs) at 3% hematocrit and 10% O+ type serum in buffered malaria culture medium (MCM). Parasites were kept in T-75 flasks, each containing 25 ml MCM. Parasites were synchronized with repeated treatments of 5% sorbitol for 10 min, and kept in a shaking incubator for all described experiments. Parasites were selected to express the PfEMP1 variant VAR2CSA, by repeated panning using chondroitin sulfate A, as previously described (25.Buffet P.A. Gamain B. Scheidig C. Baruch D. Smith J.D. Hernandez-Rivas R. Pouvelle B. Oishi S. Fujii N. Fusai T. Parzy D. Miller L.H. Gysin J. Scherf A. Plasmodium falciparum domain mediating adhesion to chondroitin sulfate A: a receptor for human placental infection.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 12743-12748Crossref PubMed Scopus (217) Google Scholar). Five μl of each sample (corresponding to 2.5 × 106 pRBC/lane) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 4–12% Bis-Tris gradient gels (Invitrogen, Carlsbad, CA), and subjected to either Western blot or Coomassie staining. For Western blot analysis, after overnight wet transfer using PVDF membranes, the membranes were blocked with 3% Bovine Serum Albumin (BSA) and probed for one hour with anti-Glycophorin C antibodies (1:250, Sigma-Aldrich), anti-ATS 6H-1 antibodies (1:100), anti-Spectrin antibodies (1:500, Abcam, Cambridge, UK), or with sera from the immunized mice described below (1:500). Mouse sera were pre-absorbed before probing for one hour with unparasitized RBCs at a 20:1 dilution. Detection by near-infrared fluorescence was performed using the LI-COR system (Lincoln, NE) after a secondary probe of anti-mouse IRDye-800CW-coupled antibodies (1:3000, LI-COR) was added for one hour. Washes were conducted with Phosphate-buffered saline (PBS) supplemented with 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO). Five μl of packed unparasitized RBCs were incubated with PBS, PBS supplemented with sucrose (5–40%), or RPMI for 15, 30, or 60 min. As a positive lysis control, the same amount of RBCs was incubated with 0.1% Triton-X 100 in PBS for 15, 30, or 60 min. Samples were subsequently centrifuged, and the supernatants collected. The amount of released hemoglobin (Hb) was measured at 415 nm by a Nanodrop spectrophotometer (Nanodrop, Wilmington, DE). The amount of lysis in each sample was calculated as follows: Lysis (test sample) = Absorbance415 (test sample)/Absorbance415 (positive lysis control), and presented as percentage. Trophozoite stage Pf2004–164/TdT (28–36 h p.i.) was harvested by magnet-activated cell sorting (MACS) and 5–6 × 108 pRBCs were washed 3× in PBS and split into two fractions. The first fraction (+) was incubated with 450 μl of PBS supplemented with 10% sucrose, 1 mg/ml trypsin and 1 mg/ml chymotrypsin (PBS-S+T/C; Sigma-Aldrich), and the second fraction (−) was incubated with 10% sucrose in PBS only (PBS-S) for 1 h while rotating at 37 °C. Cells were then collected by centrifugation, washed twice in PBS supplemented with a protease inhibitor mixture (PBS-PI; Roche, Basel, Switzerland), and washed once in PBS alone. Each cell pellet was subsequently lysed in 2 ml ice-cold hypotonic lysis buffer (5 mm KH2PO4, pH 7.4) for 20 min with repeated vortexing. The cell lysates were then centrifuged for 15 min at 20,000 × g and 80% of the resulting supernatant removed. The remaining cell pellets were lysed two more times and 80% of the supernatant removed. The remaining membrane pellets were then transferred to a new tube and washed 3× in hypotonic lysis buffer and once in PBS by centrifugation for 15 min at 20,000 × g. The final membranes were mixed with TiterMax Gold Adjuvant (Sigma-Aldrich) at a 1:1 ratio and used for animal immunization. For each membrane preparation, a group of six female BALB/c mice from Harlan (Frederick, MD) were immunized three times with 100 μl of the membrane/adjuvant-mix over a course of 6 weeks at BioQual (Rockville, MD). Three biological replicates (A, B, and C) of trophozoite stage Pf2004–164/TdT (28–32 h p.i.) pRBCs were harvested using magnetic cell sorting columns (MACS, Miltenyi Biotec, Auburn, CA) resulting in a yield of 4.8–5.3 × 108 pRBCs (88% parasitemia). The three samples were washed 3x in PBS and split into two fractions. The first fraction (+) was incubated with 450 μl PBS-S+T/C, and the second (−) with PBS-S for one hour while rotating at 37 °C. Cells were collected by centrifugation, washed twice in PBS-PI, and once in PBS alone. Membranes from each sample were subsequently prepared as above. For subsequent Western blots, all samples were normalized to contain the same number of cells (5 × 107 cells per 100 μl loading buffer). Twenty-five microliters of membrane proteins isolated from pRBCs with or without trypsin shaving in SDS sample buffer were loaded to SDS-PAGE (4–12% bis-tris gel, 10 well, 1.5 mm) (Novex by Life Technologies, Carlsbad, CA) and run in MOPS buffer for 1.5 h. A total of 72 bands resulting from six samples were excised for subsequent LC/MS/MS analysis. The 72 samples were derived from three biological replicates, each with or without trypsin shaving, and 12 bands from each sample: four bands (ABCD) over 180 kDa region, four bands (EFGH) from 100–130 kDa region, one band (I) from 70 kDa region and three bands (JKL) around 25–35 kDa region. In-gel digestion of the 72 bands was performed in three batches using an Intavis DigestPro with 32 well set-up (Cologne, Germany). Briefly, the in-gel digestion method included gel band destaining (50:50 ammonium bicarbonate/acetonitrile), shrinking of gel pieces (acetonitrile), reduction and alkylation (dithiothreitol and iodoacetamide solutions in ammonium bicarbonate), washing, in-gel trypsin digestion (8 h, Porcine sequencing trypsin, Promega, Madison, WI) and finally extraction of digested peptides with 50 ml of 1% trifluoroacetic acid/60% acetonitrile solution three times. The resulting peptide mixtures were analyzed by nano LC/MS/MS. Separation was performed using an in-house packed nano column with Magic C18 beads (Bruker, Billerica, MA; 75 μm × 10 cm) on a nanoAcquity HPLC (Waters corporation, Milford MA) in conjunction with an Orbitrap Velos (Thermo, Waltham, MA) using a top 20 data dependent acquisition (DDA) MS/MS experiment in ion trap mode with survey MS at 60 K resolution. Data base searching was performed against a combination of four databases containing 31,385 entries (5538 for P. falciparum 3D7, 5536 for P. falciparum Pf2004, 20,177 for Uniprot_human_rev and 128 for cRAP_proteomics) using Mascot search engine version 2.5.1 (Matrix Science Inc., Boston, MA). Database search parameters included fixed enzyme search with trypsin and maximum two missed cleavage events, fixed modification of cysteines with carbamidomethylation, variable modifications of methionine oxidation, asparagine and glutamine deamidation and N-terminal acetylation, mass tolerances of 10 ppm and 0.8 Da for MS and MS/MS respectively. Further filtering and summarization of the data was performed in Scaffold version 4.4.4 (Proteome Software, Portland, OR) with protein and peptide probability set at 95% with a minimum of 2 unique peptides. A false discovery rate (FDR) less than 0.5% for proteins was measured by the equation FDR = D/T where D was the number of counts of decoys and T was the target identification hits observed using the decoy database. The search results from six samples (3 biological replicates with/without trypsin shaving) of the same band were grouped as one experiment in Scaffold. Twelve sample protein reports with total unique peptide count information were exported from Scaffold and combined into one table. Single protein hits, which are only shown once in six samples or once in each arm, were removed from the table. Software R version 3.1.2 was used to calculate the average of total unique peptide counts for each protein of the treated and nontreated samples, fold changes, p values and FDR rates of an unpaired t test. Two biological replicates (A and B) of synchronized ring stage parasite cultures at 2–4 h post invasion (p.i.) were collected by centrifugation. Each replicate was resuspended in 50 ml PBS supplemented with 32.5 mg EZ-Link Sulfo-NHS-Biotin (Life Technologies) to label host (RBC) surface proteins. After room temperature incubation for 30 min on a rotating shaker, pRBCs were collected and washed twice in 100 mm glycine/PBS to neutralize the biotin, and twice in PBS alone. Each replicate was subsequently re-suspended in MCM and parasites allowed to continue their development as described above. The following day, trophozoite stage pRBCs (28–32 p.i.) were harvested by MACS, yielding 5.7 × 108 pRBCs (74% parasitemia, replicate A) and 7.1 × 108 pRBCs (91% parasitemia, replicate B). Each replicate was subsequently split into two fractions. The first fraction (+) was incubated with 500 μl PBS+10% sucrose supplemented with 70 μg/ml mass-spectrometry grade Trypsin (Sigma-Aldrich), whereas the second fraction (−) was mock-treated (PBS and 10% sucrose alone). After a one-hour incubation at 37 °C while rotating, the four samples (A+ and A−, B+ and B-) were centrifuged for 8 min at 240 × g at room temperature to separate the supernatant containing surface shaved peptides and/or pRBC lysis products from intact pRBCs. For subsequent Western blots, 5 × 107 intact pRBCs from each sample were resuspended in 100 μl SDS loading buffer. The supernatants were first cleared of any remaining cell debris by centrifugation for 20 min at 14,000 × g at 4 °C. Next, the supernatants were cleared from “contaminating” host RBC surface proteins by incubating each supernatant (500 μl) with 70 μl of washed MagnaBind Strepatavidin Beads (Life Technologies) for 30 min while rotating. The beads were removed with the MACS magnet and the supernatants were centrifuged for 20 min as 14,000 × g to remove any remaining beads. Next samples were prepared for quantitative mass spectrometry with iTRAQ. A diagram of the procedure is shown in supplemental Fig. S1. Protein content of each of the four samples (A+ and A−, B+ and B-) was measured using Pierce BCA protein assay. Twenty mm DTT was added to 100 μg of protein and samples were incubated for 30 min at 37 °C. Iodoacetamide was added at a final concentration of 50 mm and samples were incubated for 30 min in the dark at room temperature. Before trypsin digestion, urea concentration was diluted to less than 1 m by adding water and pH adjusted to 8 with 1 m Tris solution. 2 μg sequencing grade trypsin (Promega, Madison, WI) was added (1:50 enzyme to substrate ratio) and samples were incubated at 37 °C with shaking for 16 h. The reaction was stopped by addition of formic acid (FA) to a final concentration of 1% and the solution was desalted with a 1 cc (30 mg) Oasis HLB reverse phase cartridge (Waters, Milford, MA) conditioned with 3 × 500 μl acetonitrile (ACN), followed by 4 × 500 μl 0.1% FA. Samples were loaded onto the cartridges and washed with 3 × 500 μl 0.1% FA. Desalted peptides were eluted by 2 applications of 500 μl of 80% ACN/0.1% FA. Eluates were frozen, dried via vacuum centrifugation and stored at −80 °C before peptide fractionation. For each sample the de-salted peptides were labeled with iTRAQ 4-plex reagents according to the manufacturer's instructions (AB Sciex, Foster City, CA)." @default.
• W2770680654 created "2017-12-04" @default.
• W2770680654 creator A5001372647 @default.
• W2770680654 creator A5004504354 @default.
• W2770680654 creator A5011964221 @default.
• W2770680654 creator A5012050989 @default.
• W2770680654 creator A5017206147 @default.
• W2770680654 creator A5017286016 @default.
• W2770680654 creator A5035429491 @default.
• W2770680654 creator A5036255260 @default.
• W2770680654 creator A5049680626 @default.
• W2770680654 creator A5062802030 @default.
• W2770680654 creator A5064975456 @default.
• W2770680654 creator A5067632672 @default.
• W2770680654 creator A5068115789 @default.
• W2770680654 creator A5076289671 @default.
• W2770680654 creator A5081926124 @default.
• W2770680654 creator A5086201552 @default.
• W2770680654 creator A5090009935 @default.
• W2770680654 date "2018-01-01" @default.
• W2770680654 modified "2023-09-28" @default.
• W2770680654 title "Quantitative Proteomic Profiling Reveals Novel Plasmodium falciparum Surface Antigens and Possible Vaccine Candidates" @default.
• W2770680654 cites W1575837378 @default.
• W2770680654 cites W1576735252 @default.
• W2770680654 cites W1911382205 @default.
• W2770680654 cites W1950905025 @default.
• W2770680654 cites W1967117573 @default.
• W2770680654 cites W1969503178 @default.
• W2770680654 cites W1970499587 @default.
• W2770680654 cites W1973419885 @default.
• W2770680654 cites W1973541716 @default.
• W2770680654 cites W1983035189 @default.
• W2770680654 cites W1984097931 @default.
• W2770680654 cites W1985706362 @default.
• W2770680654 cites W1985782539 @default.
• W2770680654 cites W2013503056 @default.
• W2770680654 cites W2015082048 @default.
• W2770680654 cites W2021874649 @default.
• W2770680654 cites W2026745968 @default.
• W2770680654 cites W2033929194 @default.
• W2770680654 cites W2035080983 @default.
• W2770680654 cites W2035169581 @default.
• W2770680654 cites W2037173275 @default.
• W2770680654 cites W2042345412 @default.
• W2770680654 cites W2049700891 @default.
• W2770680654 cites W2051569783 @default.
• W2770680654 cites W2052523483 @default.
• W2770680654 cites W2058756577 @default.
• W2770680654 cites W2064830352 @default.
• W2770680654 cites W2064854096 @default.
• W2770680654 cites W2068819157 @default.
• W2770680654 cites W2072386798 @default.
• W2770680654 cites W2075117194 @default.
• W2770680654 cites W2081711923 @default.
• W2770680654 cites W2082400094 @default.
• W2770680654 cites W2095300212 @default.
• W2770680654 cites W2097432311 @default.
• W2770680654 cites W2099067391 @default.
• W2770680654 cites W2100962331 @default.
• W2770680654 cites W2103900678 @default.
• W2770680654 cites W2104112226 @default.
• W2770680654 cites W2108932995 @default.
• W2770680654 cites W2111443837 @default.
• W2770680654 cites W2114390237 @default.
• W2770680654 cites W2115150674 @default.
• W2770680654 cites W2116272367 @default.
• W2770680654 cites W2116416728 @default.
• W2770680654 cites W2116931446 @default.
• W2770680654 cites W2117312427 @default.
• W2770680654 cites W2121134851 @default.
• W2770680654 cites W2126296380 @default.
• W2770680654 cites W2128611502 @default.
• W2770680654 cites W2129783376 @default.
• W2770680654 cites W2130078103 @default.
• W2770680654 cites W2130366948 @default.
• W2770680654 cites W2134191208 @default.
• W2770680654 cites W2137252590 @default.
• W2770680654 cites W2139449809 @default.
• W2770680654 cites W2140729960 @default.
• W2770680654 cites W2141458291 @default.
• W2770680654 cites W2145767020 @default.
• W2770680654 cites W2146728635 @default.
• W2770680654 cites W2151602360 @default.
• W2770680654 cites W2156160408 @default.
• W2770680654 cites W2162289311 @default.
• W2770680654 cites W2165158644 @default.
• W2770680654 cites W2166068354 @default.
• W2770680654 cites W2166820820 @default.
• W2770680654 cites W2168133698 @default.
• W2770680654 cites W2436016506 @default.
• W2770680654 doi "https://doi.org/10.1074/mcp.ra117.000076" @default.
• W2770680654 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5750850" @default.
• W2770680654 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/29162636" @default.
• W2770680654 hasPublicationYear "2018" @default.
• W2770680654 type Work @default.
• W2770680654 sameAs 2770680654 @default.
• W2770680654 citedByCount "25" @default.
• W2770680654 countsByYear W27706806542018 @default.

• next