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• W2171756247 abstract "Cryptosporidiosis, caused by coccidian parasites of the genus Cryptosporidium, is a major cause of human gastrointestinal infections and poses a significant health risk especially to immunocompromised patients. Despite intensive efforts for more than 20 years, there is currently no effective drug treatment against these protozoa. This study examined the zoonotic species Cryptosporidium parvum at two important stages of its life cycle: the non-excysted (transmissive) and excysted (infective) forms. To increase our understanding of the molecular basis of sporozoite excystation, LC-MS/MS coupling with a stable isotope N-terminal labeling strategy using iTRAQ™ reagents was used on soluble fractions of both non-excysted and excysted sporozoites, i.e. sporozoites both inside and outside oocysts were examined. Sporozoites are the infective stage that penetrates small intestinal enterocytes. Also to increase our knowledge of the C. parvum proteome, shotgun sequencing was performed on insoluble fractions from both non-excysted and excysted sporozoites. In total 303 C. parvum proteins were identified, 56 of which, hitherto described as being only hypothetical proteins, are expressed in both excysted and non-excysted sporozoites. Importantly we demonstrated that the expression of 26 proteins increases significantly during excystation. These excystation-induced proteins included ribosomal proteins, metabolic enzymes, and heat shock proteins. Interestingly three Apicomplexa-specific proteins and five Cryptosporidium-specific proteins augmented in excysted invasive sporozoites. These eight proteins represent promising targets for developing vaccines or chemotherapies that could block parasite entry into host cells. Cryptosporidiosis, caused by coccidian parasites of the genus Cryptosporidium, is a major cause of human gastrointestinal infections and poses a significant health risk especially to immunocompromised patients. Despite intensive efforts for more than 20 years, there is currently no effective drug treatment against these protozoa. This study examined the zoonotic species Cryptosporidium parvum at two important stages of its life cycle: the non-excysted (transmissive) and excysted (infective) forms. To increase our understanding of the molecular basis of sporozoite excystation, LC-MS/MS coupling with a stable isotope N-terminal labeling strategy using iTRAQ™ reagents was used on soluble fractions of both non-excysted and excysted sporozoites, i.e. sporozoites both inside and outside oocysts were examined. Sporozoites are the infective stage that penetrates small intestinal enterocytes. Also to increase our knowledge of the C. parvum proteome, shotgun sequencing was performed on insoluble fractions from both non-excysted and excysted sporozoites. In total 303 C. parvum proteins were identified, 56 of which, hitherto described as being only hypothetical proteins, are expressed in both excysted and non-excysted sporozoites. Importantly we demonstrated that the expression of 26 proteins increases significantly during excystation. These excystation-induced proteins included ribosomal proteins, metabolic enzymes, and heat shock proteins. Interestingly three Apicomplexa-specific proteins and five Cryptosporidium-specific proteins augmented in excysted invasive sporozoites. These eight proteins represent promising targets for developing vaccines or chemotherapies that could block parasite entry into host cells. The protozoan parasite Cryptosporidium parvum, which causes acute gastroenteritis in both humans and animals, is considered an important pathogen all over the world (1Doganci T. Araz E. Ensari A. Tanyuksel M. Doganci L. Detection of Cryptosporidium parvum infection in childhood using various techniques..Med. Sci. Monit. 2002; 8: 223-226Google Scholar). Cryptosporidium is one of several genera in the phylum Apicomplexa, whose members share a common apical secretory apparatus mediating locomotion and tissue or cellular invasion (2Fayer R. Speer C.A. Dubey J.P. General biology of Cryptosporidium.in: Fayer R. Cryptosporidium and Cryptosporidiosis. CRC Press, Boca Raton, FL1997: 1-42Google Scholar). There are currently 16 recognized species in this genus (3Xiao L. Fayer R. Ryan U. Upton S.J. Cryptosporidium taxonomy: recent advances and implications for public health..Clin. Microbiol. Rev. 2004; 17: 72-97Crossref PubMed Scopus (719) Google Scholar), all of which have four naked sporozoites contained within a thick walled oocyst (Fig. 1A), the resistant and transmissive form of these parasites (4Blackman M.J. Bannister L.H. Apical organelles of Apicomplexa: biology and isolation by subcellular fractionation..Mol. Biochem. Parasitol. 2001; 117: 11-25Crossref PubMed Scopus (122) Google Scholar). Species of the genus Cryptosporidium have a complex life cycle. Upon ingestion, excystation of viable oocysts (4–6-μm diameter for intestinal species; gastric species are slightly larger) is triggered (5Smith H.V. Nichols R.A.B. Grimason A.M. Cryptosporidium excystation and invasion: getting to the guts of the matter..Trends Parasitol. 2005; 21: 133-142Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 6Sunnotel O. Lowery C.J. Moore J.E. Dooley J.S. Xiao L. Millar B.C. Rooney P.J. Snelling W.J. Under the microscope: Cryptosporidium..Lett. Appl. Microbiol. 2006; 42: 7-14Crossref PubMed Scopus (61) Google Scholar). Following excystation, sporozoites (Fig. 1B; the infective stage) attach to the intestinal epithelium, are enveloped by the apical membrane, and reside in an intracellular, extracytoplasmic parasitophorous vacuole (5Smith H.V. Nichols R.A.B. Grimason A.M. Cryptosporidium excystation and invasion: getting to the guts of the matter..Trends Parasitol. 2005; 21: 133-142Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 7McCole D.F. Eckmann L. Laurent F. Kagnoff M.F. Intestinal epithelial cell apoptosis following Cryptosporidium parvum infection..Infect. Immun. 2000; 68: 1710-1713Crossref PubMed Scopus (116) Google Scholar). In this peculiar niche, the parasite undergoes an endogenous asexual and sexual reproductive development (Fig. 1C), culminating in the production of an encysted stage discharged in the feces of their host (6Sunnotel O. Lowery C.J. Moore J.E. Dooley J.S. Xiao L. Millar B.C. Rooney P.J. Snelling W.J. Under the microscope: Cryptosporidium..Lett. Appl. Microbiol. 2006; 42: 7-14Crossref PubMed Scopus (61) Google Scholar, 8Fayer R. Morgan U. Upton S.J. Epidemiology of Cryptosporidium: transmission, detection, and identification..Int. J. Parasitol. 2000; 30: 1305-1322Crossref PubMed Scopus (685) Google Scholar). Coccidian oocyst stages are highly resistant to environmental stress and treatments, e.g. chemical disinfection; this is attributed to a durable oocyst wall, a complex protective barrier consisting of a double layer of a protein-lipid-carbohydrate matrix, allowing the parasite to stably persist outside hosts (2Fayer R. Speer C.A. Dubey J.P. General biology of Cryptosporidium.in: Fayer R. Cryptosporidium and Cryptosporidiosis. CRC Press, Boca Raton, FL1997: 1-42Google Scholar, 6Sunnotel O. Lowery C.J. Moore J.E. Dooley J.S. Xiao L. Millar B.C. Rooney P.J. Snelling W.J. Under the microscope: Cryptosporidium..Lett. Appl. Microbiol. 2006; 42: 7-14Crossref PubMed Scopus (61) Google Scholar). C. parvum causes self-limited watery diarrhea in immunocompetent subjects but is life-threatening in immunocompromised patients (1Doganci T. Araz E. Ensari A. Tanyuksel M. Doganci L. Detection of Cryptosporidium parvum infection in childhood using various techniques..Med. Sci. Monit. 2002; 8: 223-226Google Scholar, 9Caccio S.M. Molecular epidemiology of human cryptosporidiosis..Parassitologia. 2005; 47: 185-192PubMed Google Scholar, 10Chen X.M. O’Hara S.P. Huang B.Q. Splinter P.L. Nelson J.B. LaRusso N.F. Localized glucose and water influx facilitates Cryptosporidium parvum cellular invasion by means of modulation of host-cell membrane protrusion..Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6338-6343Crossref PubMed Scopus (77) Google Scholar). Cryptosporidiosis is also responsible for significant neonatal morbidity in farmed livestock and causes weight loss and growth retardation, leading to large economic losses (11McDonald V. Host cell-mediated responses to infection with Cryptosporidium..Parasite Immunol. 2000; 22: 597-604Crossref PubMed Scopus (58) Google Scholar). C. parvum is transmitted by fecal contamination of food or water or by accidental ingestion of infectious oocysts during water-related recreational activities (12Priest J.W. Mehlert A. Moss D.M. Arrowood M.J. Ferguson M.A. Characterization of the glycosylphosphatidylinositol anchor of the immunodominant Cryptosporidium parvum 17-kDa antigen..Mol. Biochem. Parasitol. 2006; 149: 108-112Crossref PubMed Scopus (13) Google Scholar). In 1993, the largest Cryptosporidium spp. outbreak was registered in Milwaukee, WI where 403,000 people were infected through contaminated drinking water (13MacKenzie W.R. Hoxie N.J. Proctor M.E. Gradus M.S. Blair K.A. Peterson D.E. Kazmierczak J.J. Addiss D.G. Fox K.R. Rose J.B. A massive outbreak in Milwaukee of cryptosporidium infection transmitted through the public water supply..N. Engl. J. Med. 1994; 331: 161-167Crossref PubMed Scopus (1597) Google Scholar) with associated economic costs of $31.7 million in medical costs and $64.6 million in productivity losses (14Corso P.S. Kramer M.H. Blair K.A. Addiss D.G. Davis J.P. Haddix A.C. Cost of illness in the 1993 waterborne Cryptosporidium outbreak, Milwaukee, Wisconsin..Emerg. Infect. Dis. 2003; 9: 426-431Crossref PubMed Scopus (218) Google Scholar). Over the last 2 decades increasing numbers of cryptosporidiosis outbreaks have been recorded in developed countries, and the importance of the zoonotic species C. parvum is being recognized by both government agencies and the global scientific community (15Zhu G. LaGier M.J. Hirose S. Keithly J.S. Cryptosporidium parvum: functional complementation of a parasite transcriptional coactivator CpMBF1 in yeast..Exp. Parasitol. 2000; 96: 195-201Crossref PubMed Scopus (12) Google Scholar, 16Craun G.F. Calderon R.L. Craun M.F. Outbreaks associated with recreational water in the United States..Int. J. Environ. Health Res. 2005; 15: 243-262Crossref PubMed Scopus (165) Google Scholar). Unlike many organisms belonging to the phylum Apicomplexa, such as Plasmodium spp. and Toxoplasma gondii, there is no clinically proven effective drug treatment against Cryptosporidium spp. (17Putignani L. The unusual architecture and predicted function of the mitochondrion organelle in Cryptosporidium parvum and hominis species: the strong paradigm of the structure-function relationship..Parassitologia. 2005; 47: 217-225PubMed Google Scholar). Proteomics profiling is a useful approach for obtaining a global overview of the proteins present in a system under differing conditions and can aid in understanding the molecular determinants involved with pathogenesis and vaccine development (18Adkins J.N. Mottaz H.M. Norbeck A.D. Gustin J.K. Rue J. Clauss T.R. Purvine S.O. Rodland K. Heffron F. Smith R.D. Analysis of the Salmonella typhimurium proteome through environmental response towards infectious conditions..Mol. Cell. Proteomics. 2006; 5: 1450-1461Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Compared with other apicomplexans, e.g. Toxoplasma gondii, both the limited supply of purified parasite material and the lack of transfection systems have restricted analyses of proteins in parasites of the genus Cryptosporidium (15Zhu G. LaGier M.J. Hirose S. Keithly J.S. Cryptosporidium parvum: functional complementation of a parasite transcriptional coactivator CpMBF1 in yeast..Exp. Parasitol. 2000; 96: 195-201Crossref PubMed Scopus (12) Google Scholar). The genomics analysis of C. parvum and the closely related Cryptosporidium hominis (19Xu P. Widmer G. Wang Y. Ozaki L.S. Alves J.M. Serrano M.G. Puiu D. Manque P. Akiyoshi D. Mackey A.J. Pearson W.R. Dear P.H. Bankier A.T. Peterson D.L. Abrahamsen M.S. Kapur V. Tzipori S. Buck G.A. The genome of Cryptosporidium hominis..Nature. 2004; 431: 1107-1112Crossref PubMed Scopus (426) Google Scholar) has revealed extremely streamlined metabolic pathways and a reliance on hosts for nutrients (20Abrahamsen M.S. Templeton T.J. Enomoto S. Abrahante J.E. Zhu G. Lancto C.A. Deng M. Liu C. Widmer G. Tzipori S. Buck G.A. Xu P. Bankier A.T. Dear P.H. Konfortov B.A. Spriggs H.F. Iyer L. Anantharaman V. Aravind L. Kapur V. Complete genome sequence of the apicomplexan, Cryptosporidium parvum..Science. 2004; 304: 441-445Crossref PubMed Scopus (756) Google Scholar). C. parvum has an animal-type O-linked glycosylation pathway and >30 predicted surface proteins with mucin-like segments (21Templeton T.J. Iyer L.M. Anantharaman V. Enomoto S. Abrahante J.E. Subramanian G.M. Hoffman S.L. Abrahamsen M.S. Aravind L. Comparative analysis of Apicomplexa and genomic diversity in eukaryotes..Genome Res. 2004; 14: 1686-1695Crossref PubMed Scopus (165) Google Scholar). The mechanisms involved in Cryptosporidium spp. excystation are incompletely understood; although roles for both host- and parasite-derived components are recognized, little is known about their precise involvements (5Smith H.V. Nichols R.A.B. Grimason A.M. Cryptosporidium excystation and invasion: getting to the guts of the matter..Trends Parasitol. 2005; 21: 133-142Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In vitro excystation protocols for C. parvum mimic host-derived signals, including temperature (37 °C), pH fluctuations, and bile salts (5Smith H.V. Nichols R.A.B. Grimason A.M. Cryptosporidium excystation and invasion: getting to the guts of the matter..Trends Parasitol. 2005; 21: 133-142Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). We adopted an accepted in vitro excystation method (22Gut J. Nelson R.G. Cryptosporidium parvum: synchronized excystation in vitro and evaluation of sporozoite infectivity with a new lectin-based assay..J. Eukaryot. Microbiol. 1999; 46: 56S-57SCrossref PubMed Scopus (8) Google Scholar) and analyzed C. parvum proteins at two key stages, both the transmissive (oocysts) and infective stages (sporozoites), of its life cycle (5Smith H.V. Nichols R.A.B. Grimason A.M. Cryptosporidium excystation and invasion: getting to the guts of the matter..Trends Parasitol. 2005; 21: 133-142Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 6Sunnotel O. Lowery C.J. Moore J.E. Dooley J.S. Xiao L. Millar B.C. Rooney P.J. Snelling W.J. Under the microscope: Cryptosporidium..Lett. Appl. Microbiol. 2006; 42: 7-14Crossref PubMed Scopus (61) Google Scholar). Thus, as well as evaluating the proteome of C. parvum, a major aim of our study was to identify proteins that demonstrated significant increases of expression during oocyst excystation, a vital step of the pathogenesis of these coccidian parasites (6Sunnotel O. Lowery C.J. Moore J.E. Dooley J.S. Xiao L. Millar B.C. Rooney P.J. Snelling W.J. Under the microscope: Cryptosporidium..Lett. Appl. Microbiol. 2006; 42: 7-14Crossref PubMed Scopus (61) Google Scholar). This was achieved by using LC/MS and iTRAQ™ reagent isotope labeling, providing a powerful tool for the identification and quantification (23Wu X.D. Kircher R.A. McVerry P.H. Malinzak D.A. Characterization of post-translationally modified recombinant protein using liquid chromatography/mass spectrometry..Dev. Biol. (Basel). 2000; 103: 61-67PubMed Google Scholar, 24Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents..Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3659) Google Scholar) of soluble C. parvum proteins. Using LC/MS with iTRAQ the protein expression in non-excysted and excysted soluble C. parvum oocyst fractions was compared. Then using shotgun peptide sequencing (25Kislinger T. Gramolini A.O. Pan Y. Rahman K. MacLennan D.H. Emili A. Proteome dynamics during C2C12 myoblast differentiation..Mol. Cell. Proteomics. 2005; 4: 887-901Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) the insoluble protein content of non-excysted and excysted oocysts was also analyzed. To recover high numbers of C. parvum oocysts, with minimal fecal and bacterial contaminants (26Truong Q. Ferrari B.C. Quantitative and qualitative comparisons of Cryptosporidium faecal purification procedures for the isolation of oocysts suitable for proteomic analysis..Int. J. Parasitol. 2006; 36: 811-819Crossref PubMed Scopus (31) Google Scholar), 4 × 108 C. parvum oocysts (strain code ISSC162) were recovered from experimentally infected calves using feces purification with sucrose and Percoll density gradients (27Arrowood J.M. Sterling C.R. Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients..J. Parasitol. 1987; 73: 314-319Crossref PubMed Scopus (345) Google Scholar). Purified oocysts were stored at 4 °C for no longer than 3 weeks. Using 4′,6′-diamidino-2-phenylindole-propidium iodide assays (28Bukhari Z. Marshall M.M. Korich D.G. Fricker C.R. Smith H.V. Rosen J. Clancy J.L. Comparison of Cryptosporidium parvum viability and infectivity assays following ozone treatment of oocysts..Appl. Environ. Microbiol. 2000; 66: 2972-2980Crossref PubMed Scopus (90) Google Scholar), oocyst viability was determined to be at least 92%. To obtain free sporozoites, half of the oocysts (2 × 108) were excysted as described previously (Fig. 2) (22Gut J. Nelson R.G. Cryptosporidium parvum: synchronized excystation in vitro and evaluation of sporozoite infectivity with a new lectin-based assay..J. Eukaryot. Microbiol. 1999; 46: 56S-57SCrossref PubMed Scopus (8) Google Scholar). Briefly oocysts were resuspended in 10 mm HCl (Sigma) and incubated at 37 °C for 10 min. The suspension was centrifuged at 3,000 × g for 5 min. The pellet was then resuspended in 2 mm sodium taurocholate in PBS (Sigma) and incubated at 15 °C for 10 min followed by incubation at 37 °C for 8 min, and a high level of excystation, i.e. at least 90%, was confirmed (29Mele R. Gomez Morales M.A. Tosini F. Pozio E. Cryptosporidium parvum at different developmental stages modulates host cell apoptosis in vitro..Infect. Immun. 2004; 72: 6061-6067Crossref PubMed Scopus (67) Google Scholar). All excysted and non-excysted oocysts were solubilized (30 min at 4 °C) in lysis buffer (50 mm Tris (Bio-Rad) 5 mm EDTA (Sigma), 5 mm iodoacetamide (Sigma), 0.1 mm Nα-p-tosyl-l-lysine chloromethyl ketone (Sigma), 1 mm phenylmethylsulfonyl fluoride (Sigma), 1% (w/v) octyl glucoside (Roche Applied Science)) and stored at −80 °C (30Riggs M.W. Cama V.A. Leary Jr., H.L. Sterling C.R. Bovine antibody against Cryptosporidium parvum elicits a circumsporozoite precipitate-like reaction and has immunotherapeutic effect against persistent cryptosporidiosis in SCID mice..Infect. Immun. 1994; 62: 1927-1939Crossref PubMed Google Scholar). Upon thawing, samples were sonicated with 20 10-s pulses (50 watts) on ice with 1-min intervals and were then ultracentrifuged at 4 °C (20,000 × g for 30 min) to separate the soluble and insoluble fractions (30Riggs M.W. Cama V.A. Leary Jr., H.L. Sterling C.R. Bovine antibody against Cryptosporidium parvum elicits a circumsporozoite precipitate-like reaction and has immunotherapeutic effect against persistent cryptosporidiosis in SCID mice..Infect. Immun. 1994; 62: 1927-1939Crossref PubMed Google Scholar). Insoluble fractions were then rinsed three times with ice-cold PBS and stored at −80 °C. Proteins in soluble lysate fractions were then precipitated by mixing oocyst lysates with 8 volumes of ice-cold acetone, 1 volume of TCA, and 0.7% (w/v) 2-mercaptoethanol at −20 °C for 1 h and then centrifuged at 18,000 × g for 15 min at 4 °C (31Mandal P. Chakraborty P. Sau S. Mandal N.C. Purification and characterization of a deoxyriboendonuclease from Mycobacterium smegmatis..J. Biochem. Mol. Biol. 2006; 39: 140-144PubMed Google Scholar, 32Tantipaiboonwong P. Sinchaikul S. Sriyam S. Phutrakul S. Chen S.T. Different techniques for urinary protein analysis of normal and lung cancer patients..Proteomics. 2005; 5: 1140-1149Crossref PubMed Scopus (114) Google Scholar). Pellets of precipitated soluble proteins were then washed with 1 ml of ice-cold acetone, centrifuged at 18,000 × g for 15 min at 4 °C, covered in a layer of ice-cold acetone, and stored at −80 °C. The pellets (soluble and insoluble fractions) were resuspended in 400 μl of sample preparation buffer (100 mm Tris-HCl, 8 m urea, 0.4% SDS, 5 mm tributylphosphine, pH 8.3) and placed on ice (33Ranish J.A. Yi E.C. Leslie D.M. Purvine S.O. Goodlett D.R. Eng J. Aebersold R. The study of macromolecular complexes by quantitative proteomics..Nat. Genet. 2003; 33: 349-355Crossref PubMed Scopus (311) Google Scholar). Sample mixtures were sonicated with five 20-s pulses (50 watts) with 1-min intervals on ice followed by iodoacetamide alkylation at room temperature for 1 h (34Gu Z. Kaul M. Yan B. Kridel S.J. Cui J. Strongin A. Smith J.W. Liddington R.C. Lipton S.A. S-Nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death..Science. 2002; 297: 1186-1190Crossref PubMed Scopus (831) Google Scholar). Samples were then ultracentrifuged at 100,000 × g for 20 min at room temperature. Protein concentrations were measured using a MicroBCA protein assay kit (Pierce) according to the manufacturer’s instruction (35Prozialeck W.C. Fay M.J. Lamar P.C. Pearson C.A. Sigar I. Ramsey K.H. Chlamydia trachomatis disrupts N-cadherin-dependent cell-cell junctions and sequester β-catenin in human cervical epithelial cells..Infect. Immun. 2002; 70: 2605-2613Crossref PubMed Scopus (50) Google Scholar) before reactions were quenched by the addition of DTT (33Ranish J.A. Yi E.C. Leslie D.M. Purvine S.O. Goodlett D.R. Eng J. Aebersold R. The study of macromolecular complexes by quantitative proteomics..Nat. Genet. 2003; 33: 349-355Crossref PubMed Scopus (311) Google Scholar). The protein mixtures were diluted 8-fold in 50 mm Tris-HCl. Modified trypsin (Sigma) was added to a final substrate-to-enzyme ratio of 30:1, and the trypsin digests were incubated overnight at 37 °C. The peptides from each digest solution were acidified to pH 3.0 with formic acid (FA) 1The abbreviations used are: FA, formic acid; ARM, armadillo repeat; HSP, heat shock protein; LCCL, Limulus factor C, cochlear protein Coch-5b2, and late gestation lung protein; QqTOF, quadrupole time of flight. and loaded onto a Discovery DSC-18 cartridge (Sigma). Then peptides were desalted (5 ml of 0.1% FA) and eluted with 5 ml of a solution composed of 50% ACN with 0.1% FA. Peptides derived from insoluble fractions were analyzed qualitatively using shotgun peptide sequencing as described previously (36Wolters D.A. Washburn M.P. Yates III, J.R. An automated multidimensional protein identification technology for shotgun proteomics..Anal. Chem. 2001; 73: 5683-5690Crossref PubMed Scopus (1560) Google Scholar). For peptides derived from soluble fractions, equal amounts (100 μg) of sample were labeled with two iTRAQ reagents (Applied Biosystems, Foster City, CA) following the manufacturer’s instruction: iTRAQ-114 (non-excysted) and iTRAQ-115 (excysted). Briefly after desalting on a C18 cartridge the peptide mixture was lyophilized and resuspended in 30 μl of 0.5 m triethylammonium bicarbonate (N(Et)3HCO3), pH 8.5. The appropriate iTRAQ reagent (dissolved in 70 μl of ethanol) was added, allowed to react for 1 h at room temperature, and then quenched with 300 μl of double distilled H2O. iTRAQ-labeled peptides were then concentrated, mixed, and acidified to a total volume of 2.0 ml. They were then injected into an Agilent 1100 HPLC system with a Zorbax 300-SCX column (15 cm × 4.6 mm, 5 μm) (Agilent, Waldbronn, Germany). Solvent A was 5 mm KHPO4 and 25% ACN (pH 3.0), and solvent B was 350 mm KCl in solvent A. Peptides were eluted from the column with a 40-min mobile phase gradient of solvent B. A total of 30 fractions were collected, and samples were dried by a SpeedVac prior to LC-MS/MS analysis. A nanobore LC system (Dionex, Sunnyvale, CA) that was interfaced to a QSTAR XL QqTOF mass spectrometer with a NanoSpray ion source (Applied Biosystems) was used for mass spectrometry. The Magic C18 column (100-Å pore, 75-μm inner diameter × 150 mm; Picofrit Woburn, MA) was packed in house. Solvent A was 3% ACN, 0.1% FA, and 0.01% TFA, and solvent B was 98% ACN, 0.1% FA, and 0.01% TFA. Peptide mixtures (reconstituted in 200 μl of 5% FA) were injected and eluted from the column with a 110-min mobile phase solvent B gradient (5–5% B in 5 min, 5–18% B in 10 min, 18–30% B in 65 min, 30–60% B in 10 min, 60–90% B in 10 min, and 90–90% in 5 min) at a flow rate of 250 nl/min. The mass spectrometer was operated in an information-dependent acquisition mode whereby following the interrogation of MS data (m/z 350–1500) using a 1-s survey scan ions were selected for MS/MS analysis based on their intensity (>15 cpm) and charge state (+2, +3, and +4). A total of three product ion scans (2, 3, and 3 s each) were set from each survey scan. Rolling collision energies were chosen automatically based on the m/z and charge state of the selected precursor ions. The integrated data appliance Extensions II script was set to one repetition before dynamic exclusion. Identification and quantitation was performed using Pro ID (37Smith J.A. Blanchette R.A. Burnes T.A. Jacobs J.J. Higgins L. Witthuhn B.A. David A.J. Gillman J.H. Proteomic comparison of needles from blister rust-resistant and susceptible Pinus strobus seedlings reveals upregulation of putative disease resistance proteins..Mol. Plant-Microbe Interact. 2006; 19: 150-160Crossref PubMed Scopus (30) Google Scholar) and ProQUANT software 1.1 (38Shadforth I.P. Dunkley T.P.J. Lilley K.S. Bessant C. i-Tracker: For quantitative proteomics using iTRAQ™..BMC Genomics. 2005; 6: 145Crossref PubMed Scopus (237) Google Scholar) (Applied Biosystems) using the non-redundant database C. parvum subdatabase (9811 entries) with an MS and MS/MS mass tolerance of 0.15 Da (39Tang W.H. Halpern B.R. Shilov I.V. Seymour S.L. Keating S.P. Loboda A. Patel A.A. Schaeffer D.A. Nuwaysir L.M. Discovering known and unanticipated protein modifications using MS/MS database searching..Anal. Chem. 2005; 77: 3931-3946Crossref PubMed Scopus (56) Google Scholar). Variable modification on methionine (oxidation, +16 Da) and fixed modification on cysteine (carbamidomethylation, +57 Da) residues were considered during the searching (39Tang W.H. Halpern B.R. Shilov I.V. Seymour S.L. Keating S.P. Loboda A. Patel A.A. Schaeffer D.A. Nuwaysir L.M. Discovering known and unanticipated protein modifications using MS/MS database searching..Anal. Chem. 2005; 77: 3931-3946Crossref PubMed Scopus (56) Google Scholar). Protein identification with confidence scores of >95% were considered significant, and the false positive rate was determined from decoy database (39Tang W.H. Halpern B.R. Shilov I.V. Seymour S.L. Keating S.P. Loboda A. Patel A.A. Schaeffer D.A. Nuwaysir L.M. Discovering known and unanticipated protein modifications using MS/MS database searching..Anal. Chem. 2005; 77: 3931-3946Crossref PubMed Scopus (56) Google Scholar) searching (data not shown). The identifications of all detected C. parvum proteins were then verified using BLASTP at www.ncbi.nlm.nih.gov/BLAST/ or where appropriate at CryptoDB 3.3 (cryptodb.org/cryptodb/). Using ProQUANT software, quantification was based upon the signature peak areas (m/z 114 and 115) and corrected according to the manufacturer’s instructions to account for isotopic overlap. Briefly non-excysted oocysts (control) were labeled with isobaric tag 114, and relative quantification ratios of the identified proteins were calculated, averaged, and corrected for systematic error in labeling from the iTRAQ peptides. For proteins in which the iTRAQ ratio of every peptide (labeled with tag 115) was 0, the average iTRAQ ratio was set to 0, an indication of a protein that may be highly down-regulated relative to the control. Similarly for those proteins where every (tag 115) ratio was 9999, the average was set to 9999 (indicating a protein that may be highly up-regulated). Statistically significant changes were weighted by the error factor (a measure of the variation between the different iTRAQ ratios for the reagent pair) and p value (95% confidence interval, which defined a range into which the true average iTRAQ ratio was 95% likely to fall). For instance, for a ratio of 1.0 (indicating no change in expression levels) with an error factor of 2 (and the corresponding 95% confidence interval of 0.5–2.0), the protein expression ratio and error were reported as 1.0 (p < 0.05 with an error factor of 2). Ratios in which the total peak area was <40 counts were omitted. If there were fewer than two peptides contributing toward an iTRAQ ratio average, the error factor and p values were not valid and were not calculated. Proteins that were augmented significantly during excystation had a p value <0.05 and an error factor <2. Significantly overexpressed proteins were examined using BLASTP both on the National Center for Biotechnology Information (NCBI) database and on ApiDB (www.apidb.org/blast/) to find similarity with other apicomplexan proteins or with proteins from unrelated species. Only proteins identified by the BLASTP search with an expected value lower than 10−10 were considered homologs. Searches were also performed for molecular traits and domains of the putative prote" @default.
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• W2171756247 date "2007-02-01" @default.
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• W2171756247 title "Proteomics Analysis and Protein Expression during Sporozoite Excystation of Cryptosporidium parvum (Coccidia, Apicomplexa)" @default.
• W2171756247 cites W1560624689 @default.
• W2171756247 cites W1583738858 @default.
• W2171756247 cites W1619234695 @default.
• W2171756247 cites W1897413077 @default.
• W2171756247 cites W1966919295 @default.
• W2171756247 cites W1968793425 @default.
• W2171756247 cites W1970696723 @default.
• W2171756247 cites W1972312227 @default.
• W2171756247 cites W1972658233 @default.
• W2171756247 cites W1976529775 @default.
• W2171756247 cites W1979767609 @default.
• W2171756247 cites W1986043296 @default.
• W2171756247 cites W1987617174 @default.
• W2171756247 cites W1991166057 @default.
• W2171756247 cites W1995481775 @default.
• W2171756247 cites W2010332067 @default.
• W2171756247 cites W2016777718 @default.
• W2171756247 cites W2019402469 @default.
• W2171756247 cites W2034014446 @default.
• W2171756247 cites W2041993159 @default.
• W2171756247 cites W2048860760 @default.
• W2171756247 cites W2063906120 @default.
• W2171756247 cites W2074197587 @default.
• W2171756247 cites W2075345926 @default.
• W2171756247 cites W2085357050 @default.
• W2171756247 cites W2087601646 @default.
• W2171756247 cites W2093702487 @default.
• W2171756247 cites W2101004001 @default.
• W2171756247 cites W2106127270 @default.
• W2171756247 cites W2106187536 @default.
• W2171756247 cites W2110823023 @default.
• W2171756247 cites W2114676989 @default.
• W2171756247 cites W2120016245 @default.
• W2171756247 cites W2120191747 @default.
• W2171756247 cites W2120914366 @default.
• W2171756247 cites W2141964056 @default.
• W2171756247 cites W2142878327 @default.
• W2171756247 cites W2144480059 @default.
• W2171756247 cites W2150864606 @default.
• W2171756247 cites W2157477307 @default.
• W2171756247 cites W2163344857 @default.
• W2171756247 cites W2167367944 @default.
• W2171756247 cites W2168741095 @default.
• W2171756247 cites W2169804076 @default.
• W2171756247 cites W260281820 @default.
• W2171756247 cites W3187206468 @default.
• W2171756247 cites W4235316562 @default.
• W2171756247 cites W4362208308 @default.
• W2171756247 doi "https://doi.org/10.1074/mcp.m600372-mcp200" @default.
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