de Miranda Santos, pers

de Miranda Santos, pers. blood feeding include 2 glycine-rich, cement-like proteins, 2 lipocalins, and a thyropin protease inhibitor. Among other of the 19 tick proteins recognized, albeit with undefined functions, were SPARC and cyclophilin A. This catalog provides a resource that can be mined for secreted molecules that play a role in tick-host interactions. was explained (Anatriello et al., 2010). This allows for identification of salivary proteins from tick saliva, which can be obtained by activation of partially-fed ticks with the secretagogues dopamine (DA) or pilocarpine (PC) (Kaufman, 1976; Sauer et al., 1995). Tick saliva also contains host proteins (Valenzuela et al., 2002), and the predicted proteomes of several mammals are also available in public databases. Such databases have now made it feasible to identify in greater detail the proteins secreted in tick saliva. In the present work, we describe the proteome of saliva from female ticks partially fed on rabbits and obtained by stimulating the ticks with DA and PC. Material and methods Saliva collection ticks were laboratory-reared, as previously explained (Ferreira and Silva, 1998). To obtain engorged ticks for saliva collection, rabbits (n=4) were infested with 70 pairs of adult ticks restricted by plastic feeding chambers fixed to their backs (Ferreira et al., 1998). The experiments with rabbits are in agreement with the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation (COBEA) in line with the Guidelines for Animal Users as issued by 10074-G5 the National Institute of Health, and the animal protocol was approved by the School of Medicine 10074-G5 of Ribeir?o Preto of the University or college of S?o Paulo, Institutional Animal Care and Use Committee (IACUC) under protocol number 144/2010. The procedure for collection of saliva was performed on partially-engorged (after 5C7 days of feeding) female ticks rinsed in distilled water and dried with filter paper. A solution of DA (10 L at 0.2% in phosphate-buffered saline, pH 7.4) was inoculated into each tick’s hemocoel using a micro-fine 29 Gauge (12.70.33 mm) needle (BD Biosciences, San Diego, CA). Alternatively, 2 L of PC hydrochloride (5% answer in 0.7 M NaCl) was inoculated near the border of the dorsal scutum together with a topical application of 5 L of PC hydrochloride (5% solution in methanol) to their dorsal scutum. Tick saliva was harvested from tick mouthparts using a micropipette, kept on ice, pooled, filtered through a 0.22-m pore filter (Costar-Corning Inc., Cambridge, MA), and stored at ?70C until further use. Gel electrophoresis studies Tick saliva samples (50 L) collected with Rabbit Polyclonal to MAGE-1 DA or PC (0.97 mg/mL and 1.21 mg/mL of protein, respectively) were resolved by one-dimensional (1D) sodium dodecylsulfate polyacrylamide gel electrophoresis (4C12% gradient gels) and visualized with Coomassie blue staining (Pierce, Rockford, IL). Excised gel bands were destained using 50% acetonitrile in 25 mM NH4HCO3, pH 10074-G5 8.4, and vacuum dried. Trypsin (20 g/mL in 25 mM NH4HCO3, pH 8.4) was added, and the combination was incubated on ice for one hour. The supernatant was removed, and the gel bands were covered with 25 mM NH4HCO3, pH 8.4. After overnight incubation at 37C, the tryptic peptides were extracted using 70% acetonitrile, 5% formic acid, and the 10074-G5 peptide answer was lyophilized and desalted using ZipTips (Millipore, Bedford, MA). Nanoflow reversed-phase liquid chromatography tandem mass spectrometry (nanoRPLC -MS/MS) Tryptic peptides were analyzed using nanoRPLC-MS/MS. A 75-m i.d. 360 m o.d. 10 cm long fused silica capillary column (Polymicro Technologies, Phoenix, AZ) was packed with 3 m, 300 ? pore size C-18 silica-bonded stationary RP particles (Vydac/Grace, Deerfield, IL). The column was connected to an Agilent 1100 nanoLC system (Agilent Technologies,.