Monogenoids are fish parasites which are able to quickly and reversibly attach to their host through the secretion of a proteic glue. Unlike most adhesive secretions found in the animal world which stick to abiotic substrates, monogenoidean adhesive secretion works on living tissues. Moreover, the adhesion of the parasite to its host takes place in an aqueous environment, in the presence of strong water currents. All these features make it a very promising material for exploitation in the surgical field. Another unique and very interesting feature of monogenoidean adhesion is that it can be easily reverted through the secretion of a still uncharacterized “detaching” protein (S3), likely a protease. The adhesive secretion is produced by glands located besides the pharynx or more specifically, in the antero-lateral region of the animal. Demonstration of the proteinaceous nature of the glue came in 2002 (Hamwood TE et al., Folia parasitologica; 49(1):39-49) who showed that the material is SDS insoluble without any trace of glycosylation. No further characterization of the adhesive proteins has been performed since this initial report. Besides very promising potential clinical applications, the study of these proteins represents something completely new also from the biochemical point of view. Understanding the structure of these adhesive proteins, the way they combine to yield the insoluble glue working in an aqueous environment and the mechanism of interaction with a biological substrate, like an epithelium, represents a major challenge which will widen our knowledge about protein structure/function relationship. The aim of this work is the characterization of the bio-adhesive material by means of a proteomic approach. The first step of the project was to set up the conditions to obtain and solubilise the secreted material before separation by electrophoresis and characterization by tandem mass spectrometry. OBTAINING THE SECRETED ADHESIVE MATERIAL The secreted material was obtained by electrostimulation of the parasites in a 50% PBS solution using 40 volts electric field and 2 Hz frequency. The secretion of 30 parasites was collected in a test tube and the SDS soluble components were discharged. The remaining material was solubilised in buffer A (7M Urea, 2M Tiourea, 4% Chaps, 40mM TrisHCl). Upon sonication and centrifugation the supernatant was collected and the protein content was evaluated measuring the absorbance at 280 nm before electrophoresis. PROTEIN SEPARATION BY 2D ELECTROPHORESIS The proteins soluble in buffer A were separated by 2-D electrophoresis upon reduction and derivatization by iodoacetamide. Isoelectric focusing (IEF) was performed in 7 cm immobilised pH gradient (IPG) strips with non linear pH ranges of 3-10. The second dimension was performed on 12% SDS-polyacrylamide gels using a Mini Protean cell. Running proceeded at 20 mA/gel. After running, the 2-D gels were stained with a mass-compatible silver. TANDEM MASS SPECTROMETRY AND DE NOVO SEQUENCING For mass analysis, each spot was excised, destained with 50% acetonitrile in ammonium bicarbonate 0.1 M (40 min at 25 °C), dried in a Speed Vac, soaked with ammonium bicarbonate 0.1 M and digested overnight with trypsin sequencing grade (Roche, Monza, Italy) at 37 °C. The in gel tryptic digest was extracted with 50% acetonitrile in 0.1% trifluoroacetic acid. Digested aliquots were removed and subjected to a desalting/concentration step on a µZipTipC18 (Millipore) using 40% CH3CN in 0.1% TFA as eluent. LC-ESI-MS/MS analysis was performed on a Dionex UltiMate 3000 HPLC System with a Hypersil Gold column (150 mm, internal diameter of 180 μm) filled with 3μm Reprosil-Pur C18-AQ resin. The gradient consisted of 5-15 % acetonitrile in 0.1% formic acid for 10 min, 15-40% acetonitrile in 0.1% formic acid for 52 min and 40-95 % acetonitrile in 0.1% formic for 68 min at a flow rate of 1.2 μl/min . The eluate was electrosprayed into an LTQ Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany) through a Proxeon nanoelectrospray ion source for de novo sequencing since the genome of these parasites is still unknown. Tandem mass spectrometry analysis was performed with each full scan (400-1700 m/z) followed by 5 data-dependent MS/MS scan at 35% normalized collision energy. The full scans were acquired with 2-microscan averaging at resolution 30000, AGC target 500000, maximum inection time 500 ms. All MS/MS data were analyzed with the Sequest program and the Peak Studio 5.1 program.
Characterization of a new bioadhesive material from fish parasites by a proteomic approach / E. Maffioli, A. Negri, P. Galli, P. Fusi, G. Tedeschi. ((Intervento presentato al 5. convegno European summer school in proteomic basics : from methods to clinical applications tenutosi a Bressanone nel 2011.
|Titolo:||Characterization of a new bioadhesive material from fish parasites by a proteomic approach|
MAFFIOLI, ELISA MARGHERITA (Primo)
NEGRI, ARMANDO (Secondo)
TEDESCHI, GABRIELLA (Ultimo)
|Data di pubblicazione:||ago-2011|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||Characterization of a new bioadhesive material from fish parasites by a proteomic approach / E. Maffioli, A. Negri, P. Galli, P. Fusi, G. Tedeschi. ((Intervento presentato al 5. convegno European summer school in proteomic basics : from methods to clinical applications tenutosi a Bressanone nel 2011.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|