title: Nano-LC-MS/MS for the identification of proteins trapped in sorbent cartridges used for coupled plasma filtration-adsorption treatments of healthy pigs.
abstract: A dedicated proteomic approach based on nano-Liquid Chromatography coupled with tandem mass spectrometry in ion trap is proposed for the analysis of proteins trapped in sorbent resin cartridges used to remove inflammatory mediators from blood by coupled plasma filtration adsorption (CPFA). The final purpose of the proposed proteomic approach was to obtain a reference map of plasma proteins trapped in CPFA sorbents used for the extracorporeal blood purification of healthy pigs, with the potential impact to design new bio-filters able to control the inflammatory imbalance under pathological conditions, such as severe sepsis. The five main steps of the proteomics analysis, (i) protein extraction from resin cartridges, (ii) two-dimensional gel electrophoresis (2D-PAGE) for protein separation and profiling, (iii) in-gel proteolytic digestion, (iv) tandem mass analysis of peptides resulting from enzymatic cleavage and (v) bioinformatics, for protein identification and post-processing validation of MS/MS data sets, have been carefully evaluated. Prior to electrophoresis, the efficiency of different extraction solutions and procedures to recovery plasma proteins trapped into the sorbents were tested. Then, a rapid one-step procedure for protein extraction was optimized. Protein bands corresponding to the main plasma proteins, namely porcine serum albumin, serotransferrin and immunoglobulins, were identified. In addition, the presence of haptoglobin, hemopexin, α-1 acid glycoprotein and fetuin-A, that are known as acute-phase reaction proteins, was observed, suggesting that CPFA resins led to a non-specifically protein depletion from plasma, rather than targeting specific molecules.
authors: Dipartimento di Scienze Agrarie, degli Alimenti e dell'Ambiente and CSRA- Centro Servizi di Ricerca Applicata, Università degli Studi di Foggia, Via Napoli, 25 - 71122 Foggia, Italy. Electronic address: donatella.nardiello@unifg.it.
authors: Donatella
authors: D
authors: Nardiello
authors: Dipartimento di Scienze Mediche e Chirurgiche, Sezione di Nefrologia, Dialisi e Trapianto, Università degli Studi di Foggia, Viale Luigi Pinto, 1 - 71122 Foggia, Italy.
authors: Clelia
authors: C
authors: Prattichizzo
authors: Dipartimento di Emergenza e Trapianti di Organi, Università degli Studi di Bari, Piazza G. Cesare, 11, 70124 Bari, Italy.
authors: Maria Teresa
authors: MT
authors: Rocchetti
authors: Dipartimento di Emergenza e Trapianti di Organi, Università degli Studi di Bari, Piazza G. Cesare, 11, 70124 Bari, Italy.
authors: Loreto
authors: L
authors: Gesualdo
authors: Dipartimento di Scienze Agrarie, degli Alimenti e dell'Ambiente and CSRA- Centro Servizi di Ricerca Applicata, Università degli Studi di Foggia, Via Napoli, 25 - 71122 Foggia, Italy.
authors: Diego
authors: D
authors: Centonze
mesh_terms: D000210:Acute-Phase Reaction; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D019295:Computational Biology; D045304:Cytochromes c / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005374:Filtration; D006242:Haptoglobins / Q000737:chemistry; D006466:Hemopexin / Q000737:chemistry; D007249:Inflammation; D009961:Orosomucoid / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D059748:Proteolysis; D018805:Sepsis / Q000097:blood / Q000628:therapy; D012984:Software; D016060:Sorption Detoxification / Q000379:methods; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000379:methods*; D060749:alpha-2-HS-Glycoprotein / Q000737:chemistry
chemical_list: D006242:Haptoglobins; D009961:Orosomucoid; D011506:Proteins; D060749:alpha-2-HS-Glycoprotein; D045304:Cytochromes c; D006466:Hemopexin
keywords: 2D-PAGE; Collision induced dissociation; Coupled plasma filtration-adsorption (CPFA); Nano-LC–MSMS; Pig plasma