Nano-LC–MS/MS for the identification of proteins trapped in sorbent cartridges used for coupled plasma filtration-adsorption treatments of healthy pigs

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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, serotranferrin and immunoglobulins, were identified. In addition, the presence of haptoglobin, hemopexin, -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.

Autore Pugliese

Tutti gli autori

  • Nardiello D , Prattichizzo C. , Rocchetti M.T. , Gesualdo L. , Centonze D.

Titolo volume/Rivista

JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS

Anno di pubblicazione

2016

ISSN

0731-7085

ISBN

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Numero di citazioni Wos

Nessuna citazione

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0

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Settori ERC

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Codici ASJC

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