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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Greaves, Ronda / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter


IMPACT FACTOR 2018: 3.638

CiteScore 2018: 2.44

SCImago Journal Rank (SJR) 2018: 1.191
Source Normalized Impact per Paper (SNIP) 2018: 1.205

Online
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1437-4331
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Volume 43, Issue 12

Issues

Separation of human serum proteins using the Beckman-Coulter PF2D™ system: analysis of ion exchange-based first dimension chromatography

Isabella Levreri / Luca Musante
  • Laboratorio di Fisiopatologia dell'Uremia, U.O. di Nefrologia, Istituto Scientifico Giannina Gaslini, Genova, Italy
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Andrea Petretto
  • Laboratorio di Spettrometria di Massa, Core facility, Istituto Scientifico Giannina Gaslini, Genova, Italy
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Maurizio Bruschi
  • Laboratorio di Fisiopatologia dell'Uremia, U.O. di Nefrologia, Istituto Scientifico Giannina Gaslini, Genova, Italy
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Giovanni Candiano
  • Laboratorio di Fisiopatologia dell'Uremia, U.O. di Nefrologia, Istituto Scientifico Giannina Gaslini, Genova, Italy
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Giovanni Melioli
Published Online: 2005-11-28 | DOI: https://doi.org/10.1515/CCLM.2005.227

Abstract

The ProteomeLab™ PF2D protein fractionation system is a rapid, semi-automated, 2 D-HPLC instrument that uses two different methods to separate plasma serum proteins: ion-exchange chromatography using a wide pH range in the first dimension, and non-porous reverse-phase chromatography in the second dimension. Because this methodology has only very recently been introduced in proteomic laboratories, little is known about the characteristics of PF2D fractionation of human serum proteins. To evaluate the system's application in a clinical laboratory setting, the characteristics of the ion-exchange chromatography-based separation were analyzed. Following fractionation of human serum proteins on a linear pH gradient (ranging from 8.0 to 4.0), each fraction was collected in a cool module of the instrument. Different fractions obtained from the first dimension were then pooled together and loaded on classic 2D gel electrophoresis instrumentation. The different spots obtained were then checked against the Swiss-Prot Data-base. A total of 36 human serum proteins were identified in different PF2D-generated fractions. Some important features of the separation system were observed. Different eluted fractions contained different proteins, thus demonstrating the reliability of the fractionation system. The proteins were also fractionated according to the theoretical isoelectric point (pI). This was consistent with the evidence that the vast majority of immunoglobulins, characterized by an alkaline pI, were not retained by the column and were eluted in the unbound fraction. This outcome also underlines a practical advantage: fractions eluted from pH8 to pH4 contained virtually immunoglobulin-depleted serum proteins. This finding supports an immediate use of the PF2D system in a clinical setting, where abundant proteins should be clearly identified to enable evaluation of other less abundant, but potentially relevant, species.

Keywords: 2D gels; plasma protein fractionation; ProteomeLab™ PF2D; pI value

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About the article

Corresponding author: Isabella Levreri, BD, Laboratorio Centrale di Analisi, Istituto G. Gaslini, Largo Gerolamo Gaslini 5, 16147 Genova, Italy Phone: +39-010-5636557, Fax: +39-010-3994168,


Received: 2005-05-19

Accepted: 2005-09-22

Published Online: 2005-11-28

Published in Print: 2005-12-01


Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), Volume 43, Issue 12, Pages 1327–1333, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/CCLM.2005.227.

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