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Comparison of Preparative and Analytical Two-Dimensional Electrophoresis for Isolation and Matrix-Assisted Laser Desorption/Ionization–Time-of-Flight Mass Spectrometric Analysis of Transthyretin in Cerebrospinal Fluid

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
301
Issue
2
Identifiers
DOI: 10.1006/abio.2001.5487
Keywords
  • Analytical 2-De
  • Cerebrospinal Fluid
  • Liquid-Phase Ief
  • Mass Spectrometry
  • Preparative 2-De
  • Prefractionation
  • Transthyretin
Disciplines
  • Biology

Abstract

Abstract The variety of posttranslational modifications and the relative abundance of transthyretin (TTR) in cerebrospinal fluid (CSF) make TTR a suitable model molecule when comparing the performance of different combinations of methods for characterization of CSF proteins. We have compared three different electrophoretic methods: conventional two-dimensional gel electrophoresis (2-DE), liquid-phase isoelectric focusing (IEF) as a prefractionation step in combination with analytical 2-DE, and preparative 2-DE for isolation and mass spectrometric analysis of TTR in CSF. Using liquid-phase IEF in combination with 2-DE compared with conventional 2-DE improved the sequence coverage of TTR. Only the mass spectrum from the preparative 2-DE fraction contained a tryptic peptide from the first nine amino acids, thereby yielding 100% sequence coverage. Our results show that the use of a prefractionation step before 2-DE or the use of preparative 2-DE increases the sequence coverage and provide low abundant proteins in complex biological systems in sufficient quantities for protein characterization with mass spectrometry.

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