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.
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.
Westman-Brinkmalm
Ann
A
Department of Clinical Neuroscience, Göteborg University, Mölndal, Sweden.
Davidsson
Pia
P
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D011228:Prealbumin / Q000134:cerebrospinal fluid* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D011228:Prealbumin