RESUMEN
BACKGROUND: The aim of this study was firstly, to improve and validate a cerebrospinal fluid (CSF) prefractionation method followed by two-dimensional electrophoresis (2-DE) and secondly, using this strategy to investigate differences between the CSF proteome of frontotemporal dementia (FTD) patients and controls. From each subject three ml of CSF was prefractionated using liquid phase isoelectric focusing prior to 2-DE. RESULTS: With respect to protein recovery and purification potential, ethanol precipitation of the prefractionated CSF sample was found superior, after testing several sample preparation methods.The reproducibility of prefractionated CSF analyzed on 2-D gels was comparable to direct 2-DE analysis of CSF. The protein spots on the prefractionated 2-D gels had an increased intensity, indicating a higher protein concentration, compared to direct 2-D gels. Prefractionated 2-DE analysis of FTD and control CSF showed that 26 protein spots were changed at least two fold. Using mass spectrometry, 13 of these protein spots were identified, including retinol-binding protein, Zn-alpha-2-glycoprotein, proapolipoproteinA1, beta-2-microglobulin, transthyretin, albumin and alloalbumin. CONCLUSION: The results suggest that the prefractionated 2-DE method can be useful for enrichment of CSF proteins and may provide a new tool to investigate the pathology of neurodegenerative diseases. This study confirmed reduced levels of retinol-binding protein and revealed some new biomarker candidates for FTD.
RESUMEN
By comparing the cerebrospinal fluid (CSF) proteome between Alzheimer's disease (AD) patients and controls, it may be possible to identify proteins that play a role in the disease process and thus to study the pathogenesis of AD. Two-dimensional gel electrophoresis (2-DE), SYPRO Ruby staining and mass spectrometry were used for clinical screening of disease-influenced CSF proteins in AD patients compared to controls. In order to increase the detection of CSF proteins and to improve the separation of protein isoforms in a 2-D gel, micro-narrow range IPG strips were used. The levels of eight proteins and their isoforms, including apolipoprotein A1, apolipoprotein E, apolipoprotein J, beta-trace, retinol-binding protein, kininogen, alpha-1 antitrypsin, cell cycle progression 8 protein, and alpha-1beta glycoprotein were significantly altered in CSF of AD patients compared to controls. Furthermore, we also used liquid-phase IEF, as a prefractionation step, prior to 2-DE for comparison of CSF proteins between individual AD patients and controls. The levels of 37 proteins spots were altered in AD patients. One of the identified proteins, alpha-2-HS glycoprotein, has not previously been linked to AD. Our study shows that several glycoproteins are altered in AD.