ABSTRACT
Cerebrospinal fluid (CSF) has been rediscovered in the post-genomic era as a great source of potential protein biomarkers for various diseases. The source allows rapid screening, low sample consumption, and accurate protein identification by proteomic technology. In the present study, we identified 2 candidate tumor-related proteins, N-myc oncoprotein and low-molecular weight caldesmon (l-CaD), in CSF samples of patients with primary brain tumors by using 2-dimensional polyacrylamide gel electrophoresis (2D PAGE), followed by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. N-myc and l-CaD were related to tumor cell nuclei and blood vessels, respectively, in tissue sections of the tumor biopsies taken from the same patients from whom CSF was processed. N-myc oncoprotein and l-CaD have not been detected in CSF before. The practical value of these proteins as possible tumor markers, prognosticators, or their utility in monitoring response to chemotherapy is currently a subject of investigation. It is concluded that the combination of 2D PAGE and MALDI-TOF-MS is successful as an unbiased global screening tool for CSF.
Subject(s)
Brain Neoplasms/cerebrospinal fluid , Calmodulin-Binding Proteins/cerebrospinal fluid , Neoplasm Proteins/isolation & purification , Proto-Oncogene Proteins c-myc/cerebrospinal fluid , Adolescent , Adult , Child , Child, Preschool , Databases, Protein , Electrophoresis, Gel, Two-Dimensional/methods , Female , Glioma/classification , Glioma/metabolism , Humans , Immunohistochemistry , Male , Middle Aged , Proteomics/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/instrumentation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Staining and LabelingABSTRACT
Synaptic pathology is central in the pathogenesis of several psychiatric disorders, for example in Alzheimer's disease (AD) and schizophrenia. Quantification of specific synaptic proteins has proved to be a useful method to estimate synapitc density in the brain. Using this approach, several synaptic proteins have been demonstrated to be altered in both AD and schizophrenia. Until recently, the analysis of synaptic pathology has been limited to postmortem tissue. In living subjects, these synaptic proteins may be studied through analysis of cerebrospinal fluid (CSF). In an earlier study performed by us, one synaptic vesicle specific protein, synaptotagmin, was detected in CSF for the first time using a procedure based on affinity chromatography, reversed-phase chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and chemiluminescence immunoblotting. However, other synaptic proteins were not detectable with this procedure. Therefore, we have developed a procedure including precipitation of CSF proteins with trichloroacetic acid, followed by liquid-phase isoelectric focusing using the Rotofor Cell, and finally analysis of Rotofor fractions by Western blotting for identification of synaptic proteins in CSF. Five synaptic proteins, rab3a, synaptotagmin, growth-associated protein (GAP-43), synaptosomal-associated protein (SNAP-25) and neurogranin, have been demonstrated in CSF using this method. The major advantage of liquid-phase isoelectric focusing (IEF) using the Rotofor cell is that it provides synaptic proteins from CSF in sufficient quantities for identification. This method may also be suitable for identification of other types of trace amounts of brain-specific proteins in CSF. These results demonstrate that several synaptic proteins can be identified and measured in CSF to study synaptic function and pathology in degenerative disorders.
