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Cerebrospinal fluid is home to countless biological compounds, some of which can be monitored for the diagnosis and treatment of diseases. Recent large-scale studies have identified more than 500 proteins on two-dimensional gels from CSF, including phosphoproteins. A more specific study has now identified the minor set of proteins in CSF that are phosphorylated at tyrosine residues, using a variety of isolation methods. The central nervous system (CNS) is enclosed in a liquid bath called cerebrospinal fluid (CSF), a serum-like fluid that flows through the brain and spinal cord. It cushions them against impact damage and, by providing buoyancy, reduces the net weight of the brain from about 1400 gm to about 50 gm, to lessen the pressure at the base of the brain. Apart from these physical functions, CSF also has important biological roles. It is absorbed via a one-way system into the blood stream through structures called the arachnoid villi, removing potentially toxic metabolites and unwanted chemicals. When the CSF pressure exceeds the blood pressure, it is released into the blood. The CSF also transports hormones around the brain. The total volume of CSF in a typical adult human is about 125-150 ml (equivalent to 0.21-0.26 UK pints or 0.25-0.32 US pints) but, rather surprisingly, the daily production rate is about 500-700 ml, so it is replaced several times each day. CSF contains a huge variety of compounds, including peptides, neuropeptides, hormones, proteins, sugars and enzymes. Any anomalous compounds present, or altered levels of regular compounds, may be indicative of a particular disease and CSF has been used for over one hundred years for clinical diagnosis. New biomarkers are found regularly. Unfortunately, the method of collection is by lumbar puncture, also known as spinal tap, in which a needle is inserted in the back between the vertebrae. Extreme care must be taken to avoid damaging the spinal cord. Within the bank of proteins in CSF, many are phosphorylated but only about 1% of all phosphoproteins are modified on tyrosine residues. Many of these p-Tyr proteins have significant biological properties, some being involved in signalling pathways. Although large-scale proteomics studies have identified more than 500 proteins in CSF, the presence of p-Tyr proteins is more difficult to detect compared with other phosphoproteins because they are less abundant. Dominic Desiderio has been studying neuropeptides for many years and has turned his attention to the CSF p-Tyr proteins. Working with Xianglin Yuan, both from the Charles B. Stout Neuroscience Mass Spectrometry Lab. at the Department of Neurology and the Department of Molecular Sciences of the University of Tennessee Health Science Center, they have compared three different methods of isolating these minority proteins from human lumbar CSF. The techniques were described in J. Proteome Res. 2003, 2, 476. In the first method, immunoprecipitation using anti-p-Tyr protein antibodies was attempted in one-step and two-step variations, followed by two-dimensional gel electrophoresis (2-DE) but no p-Tyr proteins were detected, probably due to their low concentrations and to non-specific binding of other proteins blocking the binding sites. The two remaining methods involved either 1-DE or 2-DE, followed by Western blotting to detect the separated p-Tyr proteins. Using the former, it was found that different individuals had different p-Tyr protein patterns, suggesting that CSF phosphoproteins might be good for diagnosing CNS disorders. However, for their identification, 2-DE was employed to ensure better separation of the proteins, followed by Western blotting, in-gel digestion with trypsin and mass spectrometric analysis. From a total of 16 spots identified on the 2-D Western blot, only four corresponded to p-Tyr proteins: kallikrein-6 precursor, complement C4 gamma-chain, gelsolin and ceruloplasmin. So even though smaller amounts of sample are used compared with immunoprecipitation, p-Tyr protein detection was possible. All four are signal proteins and may also be involved in signal transduction, that is, the transfer of signals from outside the cell to inside. In an interesting spin-off from the main thrust of the paper, four of the remaining proteins were found to be new. They are absent from all 2-D maps and were captured in this work by chance, via non-specific binding during Western blotting. The next stage is to identify p-Tyr proteins that are associated with a particular CNS disorder, so that they can be evaluated as biomarkers. CSF is drawn from several parts of the CNS, so the appearance of one particular p-Tyr protein might point to a particular problem in one specific part of the CNS. Related links:
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