We have analyzed the proteome of human cerebrospinal fluid with the help of shotgun mass spectrometry. In order to identify low-abundant proteins in these fluids, we have found it necessary to remove the abundant protein components from the mixture. Immunodepletion of the abundant proteins has allowed us to identify more than 100 proteins in cerebrospinal fluids from a patient suffering from normal pressure hydrocephalus. The identified proteins belong to a variety of different classes ranging from serum proteins to intracellular mediators that are involved in signal transduction and transcription. This work establishes a platform for future studies aimed at the comparative proteome analysis of cerebrospinal fluids from different groups of patients suffering from various psychiatric and neurological disorders.
In our efforts to improve the identification of phosphopeptides by MS we have used peptide IEF on IPG strips. Phosphopeptides derived from trypsin digests of single proteins as well as complex cellular protein mixtures can be enriched by IEF and recovered in excellent yields at the acidic end of an IPG strip. IPG peptide fractionation in combination with MS/MS analysis has allowed us to identify phosphopeptides from tryptic digests of a cellular protein extract.
: The identification of disease markers in tissues and body fluids requires an extensive and thorough analysis of its protein constituents. In our efforts to identify biomarkers for affective and neurological disorders we are pursuing several different strategies. On one hand we are using animal models that represent defined phenotypes charactersistic for the respective disorder in humans. In addition, we are analyzing human specimens from carefully phenotyped patient groups. Several fractions representing different protein classes from human cerebrospinal fluid obtained by lumbar puncture are used for this purpose. Our biomarker identification efforts range from classical proteomics approaches such as two dimensional gel electrophoresis and mass spectrometry to phage display screens with cerebrospinal fluid antibodies.
The identification of disease markers in human body fluids requires an extensive and thorough analysis of its protein constituents. In the present study, we have extended our analysis of the human cerebrospinal fluid (CSF) proteome using protein prefractionation followed by shotgun mass spectrometry. After the removal of abundant protein components from the mixture with the help of immunodepletion affinity chromatography, we used either anion exchange chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to further subfractionate the proteins present in CSFs. Each protein subfraction was enzyme digested and analyzed by tandem mass spectrometry and the resulting data evaluated using the Spectrum Mill software. Different subfractionation methods resulted in the identification of a grant total of 259 proteins in CSF from a patient with normal pressure hydrocephalus. The greatest number of proteins, 240 in total, were identified after prefractionating the CSF proteins by immunodepletion and SDS-PAGE. Immuno-depletion combined with anion exchange fractionation resulted in 112 proteins and 74 proteins were found when only immunodepletion of the CSF sample was carried out. All methods used showed a significant increase in the number of identified proteins as compared with nondepleted and unfractionated CSF sample analysis, which yielded only 38 protein identifications. The present work establishes a platform for future studies aimed at a detailed comparative proteome analysis of CSFs from different groups of patients suffering from various psychiatric and neurological disorders.
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