Generated through filtration of blood in the choroid plexus and by diffusion from the extracellular matrix of the brain into the ventricles, the CSF surrounds the brain and the spinal cord. Being in direct contact with the brain, many brain proteins diffuse into the CSF; approximately 20% of the proteins in CSF are estimated to be brain-derived. CSF is produced at a rate of 500 ml/day and turns over approximately 4 times per day by drainage into the blood. Thus, many ongoing processes in the CNS are reflected in the molecular composition of the CSF. Several CSF biomarkers have already been identified for various diseases and are used in research, clinical trials and clinical practice, including CSF-specific IgG immunoglobulins in multiple sclerosis, the 14-3-3 protein in Creutzfeld-Jakob’s disease, the tau protein, and the b-amyloid peptides in Alzheimer’s disease. While several proteomic studies have expanded our knowledge of the CSF protein composition, there are comparatively few reports on the CSF peptidome. A few recent studies have identified a large number of endogenous peptides in the CSF. Several truncated forms of these peptides and the proteins they derive from are involved in diverse biological processes, e.g., degeneration/regeneration, neuronal damage, growth, development, and learning. Others may reflect the proteolytic activity in the CNS that leads to their formation through metabolic processing. Studies have shown that peptide patterns can discriminate between different forms of cancer in serum and discriminate AD patients from controls in both serum and CSF. Compared to CSF proteomics, the pursuit of the CSF AB1010 peptidome is also motivated for analytical reasons. The CSF protein composition spans a concentration range of over ten orders of magnitude and is dominated by a small number of highly abundant proteins, most notably albumin, which accounts for over 60% of the total protein content. Compared to albumin, for example, the concentration of the tau protein, a marker of neuronal degradation, is a million-fold lower. Global proteomic workflows generally have a strong bias towards detecting proteins of high abundance. The reasons for this are both the dynamic range of the mass spectrometric instrumentation, as well as the limited loading capacity of the separation techniques used upstream of the mass spectrometer. The complexity of such workflows results in long analysis times and often compromises the analytical reproducibility, thereby hampering their use in clinical proteomic studies, in which comparative analysis of large sample sets are required. The peptides in CSF, in contrast, can be isolated relatively easily. Yuan et al demonstrated that ultrafiltration is an effective method for isolating the low molecular weight fraction of the human lumbar CSF proteome. Using this strategy, Zougman et al analyzed the CSF peptidome and proteome in depth, and found 563 endogenous peptides originating from 91 proteins. Here we employed nano-LC coupled to off-line to matrixassisted laser desorption/ionization MS for analyzing CSF peptides in the mass range 700–5,000 Da. A method based on ultrafiltration for preparation of peptide extracts from CSF was optimized. This workflow is aimed to be suitable for comparative analysis of large clinical sample sets, necessitating a fast and simple sample preparation. Nine fragments of the prion protein were identified. The detected peptides are fragments created after the signal peptide has been removed from the protein with some starting at amino acid position 23 and others starting more Cterminally in the octapeptide repeat region which is associated to prion disease.