Mass-spectrometry based assay for targeting fifty-two proteins of brain origin in cerebrospinal fluid.

Cerebrospinal fluid (CSF) is a circulatory fluid of the central nervous system (CNS) and it can reflect the biochemical changes occurring in the brain. Although CSF retrieval through lumbar puncture is invasive, it remains the most commonly used fluid in exploring brain pathology as it is less complex and contains a higher concentration of brain derived proteins than plasma (1, 2). We hypothesize that proteins produced by the brain will have diagnostic significance for brain pathologies. Hence, we expanded the previously in-house developed 31 protein panel with more proteins classified as brain-specific by the Human Protein Atlas (HPA). Using the HPA, we selected 76 protein coding genes and screened CSF using liquid-chromatography mass spectrometry (LC-MS) and narrowed the protein list to candidates identified endogenously in CSF (n=20). Next, we developed a parallel-reaction monitoring (PRM) assay for the 21 new proteins and merged it with the 31 protein assay developed earlier. In the process, we evaluated different screening strategies and optimized MS collision energies and ion isolation windows to achieve the highest possible analyte signal. Our approach allows us to measure relative levels of 52 brain derived proteins in CSF with a single LC-MS method. This new assay may have important applications in discovering CSF biomarkers for various neurological diseases.