Deep Proteome\nAnalysis of Cerebrospinal Fluid from\nPediatric Patients with Central Nervous System Cancer

The cerebrospinal\nfluid (CSF) is a key matrix for discovery of\nbiomarkers relevant for prognosis and the development of therapeutic\ntargets in pediatric central nervous system malignancies. However,\nthe wide range of protein concentrations and age-related differences\nin children makes such discoveries challenging. In addition, pediatric\nCSF samples are often sparse and first prioritized for clinical purposes.\nThe present work focused on optimizing each step of the proteome analysis\nworkflow to extract the most detailed proteome information possible\nfrom the limited CSF resources available for research purposes. The\nstrategy included applying sequential ultracentrifugation to enrich\nfor extracellular vesicles (EV) in addition to analysis of a small\nvolume of raw CSF, which allowed quantification of 1351 proteins (+55%\nrelative to raw CSF) from 400 μL CSF. When including a spectral\nlibrary, a total of 2103 proteins (+240%) could be quantified. The\nworkflow was optimized for CSF input volume, tryptic digestion method,\ngradient length, mass spectrometry data acquisition method and database\nsearch strategy to quantify as many proteins a possible. The fully\noptimized workflow included protein aggregation capture (PAC) digestion,\npaired with data-independent acquisition (DIA, 21 min gradient) and\nallowed 2989 unique proteins to be quantified from only 400 μL\nCSF, which is a 340% increase in proteins compared to analysis of\na tryptic digest of raw CSF.