Cerebrospinal Fluid Sampling and Analysis

Cerebrospinal fluid sample collection and its analysis was performed as described previously in (9 (link)). Briefly, CSF was centrifuged immediately after collection (1600 g, 4 °C, 15 minutes), aliquoted into polypropylene test tubes (each aliquot, 750 µL), frozen within 30–40 minutes after the puncture and stored at −80 C until use. After thawing, 700 µL of CSF were diluted with 700 µL buffer (pH 10.5; 2 M urea, 100 mmol/L NaCl, 0.0125% NH₃) and ultracentrifuged using Centrisart ultrafiltration devices (cut off 20 kDa) at 4 °C until 1.1 mL of filtrate was obtained. Subsequently the filtrate was applied onto a PD-10 desalting column (Amersham Biosciences, Freiburg, Germany) equilibrated in 0.01% NH₄OH in HPLC-grade water (Roth, Karlsruhe, Germany), and the eluate was lyophilized, stored at 4 °C and resuspended in 10 µL HPLC-grade water before capillary electrophoresis–mass spectrometry (CE-MS) analysis. CE-MS analysis was performed as described using a P/ACE MDQ (Beckman Coulter, Krefeld, Germany) system on-line coupled to a Micro-TOF MS (Bruker Daltonic, Bremen, Germany) (9 (link), 22–24 (link)).