CSF samples were collected by lumbar puncture from patients who received spinal anesthesia before non-neurological operations at Beijing Tiantan Hospital.

The pooled CSF sample, which contained approximately 1 mg of CSF protein, was depleted of 14 high-abundance proteins using a 4.6 × 50 mm Human 14 affinity LC column (Agilent, St. Louis, MO, USA) with a Waters HPLC system (Milford, MA, USA). The separations were performed according to the manufacturer"s instructions regarding column usage and loading capacity. The flow-through protein sample and bound protein sample were collected separately. The two samples and the non-depleted CSF sample (containing the original proteins) were subjected to the sample handling and analysis procedures described below. A filter-aided sample preparation method [21] was used to digest the proteins in the three samples (each contained 100 μg of CSF protein). Briefly, the proteins were reduced with 20 mM DTT at 95 °C for 3–5 min and washed once with 8 M urea on a 10 kDa filter at 14,000 ×g for 40 min. The samples were then alkylated with 55 mM iodoacetamide for 30 min in darkness and washed twice with 8 M urea. Next, the proteins were washed with 50 mM ammonium bicarbonate once and digested with trypsin (1 μg/50 μg protein) overnight at 37 °C. After digestion, the three peptide mixtures derived from the flow-through, the bound sample and the non-depleted CSF sample were desalted on a Waters Oasis C18 solid-phase extraction column and lyophilized for HPLC separation.

The three lyophilized peptide mixtures were fractionated with a high-pH RPLC column from Waters (4.6 mm × 250 mm, C18, 3 μm). Each peptide mixture was loaded onto the column in buffer A2 (H2O, pH = 10). The elution gradient was 5%–30% buffer B2 (90% ACN, pH = 10; flow rate, 1 mL/min) for 60 min. The eluted peptides were collected as one fraction per minute, and the 60 fractions collected were re-suspended in 0.1% formic acid and pooled into 30 fractions. A total of 90 fractions produced from the three peptide mixtures were analyzed by LC–MS/MS.

Each fractionwas analyzed with a reverse-phase C18 self-packed capillary LC column (75 μm × 100 mm, 3 μm). The elution gradient was 5%–30% buffer B1 (0.1% formic acid, 99.9% ACN; flow rate, 0.3 μL/min) for 40 min. A TripleTOF 5600 mass spectrometer was used to analyze the fractions. The MS data were acquired using the high-sensitivity mode with the following parameters: 30 data-dependent MS/MS scans per full scan, full scans acquired at a resolution of 40,000 and MS/MS scans at a resolution of 20,000, rolling collision energy, charge state screening (including precursors with charge states of +2 to +4), dynamic exclusion (exclusionduration15 s),MS/MSscan range of 100–1800 m/z, and a scan time of 100 ms.

The MS/MS spectra were searched against the Swiss-Prot human database from the UniProt website (www.uniprot.org) using Mascot software, version 2.3.02 (Matrix Science, UK).