Normal human cerebrospinal fluid proteome by 2DLC label-free approach


Knowledge about the normal human cerebrospinal fluid (CSF) proteome serves as a baseline reference for CSF biomarker discovery and provides insight into CSF physiology. In this study, high-pH reverse-phase liquid chromatography (hp-RPLC) was first integrated with a TripleTOF 5600 mass spectrometer to comprehensively profile the normal CSF proteome. A total of 49,836 unique peptides and 3256 non-redundant proteins were identified. To obtain high-confidence results, 2513 proteins with at least 2 unique peptides were further selected as bona fide CSF proteins. Nearly 30% of the identified CSF proteins have not been previously reported in the normal CSF proteome. More than 25% of the CSF proteins were components of CNS cell microenvironments, and network analyses indicated their roles in the pathogenesis of neurological diseases. The top canonical pathway in which the CSF proteins participated was axon guidance signaling. More than one third of the CSF proteins (788 proteins) were related to neurological diseases, and these proteins constitute potential CSF biomarker candidates.

In this study, high-pH reverse-phase liquid chromatography (hp-RPLC) was first integrated with a TripleTOF 5600 mass spectrometer to comprehensively profile the normal CSF proteome.

Protocol Description
Clinical materials

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

Sample preparation

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.

iTRAQ Or TMT labeling


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.

Data processing

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