Shotgun proteomics (human)

Total protein content of CSF samples was measured by Bradford assay (BioRad, Feldkirchen, Germany)and 10μg per sample were proteolysed by the commercially available in-StageTip-NHS kit (PreOmics GmbH, Martinsried, Germany) according to the manufacturer's protocol. Briefly, CSF was reduced and alkylated and incubated for 3 hrs at 37°C with Lys-C and trypsin. Resulting peptides were dried for short term storage at -80°C. Prior to measurement, peptides were resuspended in 2% acetonitrile and 0.5% trifluoroacetic acid. Mass spectrometry Peptides were analyzed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) online coupled to a UItimate 3000 RSLC nano-HPLC (Dionex). Samples were automatically injected and loaded onto the C18 trap cartridge and after 5 min eluted and separated on the C18 analytical column (Acquity UPLC M-Class HSS T3 Column, 1.8 μm, 75 μm x 250 mm; Waters) by a 90 min non-linear acetonitrile gradient at a flow rate of 250 nl/min. MS spectra were recorded at a resolution of 60000 with an AGC target of 3 x 1e6 and a maximum injection time of 30 ms from 300 to 1500 m/z. From the MS scan, the 10 most abundant peptide ions were selected for fragmentation via HCD with a normalized collision energy of 27, an isolation window of 1.6 m/z, and a dynamic exclusion of 30 s. MS/MS spectra were recorded at a resolution of 15000 with a AGC target of 1e5 and a maximum injection time of 50 ms. Unassigned charges, and charges of +1 and >8 were excluded from precursor selection.