Label-free quantitative proteomics analysis
85175
SEPARATION
To better characterize the relevant proteins, 250 µl of CSF was depleted using a Seppro IgY 14 spin column (SEP010, Sigma-Aldrich), thus allowing the removal of the 14 highly abundant proteins (Albumin, α1-Antitrypsin, IgG, IgA, IgM, Transferrin, Haptoglobin, β2-Macroglobulin, Fibrinogen, Complement C3, α 1-Acid Glycoprotein [Orosomucoid], HDL [Apolipoproteins A-I and A-II] and LDL [mainly Apolipoprotein B]), according to the manufacturer’s instructions. The recovered supernatant was analyzed to determine total protein concentration using the BCA protein assay kit from Pierce (23227, Thermo Fisher Scientific) and BSA as standard. Then, 20 µg of total proteins from each sample was in-solution digested (1,4-dithioerythritol, D8255, Sigma-Aldrich; iodoacetamide, I1149, Sigma-Aldrich; ammonium bicarbonate, O9830, Sigma-Aldrich; trypsin sequencing grade, 11418475001, Roche-Merck). Aliquots of the samples containing tryptic peptides were desalted using StageTip C18 (87784, Thermo Fisher Scientific) and analyzed by nano-scale liquid chromatographic tandem mass spectrometry (nLC–MS/MS) using a Q-Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray ion source (Proxeon Biosystems) and an nUPLC Easy nLC 1000 (Proxeon Biosystems). Peptide separations occurred on a homemade (75 µm i.d., 15-cm-long) reverse phase silica capillary column (PF360-75-10-N-5, New Objective), packed with 1.9-µm ReproSil-Pur 120 C18-AQ (r119.aq, Dr. Maisch GmbH). A gradient of eluents A (distilled water with 0.1% v/v formic acid, 85171, Thermo Fisher Scientific) and B (acetonitrile with 0.1% v/v formic acid, 85175, Thermo Fisher Scientific) was used to achieve separation (300 nl/min flow rate), from 2% B to 40% B in 88 minutes. Full scan spectra were acquired with the lock-mass option, resolution set to 70,000 and mass range from 