Lumbar CSF samples (30µL) were spiked with isotopically labeled peptides (5µL, 0.06-0.09µM), 0.5 m/v % sodium deoxycholate (SDC), 100mM ammonium bicarbonate (NH4HCO3), and reduced with 2.3mM tris[2-carboxyethyl]phosphine [TCEP] (37°C, 800 RPM,30 minutes). Free cysteines were alkylated with 4.6mM of iodoacetamide (30 minutes, at room temperature, in dark) and denatured proteins were digested with 0.3µg sequencing grade trypsin (3 hours, 37°C, 800 RPM). The digested samples were acidified using 10% v/v of formic acid and centrifuged at 4000g (4°C, 15 minutes) to facilitate SDC precipitation. Next, supernatants were filtered through 1.2µm Millipore membranes and injected onto Shimadzu LC-20AD UHPLC connected to a Sciex 6500+ triple quadrupole mass spectrometer. Peptides containing the ApoE allele variants were monitored as well as a peptide common to all ApoE forms using targeted MRM analysis.
