A pre-validation precision study was performed to evaluate the performance of the Hoofnagle Lab ApoE LC-MS/MS assay. To evaluate intra- and inter-day imprecision, two separate cerebral spinal fluid (CSF) pools were created from leftover clinical lumbar tap CSF samples: a ‘Normal’ pool and a ‘High’ pool which was spiked with 10ug/mL each of recombinant ApoE2, ApoE3, and ApoE4 (PeproTech). A 5x5 study was performed by analyzing five replicates of each sample pool prepared on five different days. Additionally, a 3x5 study was performed using five individual CSF samples (Patients 1-5) were evaluated by analyzing three replicates on five different days.
Results: Inspection of the individual Patient chromatograms for the proteotypic peptides monitored enabled determination of their ApoE genotype as indicated in the tables below. No ApoE2 was evident in the Normal Pool, which can be expected given ApoE allele frequency. Table 1 shows intra-day precision for normal and high pools was less than target
Table 1. Intra-day precision (%CV).
Table 2. Inter-day precision (Total Sum of Squares).
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.
