Parallel Reaction Monitoring (PRM) Validation of Dysregulated Proteins

Considering that most commercially available ELISA kits are developed for blood and urine samples rather than CSF samples, because of the low protein concentrations in CSF, we used an LC-MS-based PRM technique to validate the untargeted proteomic results. The high throughput in PRM analysis makes it more efficient than ELISA to use in a validation study. PRM method construction, optimization, and data processing were performed using Skyline software. For method construction and optimization, untargeted proteomic results were used as a reference for the peptide and transition selections. Targeted proteins were filtered based on the following criteria: (1) at least two unique peptides were detected; (2) at least five product ions were detected for each peptide; and (3) no post-translational modifications were observed in the peptide. After method construction and optimization, 20 proteins were selected for a final validation assay using the discovery sample cohort (used for untargeted proteomic analysis, and made up of 10 POD samples and 30 No-POD samples) and an additional 5 POD samples.

PRM analysis was performed on a nano-LC-MS system in the same way as the untargeted proteomic analysis was performed. The chromatographic separation was performed on a homemade reversed-phase C18 column (3 μm particles, 75 μm I.D. × 250 mm) at a flow rate of 300 nL/min with a 75-min gradient of 5–35% acetonitrile in 0.1% formic acid. The electrospray voltage was maintained at 2.2 kV, and the capillary temperature was set at 320°C. The Q-Exactive HF Mass Spectrometer was operated in PRM mode. All samples were analyzed in random order. After MS data acquisition, raw data were imported into Skyline and MS2-based quantification (peak areas of extracted ion chromatography of 3–5 fragment ions were calculated and used for quantification) was performed.