Proteomics of Cerebrospinal Fluid: Throughput and Robustness Using a Scalable Automated Analysis Pipeline for Biomarker Discovery

Cerebrospinal fluid (CSF) is a body fluid of high clinical relevance and an important source of potential biomarkers for brain-associated damages, such as traumatic brain injury and stroke, and for brain diseases, such as Alzheimer’s and Parkinson’s. Herein, we have implemented, evaluated, and validated a scalable automated proteomic pipeline (ASAP2) for the sample preparation and proteomic analysis of CSF, enabling increased throughput and robustness for biomarker discovery. Human CSF samples were depleted from abundant proteins and subjected to automated reduction, alkylation, protein digestion, tandem mass tag (TMT) 6-plex labeling, pooling, and sample cleanup in a 96-well-plate format before reversed-phase liquid chromatography tandem mass spectrometry (RP-LC MS/MS). We showed the impact on the CSF proteome coverage of applying the depletion of abundant proteins, which is usually performed on blood plasma or serum samples. Using ASAP2 to analyze 96 identical CSF samples, we determined the analytical figures of merit of our shotgun proteomic approach regarding proteome coverage consistency (i.e., 387 proteins), quantitative accuracy, and individual protein variability. We demonstrated that, as for human plasma samples, ASAP2 is efficient in analyzing large numbers of human CSF samples and is a valuable tool for biomarker discovery. The data has been deposited to the ProteomeXchange with identifier PXD003024.