Mass Spectrometry-Based Assay for Targeting Fifty-Two Proteins of Brain Origin in Cerebrospinal Fluid

Cerebrospinal fluid (CSF) is a circulatory
fluid of the central
nervous system and it can reflect the biochemical changes occurring
in the brain. Although CSF retrieval through lumbar puncture is invasive,
it remains the most commonly used fluid in exploring brain pathology
as it is less complex and contains a higher concentration of brain-derived
proteins than plasma (Reiber, H. Clin. Chim. Acta 2001, 310, 173−186; Macron et al. J. Proteome Res. 2018, 17, 4315−4319). We hypothesize that proteins produced by
the brain will have diagnostic significance for brain pathologies.
Hence, we expanded the previously in-house-developed 31-protein panel
with more proteins classified as brain-specific by the Human Protein
Atlas (HPA). Using the HPA, we selected 76 protein coding genes and
screened CSF using liquid chromatography–mass spectrometry
(LC–MS) and narrowed the protein list to candidates identified
endogenously in CSF. Next, we developed a parallel reaction monitoring
(PRM) assay for the 21 new proteins and merged it with the 31-protein
assay developed earlier. In the process, we evaluated different screening
strategies and optimized MS collision energies and ion isolation windows
to achieve the highest possible analyte signal resulting in the PRM
assay with an average linear dynamic range of 4.3 × 103. We also assessed the extent of Asn (N)–Gln (Q) deamidation,
N-terminal pyro-Glu (E) conversion, and Met (M) oxidation and found
that deamidation can be misassigned without high mass accuracy and
high-resolution settings. We also assessed how many of these proteins
could be reliably measured in 10 individual patient CSF samples. Our
approach allows us to measure the relative levels of 52 brain-derived
proteins in CSF by a single LC–MS method. This new assay may
have important applications in discovering CSF biomarkers for various
neurological diseases.