Shotgun proteomics for the discovery of protein biomarkers for cerebral amyloid angiopathy

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Abstract Background Oftentimes, the search for novel (protein) biomarkers starts in transgenic animal models, to minimize inter‐individual variation and confounding influences. In our search for novel biomarkers for cerebral amyloid angiopathy (CAA), we have executed shotgun proteomics analyses aimed at identifying novel biomarker candidates on the cerebrospinal fluid (CSF) of rodent models for CAA type I, and compared relative expression of proteins with wild‐type controls. Additionally, we have attempted to validate these candidates by comparing selected biomarker levels in CSF of human sporadic CAA patients (sCAA) compared to Alzheimer’s patients and control subjects. Method A Bruker TIMS‐ToF‐MS/MS instrument was used to analyze CSF samples of rTg‐DI and wild‐type rodent samples, following trypsin digestion. Protein identification was inferred from the Uniprot unreviewed rodent database using Bruker Daltonics Data Analysis. Identified biomarker candidates were additionally researched in human CAA and Alzheimer’s Dementia (AD) patients, and controls in CSF in independent validation attempts using targeted analyses including ELISA, or enzyme activity assays. Result Differential expression levels in rodents were discovered for cathepsins B (CatB; p < 0.001 and p = 0.05) and S (CatS; p < 0.001 and p = 0.06), and hexosaminidase B (HexB; p = 0.003 and p = 0.17) for uncorrected and corrected results respectively. Human CatB and CatS CSF levels measured by ELISA did not differ significantly between CAA, AD patients or controls (p = 0.77 and p = 0.09). Analytical sensitivity of the CatB and CatS activity assays was too low to quantify activity in human CSF, whereas total hexosaminidase (as a measure of HexB activity) and HexA activity analyses in human CSF did show (a trend) in significant decreases in CAA patients compared to controls (p = 0.07 and p = 0.05 respectively). Conclusion Biomarker candidates identified from untargeted protein screenings do not directly correspond to validation efforts in human biomarker studies. Increased HexB expression levels in rodents contrastingly appear to be reflected in decreased hexosaminidase activity in CSF in human CAA, but would need to be further validated in studies with greater power. This study was funded in the NIH CAFÉ project (5R01NS104147‐02).

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