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Mol Cell Proteomics. 2019 Dec;18(12):2388-2400. doi: 10.1074/mcp.RA119.001702. Epub 2019 Sep 26.

Domain-specific Quantification of Prion Protein in Cerebrospinal Fluid by Targeted Mass Spectrometry.

Author information

Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142
Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115.
Prion Alliance, Cambridge, MA 02139.
Proteomics Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142.
Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142.
Departments of Pathology and Neurology Case Western Reserve University, Cleveland, OH 44106.
Laboratory of Persistent Viral Diseases, NIAID Rocky Mountain Labs, Hamilton, MT 59840.
Environmental Health and Safety, Broad Institute of MIT and Harvard, Cambridge, MA 02142.
National Reference Center for TSE, Georg-August University, Göttingen, 37073, Germany.
Biomedical Research Networking Center on Neurodegenerative Diseases (CIBERNED), L'Hospitalet de Llobregat, 08908, Barcelona, Spain.
IRCCS Istituto delle Scienze Neurologiche di Bologna, Bologna, 40139, Italy.
Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, 40123, Italy.
Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, 40138, Italy.
Department of Chemistry & Chemical Biology, Harvard University, Cambridge, MA 02138.
Proteomics Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142


Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a multi-species targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified relative to an isotopically labeled recombinant protein standard for human samples, or isotopically labeled synthetic peptides for nonhuman species. Analytical validation experiments showed process replicate coefficients of variation below 15%, good dilution linearity and recovery, and suitable performance for both CSF and brain homogenate and across humans as well as preclinical species of interest. In n = 55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared with individuals with nonprion diagnoses. Thus, lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not an artifact of ELISA-based measurement. As a result, dose-finding studies for PrP lowering drugs may need to be conducted in presymptomatic at-risk individuals rather than in symptomatic patients. We provide a targeted mass spectrometry-based method suitable for preclinical quantification of CSF PrP as a tool for drug development.


Creutzfeldt-Jakob disease; Targeted mass spectrometry; biomarker: diagnostic; cerebrospinal fluid; multiple reaction monitoring; neurodegenerative diseases; prion

[Available on 2020-12-01]

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