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

Eric Vallabh Minikel,Eric Kuhn,Alexandra R Cocco,Sonia M Vallabh,Christina R Hartigan,Andrew G Reidenbach,Jiri G Safar,Gregory J Raymond,Michael D Mccarthy,Rhonda O'Keefe,Franc Llorens,Inga Zerr,Sabina Capellari,Piero Parchi,Stuart L Schreiber,Steven A Carr

doi:10.1074/mcp.ra119.001702

Abstract

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.

Highlights

Prion disease is a fatal and incurable neurodegenerative disease caused by misfolding of the prion protein (PrP), and may be sporadic, genetic, or acquired[1]
In addition to properly identifying the endogenous light peptides by multiple reaction monitoring (MRM), these heavy peptides control for variability in retention on the LC and the ionization on the MS, caused by the presence of a large number of peptides in the mixture, with over 4,000 peptides identified in cerebrospinal fluid (CSF) pilot study
We describe a targeted mass spectrometry assay for measuring CSF PrP

Summary

Introduction

Prion disease is a fatal and incurable neurodegenerative disease caused by misfolding of the prion protein (PrP), and may be sporadic, genetic, or acquired[1]. Therapies currently in preclinical development for prion disease seek to lower PrP levels in the brain, a genetically well-validated strategy[2]. Clinical trials of PrP-lowering agents will rely on quantification of PrP in cerebrospinal fluid (CSF) as, at a minimum, a pharmacodynamic biomarker[3]. This marker may, have even greater importance. Predictive testing of pre-symptomatic individuals harboring highly penetrant genetic mutations[4] that cause prion disease provides an opportunity for early therapeutic intervention to preserve healthy life, but randomization to a clinical endpoint in this population appears infeasible[5]. Precise quantification of PrP in CSF will be essential to the development of prion disease therapeutics

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