Prion MRM assay development and clinical study
Minikel EV, Kuhn E, Cocco AR, Vallabh SM, Hartigan CR, Reidenbach AG, Safar JG, Raymond GJ, McCarthy MD, O’Keefe R, Llorens F, Zerr I, Capellari S, Parchi P, Schreiber SL, Carr SA. -Domain-specific quantification of prion protein in cerebrospinal fluid by targeted mass spectrometry. Mol Cell Proteomics. 2019 Sep;
- Organism: Homo sapiens, Mus musculus, Rattus norvegicus, Macaca cynomolgus
- Instrument: TSQ Quantiva,Q Exactive
targeted mass spectrometry; multiple reaction monitoring mass spectrometry; prion; cerebrospinal fluid; biomarker
Lab head: Steve Carr
Submitter: Eric Kuhn
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 undetectable by ELISA, 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 by stable isotope dilution analysis. Analytical validation experiments showed intra-day and inter-day assay reproducibility coefficients of variation less than 15% (below the best practices threshold), 3 orders of dynamic linear range that encompass the entire expected range of PrP concentration in CSF, lower limits of detection near 10 ng/mL and a similar recovery response from both CSF and brain homogenate matrices. Critical for preclinical assay development, comparable assay performance was found for peptides unique to 4 species. 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 to individuals with non-prion diagnoses. Thus, lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not merely an artifact of ELISA-based measurement. As a result, dose-finding studies for PrP lowering drugs may need to be conducted in pre-symptomatic at-risk individuals rather than in symptomatic patients. We provide a targeted mass spectrometry-based method suitable for preclinical and clinical quantification of CSF PrP as a tool for drug development.
15N protein standard (32.5 fmol) was added to samples of CSF. Urea, TCEP and CHAPS were added to final concentration 6M, 20 mM and 0.03%, respectively. Samples were mixed at 800 RPM 37°C for 30 min. Samples were cooled to room temperature and incubated with 39 mM iodoacetamide 30 min in the dark. Samples were diluted to 900 mM Urea with 0.2 M Trizma pH 8.1 and trypsin (Promega V5113) was added to 1:50 (E:S) and incubated at 37°C overnight. Digestion was quenched with 5% formic acid. Digested samples were desalted using StageTips, 2 punches of Empore C18 held with an adaptor in a microcentrifuge tube and processed as follows: 50 uL 90% acetonitrile/0.1% TFA, 50 uL 0.1% TFA, addition of digestion in aliquots of 150 uL centrifuging after each load, wash twice with 50 uL 0.1% TFA, elute twice with 50 uL 40% acetonitrile/0.1% TFA and store frozen until MS analysis. Frozen samples were dried under vacuum centrifugation, resuspended in 12 µL 3% acetonitrile/5% acetic acid, vortexed for 5 min at RT, centrifuged 12,000 x g for 5 min and 10 µL were transferred into an HPLC vial (Waters 186000273). HPLC vials were centrifuged briefly (30 - 60s) at 1,200 x g to remove air bubbles and transferred into the nanoLC autosampler compartment set to 7°C. Samples were analyzed on a TSQ Quantiva triple quadrupole mass spectrometer installed with a Nanospray Flex source and Easy-nLC 1000 system (Thermo). Ion source was set to positive ion mode with capillary temperature of 300°C, spray voltage of 2,000 and sweep gas set to 0. The Easy-nLC 1000 system was primed with mobile phase A (3% acetonitrile / 0.1% formic acid), mobile phase B (90% acetonitrile / 0.1% formic acid). Samples were injected (2 µL, 20% of digested sample) onto a 0.075 mm ID PicoFrit (New Objective) column pulled to a 10 µm emitter and custom-packed to 20 cm with 1.9 µm 200Å C18-AQ Reprosil beads (Dr. Maisch). The LC gradient was 0% B to 30% B for 55 min, 30% B to 60% B in 5 min, 60% B to 90 % B in 1 min using a flow rate of 200 nL/min. Collision energies were optimized over 4 steps, 2.5 V per step in batches of less than 500 transitions per batch, 3 to 4 transitions were monitored per peptide in the final MRM method using a 1.5s cycle time.
Rat and cynomolgus monkey CSF were purchased from BioIVT. Human brain tissue was from a non-prion disease control individual provided by the National Prion Disease Pathology Surveillance Center (Cleveland, OH). Mouse brain tissue from Edinburgh PrP knockout mice backcrossed to a C57BL/10 background, and matching tissue from wild-type C57BL/10 mice were provided by Gregory J. Raymond (NIAID Rocky Mountain Labs, Hamilton, MT) Untagged recombinant HuPrP23-230 (MW=22,878) and MoPrP23-231 (MW=23,151), corresponding to full-length post-translationally modified human and mouse PrP without the signal peptide or GPI signal but retaining an N-terminal methionine, were purified by denaturation and Ni-NTA affinity from E. coli inclusion bodies as previously described (31, 32), using a vector generously provided by Byron Caughey (NIAID Rocky Mountain Labs, Hamilton, MT). 15N incorporation was achieved by growing the E. coli in 15N cell growth medium (Cambridge Isotope Laboratories CGM-1000-N) induced with 15N auto-induction medium (Millipore 71759-3).
Created on 7/29/19, 2:20 PM