Comparative profiling of highly enriched 22L and Chandler mouse scrapie prion protein preparations

Abstract

Transmissible spongiform encephalopathies (TSEs) or prion diseases are characterized by the accumulation of an aggregated isoform of the prion protein (PrP). This pathological isoform, termed PrP(Sc), appears to be the primary component of the TSE infectious agent or prion. However, it is not clear to what extent other protein cofactors may be involved in TSE pathogenesis or whether there are PrP(Sc)-associated proteins which help to determine TSE strain-specific disease phenotypes. We enriched PrP(Sc) from the brains of mice infected with either 22L or Chandler TSE strains and examined the protein content of these samples using nanospray LC-MS/MS. These samples were compared with "mock" PrP(Sc) preparations from uninfected brains. PrP was the major component of the infected samples and ferritin was the most abundant impurity. Mock enrichments contained no detectable PrP but did contain a significant amount of ferritin. Of the total proteins identified, 32% were found in both mock and infected samples. The similarities between PrP(Sc) samples from 22L and Chandler TSE strains suggest that the non-PrP(Sc) protein components found in standard enrichment protocols are not strain specific.

Figure 1

Comparison of mock, 22L or Chandler-derived PrP^Sc samples. A) Approximately 3.5 μg of total protein from mock, 22L and Chandler samples were loaded for SDS-PAGE and silver stained. B) For immunoblot, 10-fold less protein was loaded and probed with monoclonal antibody 6D11 as described in the methods. C) Venn diagram of protein distribution in mock and PrP^Sc samples (left panel). A total of 21 (68%) of the 31 total proteins could be identified in the absence of PrP^Sc. The 10 proteins that were uniquely associated with PrP^Sc and not identified in the mock samples are listed in the right panel in order of relative apparent abundance as estimated by the number of assigned spectra for each protein.

Figure 2

Proteasomal subunit 5 is present at higher levels in mock versus 22L-derived PrP^Sc samples. Proteins were subjected to western blot with anti-proteasome antibody MCP196 as described in the experimental section. Human 20 S proteasomes were loaded as a positive control.

Figure 3

Variations in protein identifications from PrP^Sc preparations do not display strain specificity. A) Samples of 22L that were prepared in 2009 share more protein identifications with each other than they do with 22L that was prepared in 2006. B) Sample 22L-09B shares more protein identifications with CH-04 than it does with 22L-06. C) CH-04 shares more protein identifications with 22L-09B than it does with CH-06 and D) The Coomassie blue-stained SDS-PAGE fingerprint of each sample appears to be more similar based upon when it was prepared rather than what strain PrP^Sc was derived from. Samples 22L-09A and B contain discernable lower molecular weight bands for myelin-associated oligodendrocyte basic protein (MOBP) and calcium/calmodulin-dependent protein kinase (CAMK-IIα), while 22L-06 and CH-06 display bands at ~50 kDa that are specific for versican core protein and an apparent PrP^Sc dimer.

Figure 4

Ferritin is the most abundant non-PrP^Sc protein found in all of the samples. A) The amount of PrP^Sc relative to ferritin and other proteins was estimated by spectral counting and was found to vary between samples. Lower relative abundances of PrP^Sc were associated with higher abundances of ferritin. In the case of CH-04, the amounts of ferritin, versican, and proteoglycan link proteins were much higher than in any of the other PrP^Sc samples. An immunoblot from representative mock and PrP^Sc samples were separated by SDS-PAGE on a 4–12% NuPAGE gradient gel and probed with B) anti-ferritin rabbit antibody, washed with TBST buffer and then probed with C) 6D11 anti-PrP mouse monoclonal antibody.