title: Transmissible spongiform encephalopathy strain-associated diversity of N-terminal proteinase K cleavage sites of PrP(Sc) from scrapie-infected and bovine spongiform encephalopathy-infected mice.
abstract: Assessment of the different conformational states of the abnormal prion protein (PrP(Sc)) in the CNS provides an established basis for distinguishing transmissible spongiform encephalopathy (TSE) strains. PrP(Sc) conformers are variably resistant to N-terminal proteinase K (PK) digestion, and analysis of the consensus products (PrP(res)) by immunoassay enables effective, but relatively low-resolution differentiation. Determination of the precise N-terminal amino acid profile (N-TAAP) of PrP(res) presents a potential high-resolution means of TSE-strain typing, and thus of differential disease diagnosis. This approach was evaluated using individual mice affected by model scrapie (22A, ME7, 87V and 79A) and bovine spongiform encephalopathy (BSE) (301V) strains. Nano liquid chromatography-mass spectrometry (LC-MS) was used to determine PrP(res) N-terminal tryptic digestion products. Four major N-terminal tryptic peptides were generated from all mouse TSE strains investigated, corresponding with predominant N-termination of PrP(res) at G(81), G(85), G(89) and G(91). Both the mass spectrometric abundance of the individual peptides and the ratios of pairs of these peptides were evaluated as markers of conformation in relation to their potential for strain discrimination. The yield of peptides was significantly greater for BSE than scrapie strains and the relative quantities of particular peptide pairs differed between strains. Thus, whereas peptide G(91)-K(105) was a dominant peptide from 301V, this was not the case for other strains and, significantly, the ratio of peptides G(91)-K(105):G(89)-K(105) was substantially higher for BSE-infected compared with scrapie-infected mice. These data support the potential of the N-TAAP approach for high-resolution TSE strain typing and differential diagnosis.
authors.affiliation: Veterinary Laboratories Agency-Weybridge (VLA), Addlestone, Surrey, UK.
authors.forename: Laurence C
authors.initials: LC
authors.lastname: Howells
authors.forename: Steve
authors.initials: S
authors.lastname: Anderson
authors.forename: Nick G
authors.initials: NG
authors.lastname: Coldham
authors.forename: Maurice J
authors.initials: MJ
authors.lastname: Sauer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D003937:Diagnosis, Differential; D016643:Encephalopathy, Bovine Spongiform / Q000175:diagnosis* / Q000821:virology; D019286:Endopeptidase K / Q000378:metabolism*; D006031:Glycosylation; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D018620:PrPSc Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012608:Scrapie / Q000175:diagnosis* / Q000821:virology; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D018620:PrPSc Proteins; D014357:Trypsin; D019286:Endopeptidase K