Absolute quantification of prion protein (90-231) using stable isotope-labeled chymotryptic peptide standards in a LC-MRM AQUA workflow.
Substantial evidence indicates that the disease-associated conformer of the prion protein (PrP(TSE)) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrP(TSE) has the ability to convert the conformation of the normal prion protein (PrP(C)) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrP(TSE) at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrP(TSE), the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.
Department of Chemistry, University of Wisconsin, Madison, WI 53705, USA.
Robert
R
Sturm
Gloria
G
Sheynkman
Clarissa
C
Booth
Lloyd M
LM
Smith
Joel A
JA
Pedersen
Lingjun
L
Li
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D002918:Chymotrypsin / Q000378:metabolism*; D006224:Cricetinae; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D016014:Linear Models; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D011328:Prions / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D012015:Reference Standards; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
D010446:Peptide Fragments; D011328:Prions; C507417:prion protein (90-231); D002918:Chymotrypsin