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Identification and removal of proteins that co‐purify with infectious prion protein improves the analysis of its secondary structure
Author(s) -
Moore Roger A.,
Timmes Andrew G.,
Wilmarth Phillip A.,
Safronetz David,
Priola Suzette A.
Publication year - 2011
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201100253
Subject(s) - protein secondary structure , fourier transform infrared spectroscopy , chemistry , absorbance , gene isoform , circular dichroism , hamster , protein structure , ferritin , biophysics , biochemistry , biology , microbiology and biotechnology , chromatography , physics , quantum mechanics , gene
Prion diseases are neurodegenerative disorders associated with the accumulation of an abnormal isoform of the mammalian prion protein (PrP). Fourier transform infrared spectroscopy (FTIR) has previously been used to show that the conformation of aggregated, infectious PrP (PrP Sc ) varies between prion strains and these unique conformations may determine strain‐specific disease phenotypes. However, the relative amounts of α‐helix, β‐sheet and other secondary structures have not always been consistent between studies, suggesting that other proteins might be confounding the analysis of PrP Sc secondary structure. We have used FTIR and LC‐MS/MS to analyze enriched PrP Sc from mouse and hamster prion strains both before and after the removal of protein contaminants that commonly co‐purify with PrP Sc . Our data show that non‐PrP proteins do contribute to absorbances that have been associated with α‐helical, loop, turn and β‐sheet structures attributed to PrP Sc . The major contaminant, the α‐helical protein ferritin, absorbs strongly at 1652 cm −1 in the FTIR spectrum associated with PrP Sc . However, even the removal of more than 99% of the ferritin from PrP Sc did not completely abolish absorbance at 1652 cm −1 . Our results show that contaminating proteins alter the FTIR spectrum attributed to PrP Sc and suggest that the α‐helical, loop/turn and β‐sheet secondary structure that remains following their removal are derived from PrP Sc itself.