Neurodegenerative diseases are characterized by an accumulation of misfolded proteins. Cells normally recognize misfolded proteins and attempt to refold or to pass them on for proteasomal degradation. Chaperone proteins are central to recognizing and in refolding client proteins. When chaperone proteins fail to fulfill this function, conformationally altered proteins accumulate and can cause cell death.

In a collaboration with Robert Rissman of UC San Diego, the Yates group at the Scripps Research Institute in La Jolla developed a method to quantitatively measure protein misfolding across a proteome. Covalent Protein Painting (CPP) is a structural proteomics approach to quantify the accessibility of lysine e-amines for chemical modification at the surface of natively folded proteins and thereby quantify changes in protein fold or altered protein-protein interaction for any protein in a proteome.

 

In short, the CPP is as follows, solvent-exposed primary amines are chemically methylated, reaction is quenched, labeling reagents are removed, and proteins are denatured and proteolytically digested. The remaining, previously unaccessible lysine residues are methylated in a second labeling step with a set of isotopically different dimethyl moieties. Using different isotopes for labeling allows to determine the fraction of protein molecules that was accessible for chemical modification at a specific lysine site based on the relative intensities of the isotope labeled peptides that include that site.

CPP was applied to survey lysine residues in HEK293T cells in vivo and it was found that mild heat shock increased rather than decreased lysine accessibility for chemical modification. CPP was able to differentiate patients with Alzheimer disease or Lewy body disease based on the relative accessibility of specific lysine residues in post-mortem brain samples.

Covalent Protein Painting Reveals Structural Changes in the Proteome in Alzheimer Disease was published by John R. Yates III and colleauges at the Department of Molecular Medicine, The Scripps Research Institute

Read the full paper in biorxiv here


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