An increasing number of proteins are known to form amyloid fibrils in vivo, and the formation of these fibrils is implicated in several diseases. One of these proteins, human (B-2-microglobulin (B2m), can form amyloid fibrils in the presence of Cu (ll), and these fibrils are presumed to be the main pathogenic process underlying dialysis-related amyloidosis (DRA). Like other amyloid systems, p2m fibril formation proceeds by partial protein unfolding, subsequent oligomerization, and eventual elongation to form mature fibrils. While aspects of general amyloid formation are understood, molecular-level information is lacking for most amyloid systems; however, this information is critical for the rational design of therapeutics against amyloid diseases like DRA. We plan to obtain amino acid-level information of the unfolding and oligomerization process associated with B2m fibrillogenesis. To do so, we will apply, optimize, and develop 3 mass spectrometry (MS)-based tools with the necessary temporal and spatial resolution. (1). Metal-catalyzed oxidation (MCO) reactions with MS detection will provide information about the evolution of Cu (ll)-B2m interactions during unfolding and oligomerization. Improvements will also be made to the MCO/MS method in order to simplify the analysis and increase the temporal resolution of this technique. (2). A new 'detuned' MCO/MS method will measure the tertiary structural changes that occur around Cu (ll) during (B2m unfolding and oligomerization. The scope of this new method will be studied, including its potential to provide information about the relative distances of amino acids from Cu (ll). (3). Radical-based protein footprinting will be used to study changes in B2m structure during unfolding and oligomerization. Peroxynitrite will be investigated as a new protein footprinting reagent. We hypothesize that Cu (ll) binding destabilizes the N-terminal region of B2m. This destabilization leads to partial unfolding, and the subsequent structural changes that lead to oligomerization will be elucidated using our 3 complementary structural analysis tools.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM075092-03
Application #
7254045
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Edmonds, Charles G
Project Start
2005-07-01
Project End
2009-06-30
Budget Start
2007-07-01
Budget End
2008-06-30
Support Year
3
Fiscal Year
2007
Total Cost
$217,581
Indirect Cost
Name
University of Massachusetts Amherst
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
Zhang, Zhe; Vachet, Richard W (2015) Kinetics of Protein Complex Dissociation Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry. Anal Chem 87:11777-83
Zhang, Zhe; Browne, Shaynah J; Vachet, Richard W (2014) Exploring salt bridge structures of gas-phase protein ions using multiple stages of electron transfer and collision induced dissociation. J Am Soc Mass Spectrom 25:604-13
Borotto, Nicholas B; Degraan-Weber, Nicholas; Zhou, Yuping et al. (2014) Label scrambling during CID of covalently labeled peptide ions. J Am Soc Mass Spectrom 25:1739-46
Dong, Jia; Callahan, Katie L; Borotto, Nicholas B et al. (2014) Identifying Zn-bound histidine residues in metalloproteins using hydrogen-deuterium exchange mass spectrometry. Anal Chem 86:766-73
Dong, Jia; Joseph, Crisjoe A; Borotto, Nicholas B et al. (2014) Unique effect of Cu(II) in the metal-induced amyloid formation of β-2-microglobulin. Biochemistry 53:1263-74
Zhou, Yuping; Vachet, Richard W (2013) Covalent labeling with isotopically encoded reagents for faster structural analysis of proteins by mass spectrometry. Anal Chem 85:9664-70
Zhou, Yuping; Vachet, Richard W (2012) Diethylpyrocarbonate labeling for the structural analysis of proteins: label scrambling in solution and how to avoid it. J Am Soc Mass Spectrom 23:899-907
Zhou, Yuping; Vachet, Richard W (2012) Increased protein structural resolution from diethylpyrocarbonate-based covalent labeling and mass spectrometric detection. J Am Soc Mass Spectrom 23:708-17
Dong, Jia; Vachet, Richard W (2012) Coordination sphere tuning of the electron transfer dissociation behavior of Cu(II)-peptide complexes. J Am Soc Mass Spectrom 23:321-9
Mendoza, Vanessa Leah; Barón-Rodríguez, Mario A; Blanco, Cristian et al. (2011) Structural insights into the pre-amyloid tetramer of β-2-microglobulin from covalent labeling and mass spectrometry. Biochemistry 50:6711-22

Showing the most recent 10 out of 21 publications