An increasing number of proteins are known to form amyloid fibrils in vivo, and the formation of these fibrils is implicated in several diseases (e.g. Alzheimer's, Parkinson's). One of these proteins, human ?-2-microglobulin (?2m), can form amyloid fibrils in the presence of Cu(II), and these fibrils are presumed to be the main pathogenic process underlying dialysis- related amyloidosis (DRA). Like other amyloid systems, ?2m fibril formation proceeds by partial protein unfolding, subsequent oligomerization, and eventual elongation to form mature fibrils. While many aspects of general amyloid formation are understood, molecular-level information is lacking for the early stages of almost all amyloid forming reactions;however, this information is critical for the rational development of therapeutics against amyloid diseases like DRA. We intend to obtain amino acid-level information about the unfolding and oligomerization of ?2m that precedes its fibril formation. To do so, we will develop, optimize, and apply three mass spectrometry (MS)-based methods with the necessary temporal and spatial resolution. (1) A new metal-catalyzed oxidation (MCO) induced deuterium labeling strategy will be investigated to complement our existing MCO/MS method. The proposed MCO-induced deuterium labeling strategy will provide Cu-??2m binding data that are more easily interpreted, contain more information about all the Cu-bound amino acids, and are less prone to false positives. (2) Covalent labeling with MS detection will be used to study changes in ?2m structure upon Cu(II) binding, unfolding, and oligomerization. The covalent labeling reactions will identify changes to the solvent accessibility of different amino acids in ?2m as this protein progresses from monomer to oligomers. (3) Electrospray ionization (ESI) with top-down sequencing will be explored as a means to separate and characterize protein oligomers formed by ?2m. The differential charging that occurs during ESI of protein oligomers will be used to rapidly separate protein oligomers, and top-down sequencing will be used to identify the solvent accessible amino acid residues that are labeled in individual oligomers. Our preliminary data on ?2m fibril formation support a model in which Cu(II) is necessary to organize the dimer and an initial tetramer but is released upon formation of a second tetramer and the hexamer. We will use these three MS-based methods to study this model and identify the structural changes associated with the formation of each oligomeric state.

Public Health Relevance

The formation of protein amyloid fibrils is associated with about 20 human diseases, including Alzheimer's, Parkinson's, and Dialysis-Related Amyloidosis (DRA), but the molecular details of how these amyloids begin to form are mostly unknown. We intend to develop new measurement tools that will provide the molecular details necessary to understand the amyloid formation of ?-2-microglobulin (?2m), which is the protein implicated in DRA. This molecular information about ?2m should inform efforts to design therapeutics against DRA but will also give insight into protein amyloid formation in general.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Edmonds, Charles G
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Massachusetts Amherst
Schools of Arts and Sciences
United States
Zip Code
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