GBMSDG Meeting Archives
March 5, 2009
Professor Richard W. Vachet
University of Massachusetts Amherst
Insight into β-2-Microglobulin Amyloid Fibril Formation Using Mass Spectrometry
Boston University School of Medicine
Amyloid fibril formation from normally soluble proteins is implicated in several human diseases including Alzheimer���s, Parkinson���s, type II diabetes, and dialysis-related amyloidosis (DRA). The main pathogenic process underlying DRA is the formation of fibrils by β-2-microglobulin (β2m), and evidence suggests that Cu(II) could play a key role in this process.
Like other amyloid systems, β2m 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 most amyloid systems; however, this information is critical for the rational development of therapeutics against amyloid diseases like DRA. Because the protein molecules involved in the early stages of amyloid formation are kinetic intermediates and are usually present as mixtures, obtaining residue-specific information for these intermediates is very challenging with existing methods. To address these challenging measurements, we are developing new mass spectrometry (MS)-based tools with the necessary temporal and spatial resolution.
Three MS-based methods are being developed and utilized to provide information about the early stages of β2m amyloid formation. First, a method based on metal-catalyzed oxidation (MCO) reactions and MS has been essential for determining the Cu(II) binding site of β2m monomers and oligomers. We have also used this MCO/MS approach to elucidate some of the changes in Cu(II)-protein binding that precede amyloid formation. Second, covalent labeling along with MS detection has been used to identify the β2m structural changes that occur upon Cu(II) binding and can be used to study the amino acids present at the interfaces of β2m oligomers. And third, the charge state distributions associated with electrospray ionization have been used to estimate the surface areas of β2m and its oligomers. These three techniques and the unprecedented insight that they provide into β2m amyloid formation will be presented.