This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The BUSM MS Resource has undertaken atomic force microscopy to complement mass spectrometry studies of individual biopolymers of high molecular weight, including, e.g., proteoglycans, collagens and molecules having collagen-type domains, and other proteins that contain high levels of glycosylation, as well as non-covalent complexes. AFM is being used to estimate the overall weight and modular composition of such species, on the basis of extrapolations that should become possible after construction of calibration curves over the range accessible by mass spectrometry.
The aim of this project is to use AFM to quickly estimate the MWs of large molecules and complexes as a supplemental tool for mass spectrometry in the high molecular weight range. This is achieved by using AFM to measure the molecular volume of single molecules of known MW as standards, extrapolating a curve using biopolymer standards at different MW, and determining the relation between molecular volume and MW. AFM is also being used to characterize amyloid fibrils, with the goal of relating fibril properties to variations in the amino acid sequence and to posttranslational modifications of the constituent proteins. By using purified patient samples as well as recombinant a-beta protein, the formation of amyloid fibrils under different conditions and protein/GAG interactions is being monitored in both this development project and in several collaborative projects (e.g., Nugent, Skinner, Spencer, Trinkaus-Randall). AFM images have also been recorded for amyloid fibrils obtained from fat aspirates of patients with primary amyloid disease.To improve resolution in AFM measurements, the use of carbon nanotubes is being explored and a device for mounting the tubes has been constructed and tested. Day-to-day operation, maintenance and supervision of the instrument are the responsibility of Dr. Hong, who completed his PhD in the Boston University Dept. of Cellular Biophysics with a focus on AFM studies of mucins. Dr. Ding, who has extensive experience in AFM of beta-amyloid and synuclein amyloid fibrils and various types of macromolecules, helped in the planning for this project and continues to provide advice and assistance to the BUSM investigators. Although he has relocated to North Carolina, he remains in email and phone contact.
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