Received on February 21, 2004
Accepted on May 13, 2004

Molecular Diagnostics and Genetics

Detection of Genetic Variants of Transthyretin by Liquid Chromatography-Dual Electrospray Ionization Fourier-Transform Ion-Cyclotron-Resonance Mass Spectrometry

¹ W.M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, Rochester, MN, and Department of Chemistry, Virginia Commonwealth University, Richmond, VA
² W.M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, Rochester, MN
³ W.M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, Rochester, MN, and Biochemistry and Molecular Biology
⁴ Hematology, Mayo Clinic College of Medicine, Rochester, MN

^* To whom correspondence should be addressed. E-mail: muddiman.david{at}mayo.edu.

Background: One of the numerous proteins causing amyloidosis is transthyretin (TTR), a protein usually responsible for the transport of thyroxine and retinol-binding protein. Variants within TTR cause it to aggregate and form insoluble fibers that accumulate in tissue, leading to organ dysfunction.

Methods: TTR was immunoprecipitated from serum by use of a polyclonal antibody and subsequently reduced with tris(2-carboxyethyl)phosphine. The purified TTR was then analyzed by fast-gradient liquid chromatography-dual-electrospray ionization Fourier-transform ion-cyclotron-resonance (FT-ICR) mass spectrometry. DNA sequencing was performed on all samples used in this study.

Results: Because of the inherent limitations in achieving high mass measurement accuracy based on the most abundant isotopic mass, we applied a fitting procedure that allowed determination of monoisotopic mass. Wild-type TTR (mean molecular mass, 13 761 Da) and its associated variant forms could be distinguished because of the high molecular mass accuracy afforded by FT-ICR (3 ppm) except for instances involving isobaric species or when isotopic distributions overlapped significantly. The [M + 11 H⁺]¹¹⁺ charge state for all samples was used to determine the mass accuracies for both wild-type and variant forms of the protein. We correctly assigned seven of seven TTR variants. Moreover, using a combination of proteomic and genomic technologies, we discovered and characterized a previously unreported cis double mutation with a mass only 2 Da different from wild-type TTR. Furthermore, DNA sequencing of the TTR gene for all individuals in this study completely agreed with the intact protein measurements.

Conclusions: FT-ICR mass spectrometry has sufficient mass accuracy to identify genetic variants of immunoaffinity-purified TTR. We believe that 91% of known TTR variants could be detected by this technique.