Journal
CLINICAL CHEMISTRY
Volume 50, Issue 9, Pages 1535-1543Publisher
AMER ASSOC CLINICAL CHEMISTRY
DOI: 10.1373/clinchem.2004.033274
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- NHGRI NIH HHS [R01HG02159] Funding Source: Medline
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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 (less than or equal to3 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. (C) 2004 American Association for Clinical Chemistry.
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