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Molecular Diagnostics and Genetics |
1 W.M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, Rochester, MN.
Departments of2
Biochemistry and Molecular Biology and3
Hematology, Mayo Clinic College of Medicine, Rochester, MN.
4 Department of Chemistry, Virginia Commonwealth University, Richmond, VA.
aAddress correspondence to this author at: Medical Sciences Building 3-115, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905. Fax 507-284-9261; 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.
The following articles in journals at HighWire Press have cited this article:
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  G. L. Hortin, N. Seam, and G. T. Hoehn Bound Homocysteine, Cysteine, and Cysteinylglycine Distribution between Albumin and Globulins Clin. Chem., December 1, 2006; 52(12): 2258 - 2264. [Abstract] [Full Text] [PDF]
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G. L. Hortin The MALDI-TOF Mass Spectrometric View of the Plasma Proteome and Peptidome Clin. Chem., July 1, 2006; 52(7): 1223 - 1237. [Abstract] [Full Text] [PDF]
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M. Kinter Toward Broader Inclusion of Liquid Chromatography-Mass Spectrometry in the Clinical Laboratory Clin. Chem., September 1, 2004; 50(9): 1500 - 1502. [Full Text] [PDF]
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H. R. Bergen III, S. R. Zeldenrust, M. L. Butz, D. S. Snow, P. J. Dyck, P. J. B. Dyck, C. J. Klein, J. F. O'Brien, S. N. Thibodeau, and D. C. Muddiman Identification of Transthyretin Variants by Sequential Proteomic and Genomic Analysis Clin. Chem., September 1, 2004; 50(9): 1544 - 1552. [Abstract] [Full Text] [PDF]
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