Detection of genetic variants of transthyretin by liquid chromatography-dual electrospray ionization fourier-transform ion-cyclotron-resonance mass spectrometry.
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 (< or = 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.
Nepomuceno
Angelito I
AI
WM Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, Rochester, MN, USA.
Mason
Christopher J
CJ
Muddiman
David C
DC
Bergen
H Robert
HR
Zeldenrust
Steven R
SR
D028226:Amyloidosis, Familial / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D017354:Point Mutation; D011228:Prealbumin / Q000737:chemistry / Q000235:genetics*; D017422:Sequence Analysis, DNA; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
D011228:Prealbumin