Characterization of Transthyretin Mutants from Serum Using Immunoprecipitation, HPLC/Electrospray Ionization and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

A mass spectrometry approach for the detection and identification of variants of the plasma protein transthyretin (TTR) is presented. The single amino acid substitutions found in TTR are closely associated with familial transthyretin amyloidosis (ATTR), a hereditary degenerative disease. A definitiv...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 1999-01, Vol.71 (2), p.452-459
Main Authors: Théberge, R, Connors, L, Skinner, M, Skare, J, Costello, C. E
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amyloidosis - genetics
Biological and medical sciences
Chemistry
Chromatography, High Pressure Liquid - methods
Errors of metabolism
Humans
Mass Spectrometry - methods
Medical sciences
Metabolic diseases
Methionine
Molecular Sequence Data
Mutation
Prealbumin - analysis
Prealbumin - genetics
Precipitin Tests - methods
Proteins and glycoproteins
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
Valine
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Summary:A mass spectrometry approach for the detection and identification of variants of the plasma protein transthyretin (TTR) is presented. The single amino acid substitutions found in TTR are closely associated with familial transthyretin amyloidosis (ATTR), a hereditary degenerative disease. A definitive diagnosis of ATTR relies on the detection and identification of TTR variants. The approach presented here is based on isolation of serum TTR using immunoprecipitation. The detection of the variant is achieved by mass measurement of the intact protein with electrospray ionization mass spectrometry (ESIMS). The liquid chromatography/ESIMS analysis of the tryptic digest of the protein followed by subsequent matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry and MALDI postsource decay of the relevant recovered chromatographic fraction containing the variant peptide allows the identification of unknown variants. The method was successfully tested using serum from ATTR patients with known variants (Val30→Met and Val122→Ile). A new TTR variant, Ser23→Asn, was detected and identified using the above method where isoelectric focusing and restriction enzyme analysis failed to identify the nature of the variant.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac980531u