Immunoaffinity chromatographic and immunoprecipitation methods combined with mass spectrometry for characterization...

Transthyretin (TTR) is a small serum protein that is involved in distinct phenotypes of amyloidosis with different tissue localization, age of onset, and rate of progression. Some types of TTR-amyloidosis (such as familial amyloid polyneuropathy) are associated with various amino acid point mutations, while cardiac amyloid myopathy may also be associated with precipitation of the wild-type molecules, e.g., in senile systemic amyloidosis. Because of the unsettled relationship between circulating and precipitated TTR we here explore on-line immunoaffinity (IA) chromatography-MS and immunoprecipitation (IP)-MS methods for characterizing the circulating TTR population in normal individuals and in patients with known TTR-amyloidosis. It was found necessary to reduce the samples, e.g., with DTT, prior to ESI-TOF-MS. This reversed oxidative modifications to sufficiently resolve the two mass peaks isolated from sera of heterozygous patients. A simple IP technique without the use of centrifugal filtration was found to be convenient for the assessment of the TTR population in serum as demonstrated for both normal and variant (the Met111Leu mutation) TTR. This approach also readily allowed the identification of oxidation, S-sulfation, and S-cysteinylation in unreduced samples, while these modifications were less well resolved in the on-line IA chromatography-MS approach.