Quantitative analysis of post-translational modifications in human serum transthyretin associated with familial amyloidotic polyneuropathy by targeted LC-MS and intact protein MS.

Abstract

Transthyretin (TTR) is an amyloidogenic tetrameric protein, present in human plasma, associated with several familial amyloidoses. Variability of TTR is not only due to point mutations in the encoding gene but also to post-translational modifications (PTMs) at Cys10, being the most common PTMs the S-sulfonation, S-glycinylcysteinylation, S-cysteinylation and S-glutathionylation. It is thought that PTMs at Cys10 may play an important biological role in the onset and pathological process of the amyloidosis. We report here the development of a methodology for quantification of PTMs in serum samples, as well as for the determination of serum TTR levels, from healthy (wt) and TTR-amyloidotic (V30M mutation) individuals. It involves an enrichment step by immunoprecipitation followed by mass spectrometry analysis of (i) the intact TTR protein and (ii) targeted LC-MS analysis of peptides carrying the PTMs of interest. Analysis of serum samples by the combination of the two methods affords complementary information on the relative and absolute amounts of the selected TTR PTM forms. It is shown that methods based on intact protein are biased for specific PTMs since they assume constant response factors, whereas the novel targeted LC-MS method provides absolute quantification of PTMs and total TTR variants.

BIOLOGICAL SIGNIFICANCE:

The study of TTR has a high clinical relevance since it is responsible for diverse familial polyneuropathies. In particular, more than 80 point mutations have been described through genetic studies. However, genetic heterogeneity alone fails to explain the diverse onset and pathological process of the TTR related amyloidosis. The use of proteomic characterization is required to gather information about the PTMs variants present in serum, which have been suggested to be relevant for the amyloidotic pathology. This article is part of a Special Issue entitled: HUPO 2014.

• Authors: Marta Vilà-Rico, Núria Colomé-Calls, Luna Martín-Castel, Marina Gay, Sebastián Azorín, Marta Vilaseca, Antoni Planas, Francesc Canals
• Journal: Journal of proteomics (J Proteomics) Vol. 127 Issue Pt B Pg. 234-46 (Sep 08 2015) ISSN: 1876-7737 [Electronic] Netherlands
• PMID: 25910794 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015 Elsevier B.V. All rights reserved.

Chemical References

• Prealbumin

Topics

• Amino Acid Substitution
• Amyloid Neuropathies, Familial (blood, genetics)
• Chromatography, Liquid
• Female
• Humans
• Male
• Mass Spectrometry (methods)
• Mutation, Missense
• Prealbumin (metabolism)
• Protein Processing, Post-Translational
• Proteomics (methods)