TY - JOUR
T1 - Quantitative analysis of post-translational modifications in human serum transthyretin associated with familial amyloidotic polyneuropathy by targeted LC-MS and intact protein MS
AU - Vilà-Rico, Marta
AU - Colomé-Calls, Núria
AU - Martín-Castel, Luna
AU - Gay, Marina
AU - Azorín, Sebastián
AU - Vilaseca, Marta
AU - Planas, Antoni
AU - Canals, Francesc
N1 - Funding Information:
The authors wish to acknowledge Dr. JM Campistol (Hospital Clínic de Barcelona) for providing the clinical samples analyzed in this study. We also acknowledge the Fundació Privada de la Marató de TV3 for grant 101431/32 to FC and AP. MVR and NCC acknowledge fellowships from the granted project by the Fundació Privada de la Marató de TV3. We thank Pierre-Olivier Schmit and Stephanie Kaspar (Bruker), for valuable technical advise and support. Top-down MS analysis were performed in the IRB Barcelona Mass Spectrometry Core Facility that participates in the BMBS European COST Action BM 1403. We thank Mar Vilanova from the IRB MS Core for the helpful assistance. Both the Proteomics Laboratory at VHIO and the IRB Barcelona Mass Spectrometry Core Facility, are members of ProteoRed, part of PRB2-ISCIII, supported by grant PT13/0001.
Publisher Copyright:
© 2015 Elsevier B.V.
PY - 2015/9/8
Y1 - 2015/9/8
N2 - 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.
AB - 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.
KW - Absolute quantification
KW - High resolution XIC quantification
KW - Intact protein analysis
KW - Post-translational modifications
KW - Targeted proteomics
KW - Transthyretin
UR - http://www.scopus.com/inward/record.url?scp=84944164987&partnerID=8YFLogxK
U2 - 10.1016/j.jprot.2015.04.016
DO - 10.1016/j.jprot.2015.04.016
M3 - Article
C2 - 25910794
AN - SCOPUS:84944164987
SN - 1874-3919
VL - 127
SP - 234
EP - 246
JO - Journal of Proteomics
JF - Journal of Proteomics
ER -