Identification of S‐sulfonation and S‐thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis

Abstract

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild-type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease. Previously, we have developed a mass spectrometry-based approach, in combination with direct DNA sequence analysis, to fully identify TTR variants. Our methodology uses immunoprecipitation to isolate TTR from serum, and electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry (MS) peptide mapping to identify TTR variants and posttranslational modifications. Unambiguous identification of the amino acid substitution is performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis. The MS and MS/MS analyses also yield information about posttranslational modifications. Using this approach, we have recently identified a novel pathologic TTR variant. This variant has an amino acid substitution (Phe → Cys) at position 33. In addition, like the Cys10 present in the wild type and in this variant, the Cys33 residue was both S-sulfonated and S-thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.

Decreased clearance of serum retinol-binding protein and elevated levels of transthyretin in insulin-resistantob/obmice

“…No information regarding insulin-glucose homeostasis was reported in either case. In addition to genetic variability, several posttranslationally modified forms of TTR have been identified (33,47,52,63) in serum of healthy humans and in subjects with familial amyloidosis. Future studies are needed to assess whether genetic or posttranslational variants of TTR contribute to elevation of RBP4 and insulin resistance in human subjects.…”

Direct targeting of human plasma for matrix‐assisted laser desorption/ionization and analysis of plasma proteins by time of flight‐mass spectrometry

“…Major spots in Fig. 1 were identified [7,23], but the low-molecular-mass proteins are not well resolved compared to the mass spectrometric results of the corresponding proteins [15][16][17][19][20][21]. The reported MALDI- MS spectra showed that the plasma lipoproteins have been dissociated into their component apolipoproteins, possibly because of the sample treatment with a matrix solution which contains TFA and ACN [19].…”

“…However, as far as we know, MS on whole plasma has not been reported. Actually, all the plasma protein studies by MS included complex separation/purification procedures prior to MS analysis, e.g., transthyretin (TTR) was normally purified by immunoprecipitation followed by LC [15][16][17], while apolipoproteins were purified using immunoaffinity chromatography or fractionated by centrifugation [19][20][21]. Electrophoresis 2005Electrophoresis , 26, 2823Electrophoresis -2834 In this paper, we describe a method for direct MALDI-TOF-MS analysis of human plasma proteins without using any prior purification or separation procedures.…”

Mass Spectrometry Approaches for the Molecular Characterization of Oxidatively/Nitrosatively Modified Proteins

“…In contrast, the occurrence of mixed disulfides with low molecular weight compounds or intramolecular disulfides, determining limited variation in molecular mass of intact proteins, has been revealed by conventional MS procedures. In the case of S-glutathionylated, S-cysteinylglycinylated, Scysteinylated, and S-sulfonated proteins, the occurrence of S-conjugated species has been ascertained by direct electrospray ionization (ESI) measurements of intact proteins, detecting the corresponding adducts presenting a mass difference of +305 Da, +176 Da, +119 Da, and +80 Da, respectively (Hanson et al, 1999;Naito et al, 2000;Lim et al, 2003). As expected, the mass spectra of species containing mixed disulfides were totally affected by reducing agent treatment.…”

“…A careful evaluation of experimental conditions suitable to avoid scrambling phenomena during protein hydrolysis is strongly recommended. Identification of the modified residues has been obtained by liquid chromatography-electrospray ionization (LC-ESI) or matrix-assisted laser desorption ionization (MALDI) mapping experiments by detecting the peptides bearing a mass difference of +305 Da (S-glutathionylated), +176 Da (S-cysteinyl-glycinylated), +119 Da (S-cysteinylated), and +80 Da (Ssulfonated), and eventually confirmed by collision-induced dissociation (CID) measurements (Vilardo et al, 2001;Lim et al, 2003). Similarly, cysteine pairing identification in species containing intra-molecular disulfides as a result of oxidative/nitrosative insult are derived by mass mapping and the tandem mass spectrometry approaches conventionally used for the assignment of disulfides in native polypeptide species.…”