Muddiman, David C.

Abstract Background: Transthyretin-associated hereditary amyloidosis (ATTR) is an inherited disease in which variants in the primary structure of transthyretin (TTR; prealbumin) lead to the extracellular polymerization of insoluble protein fibrils, causing organ failure and ultimately death when major organs are involved. We have developed an integrated approach to molecular diagnosis with initial analysis of intact plasma TTR by electrospray ionization mass spectrometry (MS) and referral of positive samples for DNA sequence analysis and real-time PCR to confirm the common Gly6Ser polymorphism. Methods: Samples from 6 patients previously diagnosed with ATTR and from 25 controls with (n = 15) or without (n = 10) polyneuropathy were analyzed in a blinded fashion for the presence of variant TTR. TTR protein was extracted with an immunoaffinity resin from 20 μL of archived plasma samples. The purified TTR was reduced with tris(2-carboxyethyl)phosphine and analyzed by MS. The appearance of two peaks (or a single peak shifted in mass indicative of a homozygous variant), including the wild-type mass of 13 761 Da, was indicative of the presence of a variant, and the individual was referred for DNA sequence analysis. Results: MS analysis of intact reduced TTR correctly identified each of six samples known to contain variant TTR. These results were corroborated by subsequent DNA sequence analysis. Additionally, all Gly6Ser polymorphisms were correctly called based on the +30 mass shift and an equal relative abundance of the +30 polymorphism relative to wild-type TTR. No false-positive results were seen. Conclusions: This referral method eliminates the necessity of sequencing most samples and allows screening for the familial forms of amyloidosis in a broad patient population in a timely fashion. This method correctly identified all previously known variants and also identified a novel variant, Val94Ala.

AbstractBackground: One of the numerous proteins causing amyloidosis is transthyretin (TTR), a protein usually responsible for the transport of thyroxine and retinol-binding protein. Variants within TTR cause it to aggregate and form insoluble fibers that accumulate in tissue, leading to organ dysfunction.Methods: TTR was immunoprecipitated from serum by use of a polyclonal antibody and subsequently reduced with tris(2-carboxyethyl)phosphine. The purified TTR was then analyzed by fast-gradient liquid chromatography–dual-electrospray ionization Fourier-transform ion-cyclotron-resonance (FT-ICR) mass spectrometry. DNA sequencing was performed on all samples used in this study.Results: Because of the inherent limitations in achieving high mass measurement accuracy based on the most abundant isotopic mass, we applied a fitting procedure that allowed determination of monoisotopic mass. Wild-type TTR (mean molecular mass, 13 761 Da) and its associated variant forms could be distinguished because of the high molecular mass accuracy afforded by FT-ICR (≤3 ppm) except for instances involving isobaric species or when isotopic distributions overlapped significantly. The [M + 11 H+]11+ charge state for all samples was used to determine the mass accuracies for both wild-type and variant forms of the protein. We correctly assigned seven of seven TTR variants. Moreover, using a combination of proteomic and genomic technologies, we discovered and characterized a previously unreported cis double mutation with a mass only 2 Da different from wild-type TTR. Furthermore, DNA sequencing of the TTR gene for all individuals in this study completely agreed with the intact protein measurements.Conclusions: FT-ICR mass spectrometry has sufficient mass accuracy to identify genetic variants of immunoaffinity-purified TTR. We believe that 91% of known TTR variants could be detected by this technique.

ABSTRACTPreviously, we identified a naturally processed and presented measles virus (MV) 19-amino-acid peptide, ASDVETAEGGEIHELLRLQ (MV-P), derived from the phosphoprotein and eluted from the human leukocyte antigen (HLA) class II molecule by using mass spectrometry. We report here the identification of a 14-amino-acid peptide, SAGKVSSTLASELG, derived from the MV nucleoprotein (MV-N) bound to HLA-DRB1*0301. Peripheral blood mononuclear cells (PBMC) from 281 previously vaccinated measles-mumps-rubella II (MMR-II) subjects (HLA discordant) were studied for peptide recognition by T cells. Significant gamma interferon (IFN-γ) responses to MV-P and MV-N peptides were observed in 55.9 and 15.3% of subjects, respectively. MV-P- and MV-N-specific interleukin-4 (IL-4) responses were detected in 19.2 and 23.1%, respectively, of PBMC samples. Peptide-specific cytokine responses and HLA-DRB1 allele associations revealed that, for the MV-P peptide, the allele with the strongest association with both IFN-γ (P= 0.02) and IL-4 (P= 0.03) secretion was DRB1*0301. For MV-N, the allele with the strongest association with IFN-γ secretion was DRB1*1501 (P= 0.04), and the alleles with the strongest associations with IL-4 secretion were DRB1*1103 and DRB1*1303 (P= 0.01). These results indicate that HLA class II MV proteins can be processed, presented, and identified, and the ability to generate cell-mediated immune responses can be demonstrated. This information is promising for new vaccine design strategies with peptide-based vaccines.