Figure 2.
Figure 2. The C-terminal mutant region predicted by Phe521Leufs is detected in renal amyloid deposits, but not its wild-type counterpart. (A) The results of LMD/MS-based proteomics analysis of amyloid plaques from 7 cases of AFib. Cases 1 to 6 carry the Aα-chain Glu526Val (E526V) variant, and case 7 the Phe521Leufs (F521fs) variant. The identified proteins are listed by relative probability score for identity, and the top 20 proteins of 103 proteins are shown. The columns show the protein name, the UnitProt identifier (protein accession number in the UniProt database, http://www.uniprot.org/), the molecular weight of the protein, and 1 microdissection from 7 patient specimens involved by AFib. The numbers indicate the number of total peptide spectra identified for each protein. Fibrinogen Aα-chain is the most abundant protein amyloidogenic in this sample set, consistent with AFib amyloidosis in each case. To show the presence of mutated proteins in the amyloid plaques, the raw mass spectrometry data files were searched using the human SwissProt database supplemented with Aα-chain variants Glu526Val and Phe521Leufs. Consistent with genetic analysis, cases 1 to 6 contain the tryptic peptide carrying the Glu526Val variant (row 7, green rectangle), whereas this variant is not present in the case with the Phe521Leufs variant. In contrast, the novel tryptic peptides generated by the frameshift in Phe521Leufs variant are only present in case 7 (row 17, red rectangle). (B) shows the Aα-chain protein coverage in 7 cases of AFib amyloidosis. Cases 1 to 6 carry the Glu526Val variant, and case 7 the Phe521Leufs variant. The top line represents the C-terminal sequence of native Aα-chain. The amino acid residues of Phe521Leufs (F521 in red) and Glu526Val (E526 in green) are indicated. The first line of rectangles (blue) labeled “WT” represents the coverage of the wild-type Aα-chain by the mass spectrometry-based proteomic method used. Two samples (S1 and S2) from 4 patients are shown. In cases 1 to 6 (Glu526Val variant), most of the coverage is identical to the wild-type Aα-chain except for the tryptic peptide carrying the point mutation (green rectangle) instead of the amino acid present in the wild-type peptide. In case 7 (Phe521Leufs), frameshift leads to a novel sequence indicated by the red rectangle and amino acid sequence in red letters. No native Aα-chain peptides are present after the frameshift mutation, indicating that only the mutant Aα-chain is present in amyloid deposits.

The C-terminal mutant region predicted by Phe521Leufs is detected in renal amyloid deposits, but not its wild-type counterpart. (A) The results of LMD/MS-based proteomics analysis of amyloid plaques from 7 cases of AFib. Cases 1 to 6 carry the Aα-chain Glu526Val (E526V) variant, and case 7 the Phe521Leufs (F521fs) variant. The identified proteins are listed by relative probability score for identity, and the top 20 proteins of 103 proteins are shown. The columns show the protein name, the UnitProt identifier (protein accession number in the UniProt database, http://www.uniprot.org/), the molecular weight of the protein, and 1 microdissection from 7 patient specimens involved by AFib. The numbers indicate the number of total peptide spectra identified for each protein. Fibrinogen Aα-chain is the most abundant protein amyloidogenic in this sample set, consistent with AFib amyloidosis in each case. To show the presence of mutated proteins in the amyloid plaques, the raw mass spectrometry data files were searched using the human SwissProt database supplemented with Aα-chain variants Glu526Val and Phe521Leufs. Consistent with genetic analysis, cases 1 to 6 contain the tryptic peptide carrying the Glu526Val variant (row 7, green rectangle), whereas this variant is not present in the case with the Phe521Leufs variant. In contrast, the novel tryptic peptides generated by the frameshift in Phe521Leufs variant are only present in case 7 (row 17, red rectangle). (B) shows the Aα-chain protein coverage in 7 cases of AFib amyloidosis. Cases 1 to 6 carry the Glu526Val variant, and case 7 the Phe521Leufs variant. The top line represents the C-terminal sequence of native Aα-chain. The amino acid residues of Phe521Leufs (F521 in red) and Glu526Val (E526 in green) are indicated. The first line of rectangles (blue) labeled “WT” represents the coverage of the wild-type Aα-chain by the mass spectrometry-based proteomic method used. Two samples (S1 and S2) from 4 patients are shown. In cases 1 to 6 (Glu526Val variant), most of the coverage is identical to the wild-type Aα-chain except for the tryptic peptide carrying the point mutation (green rectangle) instead of the amino acid present in the wild-type peptide. In case 7 (Phe521Leufs), frameshift leads to a novel sequence indicated by the red rectangle and amino acid sequence in red letters. No native Aα-chain peptides are present after the frameshift mutation, indicating that only the mutant Aα-chain is present in amyloid deposits.

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