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Fig. 2 | Immunity & Ageing

Fig. 2

From: Age-associated changes in the circulating human antibody repertoire are upregulated in autoimmunity

Fig. 2

Peptide sequence motifs in probes associated with age. a Sequence motifs in peptide probes associated with age. Peptides associated with age contain a strong N-terminus di-serine (N-di-serine) motif. Motif information content (bits, y-axis) is shown for each position (x-axis). b The N-terminus di-serine motif is much more associated with age (y-axis) than any other di-residue motif (x-axis). c The number of serine residues at the N-terminus (x-axis) is correlated with age-associated antibody binding (y-axis). d Age-associated peptide binding decreases with increased distance of di-serine from N-terminus. The starting position of di-serine residues (x-axis) relative to the N-terminus. The N-terminus is defined as N = 1. e To further characterize the peptide motifs, multiple peptide array synthesis modalities were employed (see Methods). Arrays with ~ 131 k, ~ 351 k, and ~ 3366 k probes were synthesized with peptides that had N-terminus acetyl-capping, a free N-terminus amine, or contained probes with both capped and free N-termini. f Older and younger donor sera were assayed on large microarray format with 3366 k non-control probes, which contained a broader set of peptide probes and inclusion of amino acids, including threonine and isoleucine, which were excluded in the 131 k probe microarray. The presence of multiple N-terminus serines remains the most highly significant motif, and additional serines in positions 3 and 4 may increase discrimination slightly (N = 142 probes starting with tetra-serine). Motifs including N-terminus threonine, which is biochemically similar to serine, are the second-most associated motif. Tryptophan, which is typically the ‘stickiest’ amino acid due to the aromatic indole sidechain, is shown as a negative control that is not associated with age. g Age-associated antibody binding to the di-serine N-terminus motif requires that the N-terminus be acetylated. On arrays where both acetylated and un-acetylated (uncapped free amine) probes are on present on each individual microarray, only acetylated “SS” features show age-associated binding. The number of age-associated probes with > 50% increased binding in donors > 60 yrs. vs < 40 yrs. (y-axis) is shown for uncapped free-amine probes (left) and acetyl-capped probes (right). The cutoff of 50% is representative and other cutoffs can be found in supplemental material (Figure S3G)

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