Fig 1.
Example of substitution matrix.
Figure showing overlapping peptides (left) with the valine (V highlighted in bold) being analyzed for antibody selectivity. The box contains the substitution values, xi,j, with columns representing the amino acid, i, which substitutes valine and rows representing the position, j, of the valine in the peptide (illustrated to the left) undergoing substitution. The mean substitution value, μi, of each replacing amino acid is shown in the bottom row. The global mean, μg, is calculated across all substitution values in the matrix.
Fig 2.
Schematic example of the generation of selectivity logo-plots.
Figure showing example of selectivity of two epitope positions in overlapping peptides upon substitutions with all 19 amino acids. A and B shows illustrative density plots of substitution values taken from a substitution matrix, with the mean substitution value,.μi, of each replacing amino acid shown in diamond shapes. Filled diamond is the native amino acid (E in both examples). C and D show the resulting logo-plot of the positions exemplified in A and B, respectively. The logo-plots are made using Seq2Logo [16].
Fig 3.
Example of a position specific scoring matrix.
The table shows the individual substitution values for the peptide DHVKLVNEVTEFAKT through all 15 positions (columns) with all 20 amino acids (rows). The last row shows the mean substitution value of the positions. Blank cells are native amino acids. A Dunnett’s LSD of 0.1741 was calculated on the chosen p<0.0001 level and cells exceeding this difference are highlighted with a gray gradient (weak to strong effects). The identified epitope is LVNEVTEF.
Table 1.
Example of target-specific positions of individual peptides.
The table shows output of 33 overlapping peptides mapping the positions 56 to 88 of the HSA protein sequence. Positions identified as being important for binding (identified by the Dunnett’s test of complete single-amino acid substitutions at the p<0.0001 level) are highlighted whereas dashes indicate positions not involved in binding. The median signal of copies of the corresponding native peptide is shown.
Fig 4.
Example of selectivity tables.
Tables showing substitution values of residue 518D of human serum albumin against all 20 amino acids (columns) and being represented in 10 out of 15 positions of the overlapping peptides (rows). Entire blank rows depict the native residue being represented in peptides with no positive signal. Individual blank cells are missing data. The row μ shows the mean substitution value of the replacing amino acids. Here, cells are highlighted by the effect of substitution as a color gradient from red to green through white, where white corresponds to the global mean, μg, equal to 0.240.
Fig 5.
Examples of selectivity profile of epitope.
Logo-plot representations of the selectivity of three 15-residue regions of the primary sequence of HSA. Selectivity is determined by Dunnett’s test on all substitutions of target position, being represented in overlapping peptides. Each letter represents replacing amino acids and is scaled by log(p), where the p-value is obtained from the Dunnett’s test. Positive letters denotes substitution values above the global mean, μg, while negative letters denotes substitution values below μg. The absolute height of an entire column represents the average relative change in signal of all substitutions of target residue. The average signal of native 15-mer peptides containing the epitopes is (A) 489 Au, (B) 130 Au and (C) 176 Au.
Table 2.
Table showing 18 HSA epitope regions identified by the algorithm. Dashes mark gaps of residues with no selectivity. The regions were identified from the logo plots, and defined as having a minimum of 4 residues and a maximum gap-length of 2 residues.