The last scanning method, scrambled scanning, is also used for sequence optimization but the mutants library is obtained by permuting the original sequence (Fig. ). Positions are shuffled without replacement, keeping the same amino acid composition, and the process is repeated until the desired total number of peptide sequences is reached. However, scrambled peptide libraries are mainly used as negative controls to prove the sequence specificity. If the amino acid sequence order and composition is the sole determinant of protein-peptide interaction, then none of the scrambled peptide sequences will show binding affinity, or only a few by chance.