Supplementary Figure 12: Effects of peptide composition and length on Edman cleavage efficiencies.

(A) To better understand the range of Edman efficiency across amino acids we sequenced two synthetic peptides, differing only by the first amino acid, AK^†AGAGRYG and PK^†AGAGRYG. These were chosen for their historic ease, in the case of alanine, or difficulty, in the case of proline, of Edman sequencing. Experiments were performed in triplicate (bar chart represents the mean +/- s,d. across experiments), sequencing both peptides simultaneously in a multiplexed flow cell, and results were averaged and compared with simulation to determine error rates. Error rates of 3% dye destruction, 30% surface degradation were identical across the two samples, while Edman cleavage efficiency was 95% and 91% for the alanine and proline samples respectively. (B) Consistent with the higher Edman cleavage efficiencies observed for alanine, a peptide composed of glycine/alanine repeats (GAGAGAGAGC^♦ARRYRRG) with a fluorophore at the 10^th position sequenced efficiently (top histogram, 400 fields) and was well fit by simulations with a 97% Edman cleavage efficiency, 3% dye destruction, and 4% surface degradation rate (bottom histogram). (Note that “dud" dye rates cannot be determined through simulation for singly labeled peptides.) † indicates Atto647 conjugated to lysine and ♦indicates Atto647N conjugated to cysteine.