A Theoretical Justification for Single Molecule Peptide Sequencing
Fig 7
Surface plots illustrate the consequences of differing rates of Edman efficiency, photobleaching, and fluorophore failure rates.
Each panel summarizes the consequences of varying rates of photobleaching and Edman failures for a different fixed fluorophore failure rate, ranging from 0% to 25%, as calculated after simulating 30 experimental cycles on the complete human proteome at a simulation depth of 10,000 copies per protein. Photobleaching shows the strongest negative impact on proteome coverage when compared to other errors; increasing the number of distinguishable labels strongly increases proteome coverage. Labeling and immobilization schemes are denoted as in Fig. 2. For comparison, literature evidence suggests that common failure rates of fluorophores may be about 15–20% [18,32], Edman degradation proceeds with about 94% efficiency [33], and the mean photobleaching lifetime of a typical Atto680 dye is about 30 minutes [23], corresponding to 1800 Edman cycles, assuming 1 sec exposure per Edman cycle. Thus, we expect error rates to be sufficiently low for effective fluorosequencing.