Supplementary Figure 9: Measurement and modeling of fluorophore intensities.
From: Highly parallel single-molecule identification of proteins in zeptomole-scale mixtures
(A) Fluorophore intensities follow a lognormal model. Photometries in the last frame before a dye labeled peptide permanently turns OFF predominantly represent the intensity of a single fluorophore, regardless of how many dyes a peptide initially started with. The distribution of these photometries is consistently lognormal across multiple experiments as shown by the Q-Q plot. Each point indicates a percentile of the lognormal photometry histogram of GK*AGAG, GC♦AGC♦AGAG, N-Acetyl-GK*AGAG, N-Acetyl-GC♦AGC♦AGAG, QC♦C♦TSIC♦SLYN with n=97137, n=273411, n=72905, n=155097 peptides, respectively. * indicates TMR conjugated to lysine ♦indicates Atto647N conjugated to cysteine. (B, C) Statistical models for photometry of multiple dyes were refined using forward simulation to optimize the values of the lognormal shape parameter σ* QUOTEσ * and dye-dye interaction factor Qc. Photometry distributions of peptides with the observed (blue) and simulated (orange) dye sequence: [2,2,2,2,2,1,1,1,0,0,0,0] for (B) a poor parameter choice, showing results of an overestimated σ* QUOTEσ * QUOTEσ * and underestimated Qc QUOTEQ c , and for (C) the optimized values of σ* =0.20 and Qc =0.30 QUOTEσ * =0.20 QUOTEQ 2 =0.30 . Data shown for the initial condition, 3 mock cycles, and 4 Edman cycles, n=273411 peptides. The remaining cycles, with 0 dyes, are omitted.
