Evaluating the Effect of Dye-Dye Interactions of Xanthene-Based Fluorophores in the Fluorosequencing of Peptides

Abstract

A peptide sequencing scheme utilizing fluorescence microscopy and Edman degradation to determine the amino acid position in fluorophore-labeled peptides was recently reported, referred to as fluorosequencing. It was observed that multiple fluorophores covalently linked to a peptide scaffold resulted in a decrease in the anticipated fluorescence output and worsened the single-molecule fluorescence analysis. In this study, we report an improvement in the photophysical properties of fluorophore-labeled peptides by incorporating long and flexible (PEG)₁₀ linkers at the peptide attachment points. Long linkers to the fluorophores were installed using copper-catalyzed azide-alkyne cycloaddition conditions. The photophysical properties of these peptides were analyzed in solution and immobilized on a microscope slide at the single-molecule level under peptide fluorosequencing conditions. Solution-phase fluorescence analysis showed improvements in both quantum yield and fluorescence lifetime with the long linkers. While on the solid support, photometry measurements showed significant increases in fluorescence brightness and 20 to 60% improvements in the ability to determine the amino acid position with fluorosequencing. This spatial distancing strategy demonstrates improvements in the peptide sequencing platform and provides a general approach for improving the photophysical properties in fluorophore-labeled macromolecules.

Figure 1.

Common xanthene fluorophores and overview of peptide fluorosequencing. (A) Structures of xanthene fluorophores and the two fluorophores used in the initial fluorosequencing report (2 and 3). (B) Bis-cysteine peptide with fluorophores at positions 3 and 4 being monitored with single-molecule fluorescence microscopy. A single fluorescent spot is representative of the di-labeled peptide, with corresponding loss of fluorescence for that spot occurring during the third and fourth Edman degradation cycles. (C) Initial report had short linkers between the fluorophores and the peptide backbone. The longer (PEG)₁₀ linker shown is discussed herein.

Figure 2.

Design and synthesis of short- and long-linker peptides used to study photophysical and peptide fluorosequencing properties.

Figure 3.

Solution-phase analysis of peptide QY (Φ) and FLT (τ, ns). Nomenclature for fluorophore-labeled peptides: ST = short-linker, TMR; LT = long-linker, TMR; SA = short-linker, Atto 647N; and LA = long-linker, Atto 647N.

Figure 4.

Intensity distribution and Gaussian fits for (A) short-linker Atto (18) and (B) long-linker Atto (24). (C) Percent quenching of peptides 17, 18, 23, and 24, showing a clear decrease in quenching with long linkers. (D) Percent change in correct positional assignments in fluorosequencing from short to long linkers for TMR (17 to 23) and Atto 647N (18 to 24).