Preparation of fluorescently labeled peptides

Peptides were labeled with a fluorescent dye essentially as described previously (17). In detail: Lyophilized peptides were resuspended in water to a concentration of 5 mM or 1 mM depending on their solubility and stored at −20 °C. For labeling, 500 μM peptide (500 μl) was prepared in 100 mM HEPES, pH 7.6 and the peptides reduced by the addition of 1.5 M excess of TCEP and incubation for 30 min at 30 °C. Peptides were labeled with a 5-fold molar excess of 2-(4′-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS, Molecular Probes, Invitrogen) for 2 h at RT. The peptide subsequently was separated from leftover dye on a self-packed G10 Sephadex column (20 × 1 cm) equilibrated in 100 mM HEPES, pH 7.6. Two drops per fraction were collected and only fractions containing the first fluorescent peak and displaying viscosity, while pipetting, were pooled, analyzed by high-resolution mass spectrometry, and stored at −20 °C.