Ultrasphere c18

The carotenoid profile was determined by reverse phase HPLC using an Ultimate 3000 Basic Automated System (Thermo Fisher Scientific Inc., Waltham, MA, USA) fitted with a diode arrangement detector. The chromatographic separation was performed on a Beckman Ultrasphere C18 250 × 4.6 mm silica column (5 μm dia. spheres). Elution was conducted using a gradient of methanol/acetonitrile (B: 35% 5 min, B: 35–10% 10 min, B: 10–35% 5 min, B: 35% 10 min) at a flow rate of 1 ml min⁻¹, and peak detection was performed at 480 nm. The peaks derived from the samples were identified by comparison to the spectra and retention time from astaxanthin and β-carotene standards (Sigma-Aldrich). All other reagents were purchased from J.T. Baker (Center Valley, PA, USA) unless otherwise specified.

A Gly-Gly-Gly-Cys peptide with C-terminal amidation (Genscript) was dissolved at 173 mM in degassed coupling buffer (50 mM HEPES (pH 7.4), 20 mM TCEP (pH adjusted to 7.5)). Sulfo-Cyanine5 maleimide (Lumiprobe) dissolved in degassed DMSO was mixed in a 2:1 molar ratio (dye:peptide) and incubated overnight at 4 °C. The following morning, the reaction was quenched with 10 mM DTT. The labeling reaction mixture was separated using on a C-18 reverse phase HPLC Column (Beckman Ultrasphere C-18, 4.6 25 cm) that was pre-equilibrated with Buffer 1(filtered MilliQ with 0.1% Trifluoroacetic acid (Sigma-Aldrich)). The protein was eluted at 1.5 ml/min using the following gradient- 0–1 min: 10% buffer 2 (filtered Acetonitrile (Sigma-Aldrich) with 0.1% TFA); 1–16 min: 70% buffer 2; 16–17 min: 90% buffer 2; 17–22 min: 90% buffer 2. The column eluent was monitored at 200 nm and 650 nm. Under these conditions, unlabelled GGGC peptide eluted at ~11 min and GGGC^Cy5 peptide eluted at ~15 min, respectively, under these conditions. The free Cy5 dye was eluted from the column only after washing with 100% buffer 2. The GGGC^Cy5 peptide was lyophilized and stored at −80 °C.