Agilent 1100 hplc system

Polypeptide CBP (DEDEQIPSHPPR), in which a His residue is replaced by Leu (CBP-H), was synthesized by Nanjing Peptide Industry Biotechnology (Nanjing, China). High-performance liquid chromatography (HPLC) analysis was carried out to determine the purity (98%) of CBP-H, and ESI mass spectrometry was used to confirm the peptide structure. An Agilent MALDI-TOF-MS/MS spectrometer was used to identify the eluted fractions individually (4800, SCIE). An Agilent 1100 HPLC system, electrospray ionization (ESI) interface, and an Agilent 1100 HPLC system were installed, together with the spectrometer. An automatic sampler was used to inject the sample into a C18 trap column (C18, 3 μm, 0.1020 mm), followed by injection into a C18 column (C18, 1.9 μm, 0.15120 mm). Elution was conducted with the aid of solution A (0.1% formic acid in H₂O) and solution B (0.1% formic acid in acetonitrile) using a gradient of 5–95% B. The flow rate was set at 600 mL/min for 78 min. The peptide sequences were detected using a Q-Exactive mass spectrometer after the separation.

For the analysis of nikkomycin, culture broths were centrifuged and the supernatants were filtered through a millipore membrane (pore diameter 0.22 μm). HPLC analysis was performed with Agilent 1100 HPLC system and ZORBAX SB C-18 (5 μm, 4.6 × 250 mm). Chemical reagent, mobile phase and gradient elution process were as described previously [22 (link)]. The elution was detected with photodiode array at 260 and 290 nm for nikkomycins. Bioassays against C. albicans and A. longipes were carried out by a disk diffusion method as described previously [5 (link)]. The procedure is essentially the same for both indicator strains except that an overnight culture of C. albicans was used while 5 day-old culture was used for A. longipes. The modified potato dextrose agar medium (20% potatoes, 2% glucose and 0.8% agar) was heated to dissolve and then cooled to 50°C before use. Cultures of indicator strains (50 μl for C. albicans and 10 ml for A. longipes) were well dispersed in 100 ml pre-dissolved medium and poured to a 15 cm plate. Oxford cups were placed onto the plates and antibiotics were then added into the Oxford cups. After incubation for 12–24 h at 28°C, the zone of inhibition was assessed.

The carotenoid extraction was performed in two steps. First, the concentrated LH2 complex sample was dissolved in a mixture of spectroscopic grade methanol and acetone (Sigma) in 1:1 volume ratio. The extract was spun down using bench microcentrifuge and the supernatant was collected. In the second step the remaining pellet was dissolved in tetrahydrofuran (THF) (Sigma) and centrifuged again. The supernatant was collected and mixed with the previous one; this step was repeated until the supernatant became colourless. The final extract was dried under stream of nitrogen, dissolved in HPLC grade acetonitrile (ACN):THF (8:2, v:v) (Sigma) mixture and injected into an Agilent 1100 HPLC system employing a reverse phase Zorbax Eclipse XDB-C18 column (250 mm × 4.6 mm). The HPLC protocol was programmed for a step gradient mobile phase from 100% ACN to (70:30, v:v) ACN:THF (Sigma) within 30 min as follows: (0–5 min, 100–95% ACN, 5–10 min, 96–90% ACN, 10–15 min 90–80% ACN, 15–20 min, 80–75% ACN, 20–30 min, 75–70% ACN). Carotenoids were detected using a 1024-element diode array, with a 190–950 nm wavelength range and a wavelength accuracy of ± 1 nm, self-calibrated with deuterium lines and verified with a holmium oxide filter.