Vanquish flex hplc system

Quantification of carotenoids was carried out on a Vanquish Flex HPLC system (Thermo Fisher Scientific, Waltham, MA, USA). The same column, flow rate, eluents, and gradients as for HPLC-MS measurements were used. However, detection was done by DAD at 450 nm. Pure astaxanthin (Sigma Aldrich, St. Lewis, MO, USA) was dissolved in acetone and used as external standard. All carotenoids were quantified in their absorption in reference to astaxanthin at 450 nm and expressed in μmol·g⁻¹ lyophilized biomass. Device operation and data handling was done with Chromeleon 7.2.8 (Thermo Fisher Scientific, Waltham, MA, USA).
Chlorophyll a and b were quantified as described by [22 (link)]. One milliliter of acetone extract was diluted with 0.25 mL of 2.5 mM NaH₂PO₄ buffer at pH = 7.8 to prepare samples in aqueous 80% acetone. Subsequently, absorption of these extracts at 647, 664, and 750 nm was recorded on a Nanodrop One photometer (Thermo Fisher Scientific, Waltham, MA, USA). Chlorophyll a and b concentrations were calculated by Formulas 7 and 8 in [22 (link)].

The HPLC-MS or direct MS analyses of peptide samples were conducted using the Vanquish Flex HPLC system coupled with Exactive Plus EMR MS (Thermo). The LC separation employed a mobile phase A of 0.1% formic acid (FA) aqueous solution and a mobile phase B of 0.1% FA–acetonitrile solution. Peptide samples re-dissolved into 0.1% FA (v/v) aqueous solution at a concentration of 0.1 μg μL⁻¹ were processed on a capillary HPLC column system, comprising a trap column (30 mm length × 150 μm i.d., C₁₈, 5 μm particle size, 120 Å pore diameter) and an analysis column (150 mm length × 75 μm i.d., C₁₈, 5 μm particle size, 120 Å pore diameter) using a constant flow rate of 0.1 μL min⁻¹. Samples were automatically injected (10 μL) with 5% buffer B, separation gradient increased from 5 to 35% buffer B in 10 min, flushed with 80% buffer B for 5 min, and then changed to 5% buffer B with 2 min.
A Full MS scan was conducted within the m/z range of 400–1000, employing a mass resolution of 70 000 (Exactive Plus EMR). The parameters for Exactive Plus EMR were configured as follows: ion transfer capillary at 275 °C, electrospray voltage set to 1.8 kV, and a full MS scan covering 400 to 1000 m/z with a resolution of 70 000. All MS data were acquired using the Exactive Plus EMR instrument, and subsequent data processing was carried out with an Xcalibur 2.2 (Thermo Scientific).