Captivespray ion source

Samples were re-suspended in 10 μl of 0.1% TFA and a 5 μl aliquot was injected onto an Acclaim C₁₈ PepMap trapping column (300 μm I.D. ×5 mm) (Thermo Fisher Scientific, Hemel Hempstead, UK). An 8 min wash step at 30 μl min⁻¹ using 0.1% TFA was used for online desalting or a 1 min loading/wash step for offline desalted samples. Peptide separations were performed using Buffer A: 0.1% formic acid (FA) 3% ACN. Buffer B: 0.1% FA 97% ACN. Linear gradient 3–25% B over 28 min, 25–50% B over 10 min and 50–90 over 0.9 min. Linear gradient 3–25% buffer B over 42 min, 25–50% buffer B over 10 min and 50–90 over 5 min using a 150 mm × 75 μm I.D. PepMap C₁₈ column (Thermo Fisher Scientific, Hemel Hempstead, UK) online with a maXis mass spectrometer (Bruker Daltonics, Bremen, Germany) in conjunction with CaptiveSpray ion source (Bruker Daltonics, Bremen, Germany). CaptiveSpray capillary 1400 V, dry gas 3.0 l/min, dry heater 150 °C. MS and MS/MS scans (m/z 100–2000) were acquired in positive ion mode, no active exclusion, peptide charge state selected +2 + 3 + 4, collision energy 10.0 eV, collision cell RF 1200, ion cooler pre storage pulse 5.0 μs, summation factors for MS/MS 1.6, 0.3 s, isolation widths 3–6 m/z, resolution MS and MS/MS 40,000 (m/z 1222). Lock mass calibration was performed using HP 1221.990364.

The protein hydrolysates were loaded on an Acclaim PepMap 5 mm Trap Cartridge (Thermo Fisher Scientific) and separated on a Bruker FORTY separation column (C18 ReproSil AQ, 40 cm × 75 µm, 1.9 µm, 120 A; Bruker Daltonics, Bremen, Germany) using a nanoElute UHPLC chromatography system (Bruker Daltonics) coupled on-line to a TimsToF Pro quadrupole time-of-flight mass spectrometer (QqTOF-MS) via a CaptiveSpray ion source (Bruker Daltonics). The details of the chromatographic separation method are summarized in Supplementary Information S1 (Table S1-11). The UHPLC-QqTOF-MS/MS analysis relied on data-dependent acquisition experiments performed in the positive ion mode, comprising a survey TOF-MS scans and dependent MS/MS scans for the most abundant signals in the following 3 s (at certain t_R) with charge states ranging from 2 to 5. The mass spectrometer settings are summarized in Table S1-12.

Proteomic analysis was conducted using the rhodopsin-containing 25-kDa protein bands resolved by SDS-PAGE. These were excised from Coomassie-stained gels, washed, and tryptically digested as described by Wöhlbrand et al. (2016 (link)). Generated peptides were separated via nanoLC (UltiMate 3000 RSLCnano; Thermo Fisher Scientific, Germering, Germany) and analyzed by online coupling to electrospray ionization (CaptiveSpray ion source; Bruker Daltonik GmbH, Bremen, Germany) and mass analysis by a 3D ion trap (amaZon speed ETD; Bruker Daltonik GmbH) operated as described (Wöhlbrand et al. 2016 (link)). Protein identification was performed via the ProteinScape platform (version 3.1, Bruker Daltonik GmbH) on a Mascot server (version 2.3; Matrix Science, London, UK) against available rhodopsin sequences of O. marina also including translated EST data (obtained from NCBI, February 2016) and a target-decoy strategy applying described parameters (Wöhlbrand et al. 2016 (link)).

Steroid affinity-purified CBG (6 µg) was digested (100:1 w/w) with trypsin (Promega) for 16 h in ammonium bicarbonate buffer, pH 8. As a positive control, purified CBG was incubated with human NE (10 min, 37°C) prior to trypsin digestion. Tryptic peptides were desalted using C18 stage tips (Supelco) and analyzed by liquid chromatography using an Easy nLC-1000 (Thermo Fisher Scientific) coupled to an Impact II QTOF tandem mass spectrometer equipped with a CaptiveSpray ion source (Bruker Daltonics). Desalted peptides (1 µg) were loaded onto an in-house packed column with 1.9 µm C18 ReproSil-Pur (Dr Maisch GmbH) and resolved using a linear gradient of 5–24% buffer B (100% acetonitrile) over 60 min. For peptide identification, a top 17 method was used, with the normalized fragmentation energy at 27%. Survey and fragment spectra were analyzed with Byonic v 2.16.11 (Protein Metrics).

The purified proteins were identified using a nanoflow UPLC system (UltiMate 3,000 RSLCnano system, Dionex, Netherlands) coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer (maXis impact, Bruker, Germany). The peptides were separated using an RP C18 capillary column (25 cm × 75 µm id) at a 300 nL/min flow rate, and eluted with a linear ACN gradient from 10 to 50% ACN in 0.1% formic acid for 90 min. The eluted peptides from the capillary column were sprayed into the MS using a captive spray ion source (Bruker, Germany). Data acquisition from Q-TOF was performed using Data Analysis software (version 4.1, Bruker, Germany). Proteins were identified by the nanoLC-MS/MS spectra (Table 1) by searching the SwissProt database using the MASCOT search algorithm (version 2.3.02).