Tunemix mixture

Most of the chemicals and solvents were obtained from Sigma Aldrich (Poznan, Poland). Protected amino acids were provided by Trimen Co (Lodz, Poland), and MBHA Rink-Amide peptide resin (100–200 mesh, 0.8 mmol/g) was provided by NovaBiochem. Opioid radioligands, [³H]DAMGO, [³H]deltorphin-2, and [³H]U-69593, and human recombinant ORs and NK1 receptor came from PerkinElmer (Krakow, Poland). GF/B glass fiber strips were purchased from Whatman (Brentford, UK). Analytical and semi-preparative RP HPLC was performed using a Waters Breeze instrument (Milford, MA, USA) with a dual absorbance detector (Waters 2487). The ESI-MS experiments were performed on a Bruker FTICR (Fourier transform ion cyclotron resonance) Apex-Qe Ultra 7 T mass spectrometer equipped with a standard ESI source. The instrument was operated in the positive ion mode and calibrated with the Tunemix™ mixture (Agilent Technologies, CA, USA). The structures of peptides were confirmed using a Shimadzu LCMS-IT-TOF (ion trap–time-of-flight) hybrid mass spectrometer with auto-tuning in the positive ion mode.

All ESI-MS experiments were performed on a micrOTOF-Q mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a standard ESI source. The instruments were operated in the positive-ion mode and calibrated with the Tunemix™ mixture (Agilent Technologies, Palo Alto, CA, USA). The mass accuracy was better than 5 ppm. Analyte solutions (70 μL) were introduced at a flow rate of 3 μL/min. The instrument parameters were as follows: for micrOTOF-Q MS: scan range: 50–3000 m/z; drying gas: nitrogen; flow rate: 4.0 L/min, temperature: 200 °C; potential between the spray needle and the orifice: 4.2 kV.

In a final volume of 150 μl of 30 mM Tris–HCl (pH 8.0), 200 mM NaCl buffer, 150 μM of prereduced STR18, STR18 C47S, and STR18 C89S were incubated 30 min in the presence of 300 μM l-cysteine, 2 μM ABA3, and 5 μM PLP at 25 °C. After extensive dialysis, samples were split in two parts and treated or not with 1 mM DTT. Mass spectrometry analysis of these samples was performed using a Bruker microTOF-Q spectrometer (Bruker Daltonik), equipped with Apollo II electrospray ionization source with ion funnel, operated in the negative ion mode. The concentrated samples in formic acid were injected at a flow rate of 10 to 20 μl min⁻¹. The potential between the spray needle and the orifice was set to 4.5 kV. Before each run, the instrument was calibrated externally with the Tunemix mixture (Agilent Technologies) in quadratic regression mode. Data were analyzed with the DataAnalysis software (Bruker).

ESI-MS experiments were performed on a micrOTOF-Q mass spectrometer (Bruker Daltonics, Bremen, Germany) with a standard ESI source. The instruments parameters were as follows: Positive-ion mode, calibration with the Tunemix™ mixture (Agilent Technologies, Palo Alto, CA, USA), mass accuracy was better than 5 ppm, scan range: 50–1600 m/z; drying gas: Nitrogen; flow rate: 4.0 L/min, temperature: 200 °C; potential between the spray needle and the orifice: 4.2 kV analyte (flow rate: 3 μL/min.

The CID experiments were performed using an Apex-Qe 7T Bruker instrument (Bremen, Germany) equipped with a standard ESI source. Spectra were recorded using aqueous solutions of acetonitrile (50%) and formic acid (0.1%), at the peptide concentration of 5 μM. The instrument’s parameters were as follows: Positive-ion mode, calibration with the Tunemix™ mixture (Agilent Technologies, Palo Alto, CA, USA), mass accuracy was better than 5 ppm, scan range: 50–1600 m/z; drying gas: Nitrogen; flow rate: 4.0 L/min, temperature: 200 °C; potential between the spray needle and the orifice: 4.5 kV, analyte was introduced to the ESI source by KDScientific pump (Holliston, MA, USA) with a flow rate: 3 µL/min).
In the MS/MS mode, the quadrupole was used to select the precursor ions, which were fragmented in the hexapole collision cell applying argon as the collision gas. The obtained fragments were subsequently analyzed by the ICR mass analyzer. For the MS/MS measurements, the voltage over the hexapole collision cell varied from 15 to 40 V.

All ESI-MS experiments were performed on a compact mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a standard ESI source. The instruments were operated in the positive-ion mode and calibrated with the Tunemix mixture (Agilent Technologies, Palo Alto, CA, USA) in a quadratic method. Spectra were recorded for samples dissolved in H₂O with an M(II):L molar ratio of 1:0.5, 1:1.1, and 1:2. pH was adjusted by adding NaOH or HCOOH and checked by a Metrohm 913 pH-meter (Herisau, Switzerland). Analyte solutions (150 μL) were introduced at a flow rate of 180 μL/h. The instrument parameters were as follows: scan range: 50–3000 m/z, drying gas: nitrogen, flow rate: 4.0 L/min, temperature: 200 °C, and potential between the spray needle and the orifice: 4.0 kV.
For MS spectra analysis, Bruker Compass Data Analysis 4.2 software was used. A sophisticated numerical annotation procedure (SNAP) algorithm was used for finding peaks. The relative intensities (Tables S1–S4, Supplementary Materials) were read directly from the MS spectra in the Data Analysis 4.2 program. Figures S1–S9 contain raw MS spectra with intensities on the y-axis and isotopic pattern enlargements for the main species present.