Esi it tof

For mass spectrometric de novo peptide sequencing the samples were directly injected on the ESI-IT-TOF (Shimadzu Co., Japan), at 0.05 mL/min constant flow rate, in positive mode, for MS, MS² and MS³ analyses. The interface voltage was kept at 4.5 kV, the detector voltage at 1.8 kV and the capillary temperature at 200 °C. Data were collected at a range of 50-1800 m/z. For fragmentation, the precursor ions were selected under a 0.5 m/z window, and the argon collision energy was kept at 50%. The instrument control and data acquisition were performed with the LC-MS Solutions software (Shimadzu Co., Japan).

Liposomes were obtained and analyzed as described by Sciani et al. [33 (link)]. Briefly, liposomes (10 µL) and
bufotenine (10 µg/10 µL) were separately analyzed for retention time
determination. Next, bufotenine and liposomes were incubated (20 µL, 1:1 V/V)
for 10 minutes at room temperature, and the mixture was analyzed under the same
conditions. The liposome peak was collected and lysed with 50% acetonitrile
containing 0.1% formic acid. This solution was then sonicated and centrifuged
for 10 minutes at 10840 x g at 4°C. The supernatant was analyzed by
electrospray-ion trap-time of flight (ESI-IT-TOF) (Shimadzu Co., Japan) equipped
with binary ultra-fast liquid chromatography system (UFLC) (20A Prominence,
Shimadzu Co., Japan), in order to identify bufotenine inside the liposomes.

In preliminary experiments, estimated different enzymatic hydrolysis systems were estimated: hydrolysis of 3% T20, T40, and T70 for 1 h (3 U/ml), hydrolysis of 3% T20 for 30 min, 1 and 3 h (3 U/ml), and 0.5% T20 hydrolyzed by different concentrations of dextranase (0.5, 1, and 5 U/ml) for half an hour. All the above reactions were implemented at 55°C and pH 7.5. Finally, hydrolysates were filtered by a 0.22-μm membrane and subjected to HPLC and LC-MS. The products were detected and analyzed by Waters Sugar-Pak1 (300 mm × 7.8 mm; Milford, United States) HPLC with a differential refraction detector. Methods of HPLC were based on previous experiments (Ren et al., 2018 (link)). In order to further identify the hydrolysates, LC-MS was performed. The liquid chromatography was Agilent 1100 Series LC Chem Station and analyzed by Zorbax SB-C₁₈ (5 μm, 0.5 mm × 250 mm, Agilent Technologies, United States). The mobile phase was 15 mM PTA acetonitrile solution (70%, pH 7.0) at 10 μl/min, the column temperature was adjusted to 35°C, and the injection volume was 2 µl. ESI-IT-TOF (SHIMADZU, JAPAN) high-resolution tandem resolution mass spectrometry was used for detection under the following conditions: in positive ion mode, the spray voltage and current were 3.6 kV, and 0.5 L/min, respectively; the ion source temperature was 200°C, the detection voltage was 1.8 kV.

The conditions for high-performance liquid chromatography were consistent with the aforementioned Section 2.5. The analysis protocol was carried out on a Shimadzu LC-MS instrument (ESI-IT-TOF) with scanning performed in both positive and negative ion modes. The mass spectrometry scanning range was set between 90 and 1000 m/z. Other MS parameters, modified according to the method outlined by Wang et al. [21 (link)], included ESI as the ion source, with detector and interface voltages set at 1.57 and 4.5 kV, respectively. The capillary discharge line (CDL) and heating block temperatures were maintained at 200 °C. Nitrogen gas, at a flow rate of 1.5 L/min, was utilized as the nebulizing gas, and the mobile phase consisted of methanol and water with a flow rate of 0.2 mL/min.

Immobilization of protein in a microcolumn, and affinity tests, were performed as described previously by Spodzieja et al. [55 (link)]. The IM peptide was added onto an albumin-Sepharose column equilibrated in ammonium hydrogen carbonate, pH 7.4, and incubated for 2 h at 25 °C with gentle shaking. After 2 h, the column was washed with 100 mL of ammonium hydrogen carbonate, collecting the first and last milliliter of the solution (supernatant and last wash fraction). The albumin-peptide complex was dissociated by incubation for 15 min in 0.1% TFA, with gentle shaking. The procedure was repeated twice, and the elution fraction was collected each time. All fractions were lyophilized and analyzed by mass spectrometry (ESI-IT-TOF, Kyoto, Shimadzu Japan).

Liquid chromatography coupled to mass spectrometry analysis was used and performed in a mass spectrometer ESI-IT-TOF (20A Prominence, Shimadzu Co., Japan). Samples (RP-HPLC sample, 3 ug) were diluted in 0.1% acid formic and loaded in the spectrometer at a flow of 0.2 mL min⁻¹ at 40 °C, and an isocratic profile of 50% acetronile solvent (B) with 50% water acid at 0.1% acid formic as solvent (A), spectra were collected in the range of m/z: 200 to 1950^{43 (link)}. The raw data (mzxml) was uploaded to Peaks Studio 8.5 (Bioinformatics Solution Inc., Waterloo, Canada) and processed with new peptide sequencing and Peaks DB. The MS/MS spectra were searched in a custom database GEWZ00000000.1 compiled from the UniProt database.