Proton NMR spectra of all compounds were recorded with a JEOL ECA 500 II (500 MHz) spectrometer. The exact mass of all compounds was obtained using a Triple TOFTM 5600 System (AB SCIEX). All data are listed in the Supplementary Data Sheet 1 .
Triple toftm 5600 system
Manufactured by AB Sciex
Sourced in Canada, United States
The Triple TOFTM 5600+ system is a high-resolution mass spectrometer designed for advanced analytical applications. It features a triple quadrupole configuration and a time-of-flight (TOF) analyzer, providing accurate mass measurement and high-throughput capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Eca 500ii, by JEOL (1 mentions)
Waters e2695, by Waters Corporation (1 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (1 mentions)
Duo spray source, by AB Sciex (1 mentions)
Circular dichroism, by Applied Photophysics (1 mentions)
Avance 3 hd 400 mhz digital nmr spectrometer, by Bruker (1 mentions)
Duo spray tm, by AB Sciex (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
7 protocols using triple toftm 5600 system
1
Proton NMR and Mass Spectrometry
2
Quantification and Identification of ZEN Degradation
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The amount of ZEN was determined by high-performance liquid chromatography (HPLC, Waters e2695, Waters Corp., Milford, MA, USA) with an ultraviolet (UV) detector at 236 nm. An Eclipse XDB-C18 column (ODS, 4.6 mm × 250 mm, Zorbax) was used for chromatographic separation, and the elution was water-methanol (20:80, v/v) at 0.8 mL/min. The ZEN was eluted at 6.5 ± 0.2 min. The quantification of ZEN was accomplished by the external standard method and all the experimental data were presented as the means ± SD of three replicates. The ZEN degradation rate was calculated according to the following formula: De = (1 − Ct/Co) × 100%
De: Biodegradation rate of ZEN; Co and Ct: ZEN concentrations in control groups and experimental treatments, respectively.
To identify the ZEN degradation products, strain HQ was incubated in 10 mL MSM containing 15 μg/mL ZEN, and samples were collected at regular intervals. For mass spectral analysis, the degradation products were lyophilized and dissolved in 2 mL of acetonitrile before being filtered through a 0.22 mm filter. The LC-TOF-MS/MS analysis was operated with an AB SCIEX Triple-TOFTM 5600 system (Ontario, Canada). TOF-MS/MS scan was acquired with a collision energy (CE) of 40 eV over the mass range of 100–1000 Da.
De: Biodegradation rate of ZEN; Co and Ct: ZEN concentrations in control groups and experimental treatments, respectively.
To identify the ZEN degradation products, strain HQ was incubated in 10 mL MSM containing 15 μg/mL ZEN, and samples were collected at regular intervals. For mass spectral analysis, the degradation products were lyophilized and dissolved in 2 mL of acetonitrile before being filtered through a 0.22 mm filter. The LC-TOF-MS/MS analysis was operated with an AB SCIEX Triple-TOFTM 5600 system (Ontario, Canada). TOF-MS/MS scan was acquired with a collision energy (CE) of 40 eV over the mass range of 100–1000 Da.
3
Semisynthesis of Insulin Analogs
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Previously described insulin analog [d -HisB24]-insulin (29 (link)) and five newly prepared analogs ([d -HisB24]-DTI-NH2, [TyrB25, PheB26, AsnB27, LysB28, ProB29]-insulin, [TyrB25, PheB26, AsnB27, LysB28, ProB29, GlyB31, TyrB32]-insulin, [GlyB31, TyrB32]-insulin, and [d -HisB24, GlyB31, TyrB32]-insulin), together with des-PheB24-insulin (37 (link)), were prepared by enzymatic semisynthesis of des-(B23-B30)-octapeptide insulin and corresponding tetra-, hepta-, octa-, and decapeptides. All semisynthetic procedures were described in detail previously (22 (link)), except that we used Fmoc-Lys(Pac)-OH, which was prepared by a modification (see supporting information ) of a method described previously (43 (link)). The identities of peptides and insulin analogs were confirmed, using mass spectrometer LTQ-orbitrap XL (Thermo Fisher) or the TripleTOFTM 5600 system (AB SCIEX), and their purities (≥95%) were controlled by analytical HPLC. A schematic presentation of the analogs is shown in Fig. 3 .
4
Identification of Pulsatilla Saponins Constituents
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To identify the constituents of Pulsatilla Saponins, the extract was screened on a Triple TOFTM 5600+ system with a Duo Spray source (AB SCIEX, Foster City, CA, USA). Briefly, the parameters’ negative ion mode was set as follows: ion spray voltage, −4500 V; ion source temperature, 550 °C; curtain gas, 30 psi; nebulizer gas (GS1), 50 psi; heater gas (GS2), 50 psi; and decluster potential (DP), −100 V. The mass ranges of TOF-MS and TOF-MS/MS experiments were all set at 50–1250 Da. In the TOF-MS/MS experiment, the most intensive eight ions from each TOF-MS scan were selected as MS/MS fragmentation. The collision energy (CE) was set at −35 eV, and the collision energy spread (CES) was (±) 15eV for the UHPLC-QTOF-MS/MS detection. The mobile phase was composed of water containing 0.1% formic acid (solvent A, v/v) and acetonitrile (solvent B) at a flow rate of 0.3 mL/min. A 15 min binary gradient elution as follows was performed for the separation: 0–3 min, 1% → 67% solvent B; 3–9 min, 67% solvent B; 9–12 min, 67% → 90% solvent B; 12.01–15 min, 1% solvent B; 12.01–15.0 min, followed by 3 min of column re-equilibration. In total, ten compounds were identified. The TIC profiles of those compounds are revealed in Supplementary Figure S1 . Detailed information on the chemical compounds is shown in Supplementary Table S1 .
5
Mass Spectrometry for Metabolite Analysis
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Mass spectrometric analysis was performed using a Triple TOFTM 5600+ system (AB SCIEX, Concord, ON, Canada) equipped with a Duo-Spray source operated using the positive-ion (ESI+) and negative-ion (ESI−) modes to detect many ions. The ion spray voltage capillary was 4500 eV with declustering potential voltages of 80 V in positive and −80 V in negative modes. The source temperature was set at 600 °C, the curtain gas was 25 psi, and gas 1 and 2 were 40 psi. The collision energies were 35 V and −35 V for positive and negative modes, respectively, with CE spreading 20 V. Sequential windowed acquisition of all theoretical MS (SWATH) methods was utilized to capture all metabolites within the samples [78 (link),79 (link)]. The SWATH Acquisition method consisted of a single TOF scan from 50 to 1100 Da accumulated in 30 ms and a fragment ion TOF scan from 50 to 1100 Da using fixed 50 Da transition windows [78 (link)]. Batches for MS and MS/MS data collection were created using Analyst TF (v 1.7.1).
6
Structural Elucidation of Novel Gorgonian Metabolites
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Optical rotation data were measured on a MCP 200 dig-ital polarimeter (Anton Paar, Austria). UV and Circular Dichroism spectra were measured on a chirascan Circular Dichroism (Applied Photophysics, UK). HR-ESI-MS data were performed on a Triple TOFTM 5600 ＋ system (AB SCIEX, USA). NMR spectra were recorded on a Bruker avance III HD 400 MHz digital NMR spectrometer (Bruker Optics, Karlsruhe, Germany) with TMS as an internal standard. Column chromatography (CC) was performed with 200～300 mesh silica gel (Qingdao Marine Chemical Factory, China). TLC was performed on silica gel GF254 (Qingdao Marine Chemical Factory, China), spots were visualized under Ultraviolet transmission reflectometer (Shanghai Jingke Industrial, Shanghai). The preparative HPLC was performed on a Semi-preparative HPLC (QuikSep, Beijing, China) with Kromasil 100-5 C 18 column (10 mm×250 mm, 5 μm, Sweden). Subergorgic acid and 2-deoxy-2β-hydroxysubergorgic acid were used to establish GNPS, and their structures were determined by NMR and MS. 2-Deoxy-2β-hydroxysubergorgic acid was also from gorgonian Leptogorgia rigida. [13] The strains in this work were cultured in eight liquid media, eleven bioconversion and four rice media. All of culture media compositions are available in the Supporting Information (Tables S1～S3).
7
Nano-HPLC-MS/MS Peptide Separation
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Peptides were chromatographically separated using an Ultimate 3000 nano-high performance liquid chromatography (HPLC) system (LC Packings, DIONEX, Thermo Fisher Scientific, Sunnyvale, CA, USA). Samples were pre-concentrated in a pre-column cartridge (PepMap-100 C18 5 mm 100 A, 30 mm id  5 mm, LC Packings) by the loading pump. Chromatographic separation of peptides was performed using a C18 PepMap-100 column (15 cm  75 mm id, LC Packings) equilibrated at 30 8C with a solvent A (water/acetonitrile 98/2 (vol/vol), 0.1% formic acid) at a flow rate of 300 nL min À1 . Runs were performed under 60 min linear gradient from 10% to 45% of solvent B (water/acetonitrile 2/ 98 (vol/vol), 0.1% formic acid) followed by 10 min of a purge step and 20 min re-equilibration step. The HPLC system was directly coupled to TripleTOF TM 5600 System (AB SCIEX, Toronto, Canada), equipped with a DuoSpray TM ion source (AB SCIEX). Eluted peptides were directly processed through the mass spectrometer, controlled by Analyst 1 1.6.1 software (AB SCIEX). For information dependent acquisition (IDA) analysis, survey scans were acquired in 250 ms and 25 product ion scans exceeding a threshold of 125 counts per second (counts/s) were collected. Dynamic exclusion was set for 1/2 of peak width ($8 s), and then the precursor was refreshed off the exclusion list.
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