Tripletof 5600 mass spectrometer

The mass spectroscopy analysis was performed using a AB SCIEX TripleTOF™ 5600 mass spectrometer (AB SCIEX, Framingham, MA, USA), coupled with online micro flow HPLC system (Shimadzu Co, Kyoto, Japan) as described previously. Genome sequence and annotation information of Synechocystis sp. PCC 6803 were downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes). The details for the experimental design, execution, and proteomic data analysis can be found in the original publications (Liu et al., 2012 (link); Qiao et al., 2012 (link), 2013 (link); Huang et al., 2013 (link); Tian et al., 2013 (link)).

Mouse primary preadipocytes were differentiated for 2 days. Cell lysate was incubated with anti-Senp7 beads or mouse IgG individually. Then proteins were eluted and collected followed by SDS-PAGE. Proteins were digested into peptides by trypsin. Tryptic peptides were desalinated and then subjected to MS analysis. Mass spectral analysis was performed on the AB Sciex Triple TOF 5600+ mass spectrometer (AB Sciex) with an electrospray ionization probe operated in positive ion mode. The raw data were processed using AB SCIEX ProteinPlot software (version 4.5, https://sciex.com/products/software/proteinpilot-software) in its standard mode. Data were searched against the February 2018 UniProt mouse database (61,314 entries). Peptides with confidence >95% were considered for further analysis. Three trypsin missed cleavages were considered. The minimum length of peptides was seven amino acids. Identified proteins in IgG control sample were excluded from protein result of anti-Senp7 pulldown sample. The analysis results were listed in Table S1.

The extract of ZWT was used for UPLC-Q-TOF-MS analysis according to our previous study (Liang et al., 2019 (link)). The chromatographic separation was achieved by Shimadzu UPLC-30AD (Shimadzu Corporation, Kyoto, Japan) equipped with a Phenomenex Genmini 3u-C18-110 column (150 × 2 mm, 3 μm). The mobile phases included 0.025% formic acid in water (A) and acetonitrile (B) at an ambient temperature of 35°C. The linear gradient elution is as follows: 0–10 min, 5% B; 10–12 min, 8% B; 12–15 min, 20% B; 15–30 min, 35% B; 30–35 min, 45% B; 35–45 min, 95% B. The flow rate was set at 0.3 ml/min. The sample injection volume was 5 μl. Mass spectrometry was measured by the ABsciex Triple TOF 5600 mass spectrometer (ABsciex, Framingham, MA, USA), and the data were analyzed using Peakview software (Version 2.0, ABsciex). The mass parameters were as follows: ion source in electrospray mode: negative; electrospray ionization: 55 psi; IonSpray Voltage Floating: 5,500 V; declustering potential: 100 V; collision energy: 45 eV.

All samples were analyzed using the HPLC-MS/MS system following the manufacturer's instructions. First, all chromatographic separations were performed using an HPLC system. The sample was collected on a Waters BEH (100 ∗ 2.1 mm, 1.7 um column). The column oven was maintained at 40°C, and the flow rate was 0.4 ml/min. In the positive ion mode, the mobile phase consisted of solvent A (water + 0.1% formic acid) and solvent B (organic phase acetonitrile). In the negative ion mode, the mobile phase consisted of solvent A (0.1% formic acid +5 mM ammonium acetate) and solvent B (organic phase acetonitrile). The specific chromatographic conditions are listed in Table 2.
An AB SCIEX Triple TOF 5600 + mass spectrometer was used to collect data in the positive and negative ion modes separately. The scanning method was a classic data-dependent scanning (IDA), and one primary mass spectrometry scan (100 ms) triggers 10 secondary mass spectrometry scans (500 ms). Dynamic background deduction (DBS) was turned on, first-level scanning range: 50 m/z–1000 m/z; secondary scanning range: 50 m/z–1000 m/z; air curtain gas: 35 psi; atomizing gas: 55 psi; auxiliary atomizing gas: 55 psi; ion source temperature: 550°C; decluster voltage: 80 V; and collision voltage: 35 ± 15 V.

The optical rotations were detected at 20°C using MCP 5100 digital polarimeter (Anton Paar, Graz, Austria). The UV data were recorded on a Shimadzu UV-2500 spectrophotometer (Shimadzu, Kyoto, Japan). The Chirascan CD spectrometer (Applied Photophysics Ltd., Surrey, UK) was used to acquire ECD spectra. 1D and 2D NMR spectra were recorded on a Bruker AVANCE III 500 M NMR spectrometer (Bruker BioSpin Corporation, Billerica, USA). UPLC-Q-TOF/MS analysis was carried out on a Waters ACQUITY UPLC system (Waters Corporation, Milford, USA) equipped with an AB SCIEX Triple TOF 5600 mass spectrometer with electrospray ionization source (ESI; Framingham, MA, USA). Silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), RP-C₁₈ silica gel (Merck KGaA, Darmstadt, Germany), and Sephadex LH-20 gel (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were employed for column chromatography (CC). High-performance liquid chromatographies (HPLCs) were performed on Agilent 1260 series (Agilent Technologies, Santa Clara, USA) with C₁₈ reversed-phase columns (YMC, Kyoto, Japan; 250 × 4.6 mm i.d., 5 μm, for analysis; 250 × 10 mm i.d., 5 μm, for separation).

The extraction, separation, concentration, and validation of IBU from experiment samples were analyzed according to Picó et al. [26 (link)] and Andreotti et al. [64 (link)] using ultra-high performance liquid chromatography (Agilent 1260 Infinity, Waldbronn, Germany) and an AB SCIEX TripleTOF™ 5600 mass spectrometer (AB SCIEX, Foster City, CA, USA). Data acquisition processing and instrument control were performed using Analyst, Peak View 1.0, and MultiQuant 2.0. software [26 (link)].
The bioconcentration factor (BCF), bioaccumulation factor (BAF), and translocation factor (TF) of IBU in V. unguiculata plants were calculated according to Wang [65 ]: $BCF\left(\mathrm{L}/\mathrm{K}\mathrm{g}\right)=\frac{IBUconcentrationinmedia\left(\mathrm{m}\mathrm{g}/\mathrm{L}\right)}{IBUconcentrationinplanttissue\left(\mathrm{m}\mathrm{g}/\mathrm{K}\mathrm{g}\right)},$
$BCF\left(\mathrm{L}/\mathrm{K}\mathrm{g}\right)=\frac{IBUconcentrationinmedia\left(\mathrm{m}\mathrm{g}/\mathrm{L}\right)}{IBUconcentrationinwholeplanttissue\left(\mathrm{m}\mathrm{g}/\mathrm{K}\mathrm{g}\right)},$
$TF=\frac{IBUconcentrationinshoot\left(\mathrm{m}\mathrm{g}/\mathrm{K}\mathrm{g}\right)}{IBUconcentrationinroot\left(\mathrm{m}\mathrm{g}/\mathrm{K}\mathrm{g}\right)}.$