Hypersep c18 column

Oxytocin was pretreated to solid phase extraction using a Hyper Sep C18 column (3 mL/200 g, 608–303, Thermo Fisher Scientific, Waltham, MA, USA). Oxytocin concentrations were quantified using a DetectX^® Oxytocin Enzyme Immunoassay Kit (K048-H5, Arbor Assays LLC, Ann Arbor, MI, USA, goat anti-rabbit IgG). The standard curve ranged from 16.38 to 10,000 pg/mL, with a sensitivity and detection limit of 17.0 pg/mL and 22.9 pg/mL, respectively. The absorbance was measured at 450 nm using an iMark microplate reader (Bio-Rad, Tokyo, Japan). The intra-assay and inter-assay coefficient of variation (%CV) were 2.86% and 9.71%, respectively.

To the homogenate supernatant fractioned for quantification of LTB₄, a solution with internal deuterated standards (Cayman Chemical Co Ann Arbor, MI, USA) was added in a known concentration for the identification of LTB₄. Samples were acidified with 50 µL of HCl (1N), centrifuged and applied in HYPERSEP C18 column (Thermo Scientific, Rockwood, TN, USA) as previously described [41 (link)]. The HPLC/MS/MS (LC Shimadzu—ESI Triple TOF 5600+ SCIEX) method for identification and quantification of lipid mediators was optimized using stationary phase chromatographic column C18 Ascentis EXPRESS (Supelco—St. Louis, MO, USA) [47 (link)]. The data processing was obtained by the PeakView 2.1 and MultiQuant 3.0 programs (AB Sciex, Foster, CA, USA).

Proteins in the eluate were analyzed on a 6 to 15% SDS-PAGE gradient gel. Each lane was diced, and gel pieces were put into 1.5-mL microcentrifuge tubes. The samples were prepared by adapting standard protocols for in-gel mass spectrometry sample preparation (84 (link)). Incubation with 10 mM dithiothreitol (DTT) was carried out for 45 min at 55°C in order to reduce disulfide bonds. Afterwards, carbamidomethylation of cysteines was with 55 μM iodoacetamide for 30 min at room temperature (in the dark). Gel pieces were washed with gel wash buffer (25 mM ammonium bicarbonate and 50% acetonitrile) and dehydrated with 100% acetonitrile prior to drying in a SpeedVac vacuum centrifuge (Thermo). Trypsin digestion was carried out in 25 mM ammonium bicarbonate containing 10 μg mL⁻¹ MS-grade trypsin protease (Pierce), and the mixture was incubated overnight at 37°C. Peptides were subsequently extracted by a two-step process, which was repeated twice: incubation of gel pieces with 5% formic acid (Sigma-Aldrich), followed by 100% acetonitrile. To remove residual salts, the peptide samples were desalted using a HyperSep C₁₈ column (Thermo Fisher Scientific) according to a previously described procedure (84 (link), 85 (link)). C₁₈ column-eluted samples were dried by vacuum centrifugation and frozen prior to mass spectrometry.

Samples (5 mg) were precipitated with chilled acetone (−20 °C, overnight). After centrifugation (10 min, 4 °C, 16,000g) and removal of the detergent-containing supernatant, pellets were resuspended in 6 M Urea with 200 mM ABC. Samples were reduced with dithiothreitol (5 mM, 37 °C, 60 min) and alkylated in the dark with iodoacetamide (10 mM, 25 °C, 30 min). The resulting protein extract was first diluted to 2 M urea with 200 mM ABC for digestion with endoproteinase LysC (1:100 w:w, 37 °C, o/n, Wako, cat #129-02541) and then diluted twofold with 200 mM ABC for trypsin digestion (1:100 w:w, 37 °C, 8 h, Promega cat #V5113). After digestion, peptide mix was acidified with formic acid 0.1% and desalted with a Hypersep C18 column (Thermo Fisher P/N 60108-305). Immunoaffinity purification of K-ε-GG peptides was performed using PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit (Cell Signaling, P/N 5562S) following manufacturer’s instructions^{75 (link)}. Enriched peptides were desalted using MicroSpin C18 column (The Nest Group, Inc) prior to mass spectrometric analysis.

The reference substances of isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, quercetin, rutin, kaempferin, neochlorogenic acid, hesperidin, and cryptochlorogenic acid were precisely weighed and added to 2 ml of chromatographic methanol. The reference substances were fully shaken, and the reference substance reserve solution was obtained. The liquid phase conditions were as follows: mobile phase A: 0.5% acetic acid water; mobile phase B: acetonitrile; flow rate: 0.5 ml/min; chromatographic column: HyperSep C18 column (Five micron, 4.6 × 150 mm, Thermo Fisher Scientific, Inc.); injection volume: 10 µl; column temperature: 30°C; detection wavelength: 280 nm; and gradient conditions: 0–30 min, 12%–45% (acetonitrile); 30–35 min, 45%–100% (acetonitrile); and 35–40 min, 100% (acetonitrile). The composition of Insect tea extract was determined by using the UltiMate3000 HPLC System (Thermo Fisher Scientific, Inc.).

We dissolved ITPL in dimethyl sulfoxide to obtain a 10 mg/ml solution, which we diluted with 50% methanol to obtain a 2 mg/ml ITPL solution sample. We filtered the ITPL sample and the standard solution through a 0.22 μm membrane and assayed a sample volume of 10 μl. The chromatographic conditions included a Hypersep C18 column (4.6 mm × 150 mm, 5 μm); mobile phase A, 0.5% acetic acid water; mobile phase B, acetonitrile; flow rate, 0.5 ml/min; column temperature, 25°C; and detection wavelength, 280 nm (Ultimate 3000, Thermo Fisher Scientific) (Li et al., 2019).