Xcalibur version 2

The purified peptide samples were loaded onto Orbitrap Elite hybrid mass spectrometer (Thermo Scientific) coupled to EASY-nLC II system (Thermo Scientific) using the Xcalibur version 2.7.0 SP1 (Thermo Scientific). The MS analysis was conducted in data-dependent acquisition where one high resolution (120000) FTMS full scan (m/z 300–1700) was followed by top20 CID-MS2 scans in ion trap (energy 35). Only the precursor ions with over 500 ion counts were allowed for MSⁿ. Charge state rejection was enabled as well as dynamic exclusion which was fixed at 30 s for the selected ions.
The MS1 intensities of peptides for label-free quantification were acquired by the Progenesis LC-MS software (v 4.1, Nonlinear Dynamics Limited, Tyne, UK). For protein identification, the MS2-scan data obtained from Progenesis LC - MS were searched against the human component of the UniProtKB database using the SEQUEST search engine in Proteome Discoverer software (version 1.4, Thermo Scientific). The results were filtered to a maximum false discovery rate (FDR) of 0.05. Afterwards, spectral counts for each protein were extracted from the search results of the SEQUEST database and used to quantify protein abundance differences [20 (link)].

All crude extracts were prepared to a concentration of 1 mg/mL in methanol (MeOH, HPLC-grade). A blank solvent was also included. A silica C-18 HPLC column with the size of 70 × 3.0 mm², particle size of 5 µm and pore size of 100 °A (Hichrom Limited, Reading, UK) was used. The experiment was carried out according to an established standard operating procedure [13 (link)] using the ThermoFinnigan Exactive Orbitrap Mass Spectrometry (Thermo Fisher Scientific, GmbH, Bremen, Germany) in both positive and negative ionisation on switch mode. Please also see supplementary information for details. LC-MS data was recorded using Xcalibur version 2.2 (Thermo Fisher Scientific, GmbH Bremen, Germany). The LC-MS Xcalibur raw data from both positive and negative ionization modes were sliced using the MassConvert file converter to separate both positive and negative masses and processed using Mzmine 2 following earlier published protocol [13 (link)]. Please see Supplementary Information for details. Dereplication was done using an In-house EXCEL Macro as earlier described [13 (link)]. The EXCEL Macro file was coupled with the Dictionary of Natural Product (DNP) database for peak identification and dereplication. The data were then converted into a CSV file that was exported into SIMCA 17.0 (Umetrics, Umeå, Sweden) for multivariate analysis.

Mass spectrometer fitted with a heated electrospray ionization source (HESI) was operated in the negative ionization mode. Electrospray voltage was set at −4 kV. Sheath gas and auxiliary gas flow rates were set at 60 arbitrary units (a.u.) and 44 a.u., respectively, and the drying gas temperature was set at 275°C. The mass resolution power of the detector was 60,000 m/Δm (full width at half maximum, FWHM at m/z 400). Mass spectra were acquired over an m/z range from m/z 50 up to m/z 1000. Raw data were analyzed using the Qual-browser module of Xcalibur version 2.2 (Thermo Fisher Scientific, Courtaboeuf, France).

Discovery raw data files were converted to .cdf format using the file converter tool in Xcalibur version 2.2 (Thermo Scientific). Peak detection, integration and alignment were performed with the open-source software XCMS version 1.30.3 (Smith et al. 2006 (link)). The following XCMS settings were used: For peak detection and integration with the centWave algorithm, ppm = 10, snthr = 5, peakwidth = c(5.15), mzdiff = −0.01, prefilter = c(6,100), fitgauss = TRUE; for alignment with Obiwarp, distFunc = “cor”, profStep = 1, grouping parameters bw = 1, mzwid = 0.005, minfrac = 0.5. Isotopes and adducts were not removed.
Sample-wise median normalization (Trezzi et al. 2015 (link)) was used to correct for technical variation in e.g. sample preparation and MS signal intensity. Next, a two-step peak filtering approach was applied. First, peaks with an intensity level ≥ 60,000 were retained. Second, only peaks present in at least 75% of samples in at least one group were used for the following statistical analyses.

Filtrated extract (10 μL) was injected in an Accela Series UHPLC (ThermoFisher Scientific, Waltham, MA, USA) coupled to an Exactive mass spectrometer (Thermo FisherScientific, Waltham, MA, USA). A chromatographic C18 Accucore column (2.1 × 100 mm, 1.5 μm, ThermoFisher Scientific, Waltham, MA, USA) was used. Flow rate was set to 300 μL/min. Run time was 10 min, with the elution gradient starting at 20% MeOH (0–1 min), linearly increasing to 80% MeOH (1–6 min), remaining at 80% MeOH (6–8 min), then decreasing and staying at 20% MeOH for re-equilibration. Mass detection was performed in negative mode at a scan range 90–500 m/z. A heated electrospray ionisation source was used with spray voltage of 4 kV and capillary temperature of 275°C. Mass spectra were obtained using Xcalibur version 2.2 (ThermoFisher Scientific, Waltham, MA, USA). For JH and 20HE quantification, calibration curves were constructed with 1× weighted linear fitting for each component (0.01, 0.1, 1, and 10 μg l^-1). Recovery percentages (92–95%) were calculated from the internal standard methoprene.

Liquid chromatography/mass spectrometry analysis was conducted using a Micro-LC Dionex RLSC nano Ultimate 3000 adapted with a micro flow meter coupled with a LTQ-XL-Orbitrap XL (Thermo Fisher Scientific) mass spectrometer. Same gradient was used as per except for the addition of an acidic modifier (formic acid 0.01% (v/v)) to both mobile phases and micro LC Column Acclaim RSLC 120 C18 2.2 μm 120 Å1.0 × 50 mm (Thermo Fisher Scientific) was used at a constant flow of 45 μL/min. Ionization was electrospray (ESI) in positive ion mode with a mass range of 50–200 Da. ESI conditions were as follows: ion spray voltage: 4.6 (V), capillary temperature: 280°C, sheath gas flow: 20 (Arb) auxiliary gas flow: 8 (Arb), collision energy (for collision induced dissociation (CID) MSn) ramp mode from 15–50 (V), and mass resolution mode IT-FT: 30,000 resolution power (RP). Mass spectra were processed using Xcalibur version 2.2 (Thermo Fisher Scientific).

For direct injection, oligosaccharides were analyzed using an LTQ-Velos Pro linear ion trap mass spectrometer (Thermo Scientific, San Jose, CA, USA) connected to an Ultimate 3000RS HPLC (Dionex, Sunnyvale, CA, USA). The setup was used for direct injection without a column; the pump delivered 200 µL/min of 0.03 µM formic acid in 70% acetonitrile and data was acquired for 24 s after injection. For the MS, the capillary voltage was set to 3.5 kV and the scan range was m/z 150–2,000 using two micro scans. The automatic gain control was set to 10,000 charges and the maximum injection time was 20 ms. For fragmentation of selected precursor ions by MS/MS, the normalized collision energy was set to 37 and three micro scans were used. For PGC-MS, the same MS-parameters were used as for direct injection with the exception that the scan range was m/z 250–2,000. For HILIC-FLD-MS the instrument was operated in positive mode with an ionization voltage of 3.5 kV, auxiliary and sheath gas settings of 5 and 30 respectively (arbitrary units) and with capillary and source temperatures of 300 °C and 250 °C, respectively. The scan range was set to m/z 110–2,000 and MS/MS analysis was performed with CID fragmentation with helium as the collision gas. All data were recorded with Xcalibur version 2.2 (Thermo Scientific).