Sample preparation for LCMS

Safety measures

• The work with liquid nitrogen should be conducted with thick gloves and

protection glasses

Equipment

• Thick gloves
• Protection glasses
• Ice bucket
• Liquid nitrogen (in special canister)
• Cooling centrifuge
• filters 0.22 ul

Materials

• Methanol (LCMS grade)
• Formic acid
• LCMS grade water

Protocol

Extraction buffer

• To prepare working stock for 50 ml of extraction buffer, add 40 ml of HPLC grade methanol, 0.05 ml formic acid and 9.95 ml water DDW or LC-MS grade water.

Sample extraction

1. 10 BSF larvae from each replicate and treatment should ground with liquid nitrogen.
2. The larval powder should be freezed immediately in -80°C until lyophilization.
3. After lyophilization, 100 mg of larval powder dissolved in 700 microliters of precooled metabolite extraction buffer, resuspended by vortex, incubated on ice for 15 min at 4 °C at 300rpm.
4. Sonicate the extracts for 1.15 min with 15 s pulse on and 30 s pulse off. The sample tubes should be kept on the ice while performing sonication to avoid heating of the samples.
5. The lysate should be centrifuged for 20 min at 15,000 rpm and 4°C.
6. The supernatant portion should be diluted with LC-MS grade water to obtain final concentration of 53% methanol (i.e., To prepare 1 ml of extract for LC-MS, take 660 ul of larval extract and add 340 ul DDW).
7. The mixture should be centrifuged again at 15,000 rpm and 4°C for 20 minutes.
8. The supernatant will be filtered using a 0.22-µm membrane filter and kept at -20°C until further use.

sample preparation to LCMS (Tel-Hai)

1. To prepare the internal control of samples: take 100 microliters from each sample and put in to a fresh tube. Then mix and divide into 5 test tubes on which are written QC1-QC5 (at least 1 QC for 7 samples)
2. To prepare experimental BLANK (EXT-BLK), just use the extraction buffer alone and follow the steps from 3 to 8 (At least 1 EXT-BLK for 10 samples).
3. Transfer the sample tubes to Tel-Hai in ice for LC-MS.

• The QC and blank samples, and the samples from the treatments will be analysed by injecting 5 μL of the extracted solution into a UHPLC (ultra-high performance liquid chromatography) apparatus connected to a photodiode array detector (Dionex Ultimate 3000), with a reverse-phase column (ZORBAX Eclipse plus C18, 3.0*100 mm, 1.8 μm) as reported in Mani et al., 2023.
• Specifically, the QC samples will be injected at the beginning of the sequence, after each 10 experimental samples, and at the end of the sequence, to ensure data quality.
• The mobile phase comprised (A) DDW with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The gradient began with 2% B then increased to 30% B over 4 min, then increased to 40% B over 1 min and will be kept isocratic at 40% B for 3 min. It will be then increased to 98% B over 6 min and kept isocratic at 98% B for 9 min. Phase B will be then returned to 5% over 3 min and the column will be allowed to equilibrate at 5% B for 5 min before the next injection.
• The flow rate will be 0.4 mL/min. The LC–MS/MS analysis will be performed with a heated electrospray ionization (HESI-II) source connected to a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Scientific™, UK). The ESI capillary voltage will be set to 3900 V, capillary temperature to 350°C and gas (nitrogen) temperature to 350°C.
• The flow rates of sheath gas, aux gas, and sweep gas will be maintained at 35 L/min, 10 L/min and 1 L/min respectively. For MS2 (tandem mass spectrometery) analysis, the collision energy will be set to 15, 50 and 100 EV.
• The mass spectra (m/z 100–1000) will be obtained in negative and positive-ion mode with high resolution (FWHM = 70,000 at m/z = 200) and mass tolerance = 5 ppm.

LC-MS data pre-processing and untargeted metabolite identification

• Peak determination and peak area integration will be performed with Compound Discoverer 3.3 (Thermo Xcalibur, UK).
• Auto-integration of peak areas will be manually inspected and corrected where necessary.
• Peak areas from each sample will be normalized to the quality controls as follows: peak areas exhibiting RSD > 50% in the pooled QC samples will be removed from the analysis.
• Peak areas exhibiting RSD < 50% will be corrected using the QC samples. The QC correction will be performed as reported previously (Dunn et al., 2011).
• For some compounds, identification will be based on the MzCloud database using MS2 data (to obtain accurate qualitative and relative quantitative results) and the ChemSpider database using HRMS (high resolution mass spectrometry).

Reference:

1. Zhou, F., Liu, B., Liu, X., Li, Y., Wang, L., Huang, J., Luo, G. and Wang, X., 2021. The impact of microbiome and microbiota-derived sodium butyrate on Drosophila transcriptome and metabolome revealed by multi-omics analysis. Metabolites, 11(5), p.298.
2. Mani, K., Tzach, V., Lilach, B.M., Soliman, K., & Itai O. (2023). Effect of yeast supplementation on growth parameters and metabolomics of Black soldier fly larvae, Hermetia illucens (L.) (Diptera: Stratiomyidae). Journal insect as Food and Feed (In press)