C18 spin column

Precipitated proteins were dissolved in 100 µL of 8 M urea, with a 50 mM Tris-HCl (pH 8.0) buffer. The sample was treated by 2 µL of 1 M Dithiothreitol at RT for 1 h and 8 µl of 500 mM Iodoacetamide at RT for 1 h with shading. The alkylation was stopped by 1 µL of 1 M Dithiothreitol and then diluted eight times by 50 mM Tris-HCl (pH 8.0). For the digestion, 1 µg of trypsin (Agilent, Santa Clara, CA, USA) was added to the sample and incubated at 37 °C for 16 h with shaking. The digestion was stopped by the addition of 1 µL of 50% Trifluoro acetic acid (TFA). The digested sample was purified by C18 spin column (GL Science, Tokyo, Japan) according to the manual. Briefly, a C18 spin column was activated by 100% and 50% acetonitrile sequentially and then equilibrated by 0.2% TFA with centrifuging at 3000× g for 30 sec. After conditioning, the sample was loaded into a spin column and centrifuged at 3000× g for 90 sec. Then, trapped peptides were washed by 0.2% TFA twice and eluted by 95% acetonitrile with 5% formic acid. The eluted sample was dried up by a VEC-260 vacuum dryer (Iwaki, Tokyo, Japan). The sample was re-suspended by 0.1% formic acid and the peptide concentration was measured by Nano drop 1000 (Thermo, Bremen, Germany). The sample was stored at −80 °C until use.

Each tissue of G. sinensis was immediately frozen in liquid nitrogen and ground to powder using a mortar and pestle. Powdered samples (100 mg) were extracted with three volumes of methanol. After two homogenizations using a TissueLyser (Qiagen) at 27 Hz for 2 min each, the homogenates were centrifuged (12,000 × g, 10 min, 4 °C). The supernatant was filtered through a C18 Spin column (GL Sciences, Tokyo), and the filtrate was used for LC-Orbitrap-MS analysis.

Precipitated proteins were dissolved in 100 µL of 8 M urea/50 mM Tris-HCl (pH 8.0) buffer. The sample was treated by 1 µL of 1 M dithiothreitol at RT for 1 h and 8 µL of 500 mM iodoacetamide at RT for 1 h with shading. The alkylation was stopped by 1 µL of 1 M dithiothreitol and then diluted eight times by 50 mM Tris-HCl (pH 8.0). For the digestion, 1 µg of trypsin (Agilent, Santa Clara, CA, USA) was added to the sample and incubated at 37 °C for 16 h with shaking. The digestion was stopped by 50% of trifluoro acetic acid (TFA).
The digested sample was purified by C18 spin column (GL Science, Tokyo, Japan) according to the manual. Briefly, a C18 spin column was activated by 100% and 50% acetonitrile sequentially and then equilibrated by 0.2% formic acid with centrifuging at 3000 g for 30 s. After conditioning, the sample was loaded into the spin column and centrifuged at 3000 g for 90 s. Then, trapped peptides were washed with 0.2% TFA twice and eluted by 95% acetonitrile with 5% formic acid. The eluted sample was dried up by VEC-260 vacuum dryer (Iwaki, Tokyo, Japan). The sample was re-suspended by 0.1% formic acid and the peptide concentration was measured by Nano drop 1000 (Thermo Fisher Scientific, Bremen, Germany). The sample was stored at −80 °C until use.