L methionine sulfone

The dialyzer with low flux used in this study was the Fresenius F6, and the dialyzer with high flux was the Fresenius F60. Liquid chromatography grade methanol and chloroform were purchased from Merck (Darmstadt, Germany). Liquid chromatography grade ammonium acetate was purchased from Sigma-Aldrich (St. Louis, Mo, USA), and 98% formic acid was purchased from Fluka (Darmstadt, Germany). Ultrapure Water (18.2 MΩ-cm, TOC = 6 ppb) was prepared with a Milli-Q system (Millipore, Billerica, MA, USA). 10 mM D-camphor-10-sulfonic acid sodium salt (Wako, Japan) and 10 mM L-methionine sulfone (Wako, Japan) in methanol were used as internal standard solution 1 (ISS1). A mixture of 1 mM disodium 3-hydroxynaphthalene-2,7-disulfonate (Wako, Japan), trimesic acid (Wako, Japan), N,N-diethyl-2-phenylacetamide (Wako, Japan), and 3-aminopyrrolidine dihydrochloride (Aldrich, USA) in water was used as internal standard solution 3 (ISS3). ISS1 was used to standardize the metabolite intensity and ISS3 was used to adjust the migration time of the metabolites.

Cells from 10 cm dishes were washed twice with 10 ml 5% mannitol in MilliQ water before metabolites were extracted from melanoma cell pellets using 1 ml methanol for 10 min and samples were deproteinized using 400 µl CHCl₃ and 200 ml MilliQ water followed by centrifugation at 10,000 g for 3 min at 4°C. 400 µl of the aqueous layer was then filtered using a 5 kDa ultrafiltration tube and analysed by capillary electrophoresis mass spectrometry (CE‐MS) after addition of 25 µl 200 mM internal standards: L‐methionine sulfone (Wako 502–76641), 2‐(N‐morpholino) ethanesulfonic acid (Dojindo 349–01623), D‐Camphor‐10‐sulfonic acid (Wako 037–01032), 3‐aminopyrrolidine (Aldrich 404624) and trimesate (Wako 206–03641) as described (Kami et al., 2013) on an Agilent capillary electrophoresis system consisting of an Agilent G6220A LC/MSD TOF, an Agilent 1100 series isocratic HPLC pump, a G1603A Agilent CE‐MS adapter kit, and a G1607A Agilent CE‐ESI‐MS sprayer kit (Agilent Technologies, USA).

Plasma (40 microliters [μL]) was extracted with the addition of 400 μL of ice-cold methanol containing the internal standards (10 millimolars [mM] l-methionine sulfone [Wako], 100 mM 2-morpholinoethanesulfonic acid [Dojindo], 100 mM D-10-camphorsulfonic acid [Wako]), 400 μL of chloroform, and 120 μL of water. After centrifugation at 10,000 × g for 3 min at 4 °C, the separated aqueous layer was filtered through a 5 kilodalton (kDa) cutoff filter (Millipore) to remove protein contamination. The filtrate (300 μL) was lyophilized and dissolved in 20 μL water containing the 2 types of reference compounds (200 μM each of trimesate [Wako] and 3-aminopyrrolidine [Sigma-Aldrich]) for migration time and then injected into the capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) system (Agilent Technologies)^{36 (link)–38 (link)}. Among the measured molecules, GIP (active) was measured using an ELISA kit. Blood hormones and some metabolites were measured according to methods developed by LSI Medience Co., Ltd. The methods used to measure each of these molecules are listed in Supplementary Data 5; among these, the amino acid fractions measured by liquid chromatography–mass spectrometry (LC–MS) are listed in Supplementary Data 6, and the metabolites measured by CE-TOFMS are listed in Supplementary Data 7.

Cells in a 100-mm dish were washed by 5% mannitol, and then 1 mL methanol(Wako, Tokyo, Japan) with 25 μM each l-methionine sulfone (Wako, Tokyo, Japan), 2-morpholinoethanesulfonate (Dojindo, Kumamoto, Japan), and D-Camphor-10-sulfonic Acid Sodium Salt (Wako, Tokyo, Japan) was added. Collected cell and solution with scraper, and the transferred solution was mixed with Milli-Q water and chloroform. The solution was centrifuged, and the separated aqueous layer was centrifugally filtered through an HMT 5-kDa ultrafiltration tube (Human Metabolome Technologies Inc., Japan). Samples were dried using an evacuated centrifuge, and Milli-Q water containing 3-aminopyrrolidine (Sigma Aldrich, USA) and Trimesate (Wako, Tokyo, Japan) was added. Metabolomic analyses were performed by capillary electrophoresis time-of-flight mass spectrometry at the Institute for Advanced Biosciences, Keio University (Tsuruoka, Japan) as previously reported^{43 (link)}. The measured metabolite concentrations were normalized using cell number to obtain the amount per cell. Raw data were processed using software (MasterHands) developed in-house as previously described^{43 (link)}.