5 kda cutoff filter

Raw264.7 cells were transfected with scramble or Flcn‐targeted siRNA, followed by a medium change to MEMα containing 10% FCS (HyClone) and 40 ng/mL of RANKL at 24 hours after transfection. After 48‐hour culture, the cells were washed twice with 5% (w/v) aqueous mannitol solution. Then, 1 mL of methanol containing the internal standards (25 µmol/L each of methionine sulfone and D‐camphor‐10‐sulfonic acid) was added. After 10‐minute incubation, the 400 µL of dissolved solution was mixed with 200 μL of Milli‐Q water and 400 μL of chloroform. After centrifugation, the separated methanol‐water layer was ultrafiltered using a 5‐kDa cut‐off filter (Human Metabolome Technologies, Tsuruoka, Japan) to remove proteins. The filtrate was dried using an vacuum centrifuge and dissolved in 50 µL of Milli‐Q water containing 200 µmol/L reference compounds (3‐aminopyrrolidine and trimesic acid) before CE‐MS analysis. CE‐MS‐based metabolomic profiling and data analysis were performed essentially as described.22, 23, 24, 25, 26

Frozen serum samples were thawed, and 40 µL aliquots were added to 360 µL of methanol containing internal standards (20 µmol/L each of methionine sulfone and D-camphor-10-sulfonic acid). Solutions were mixed well, and then both 400 µL of chloroform and 160 µL of Milli-Q water were added, followed by centrifugation at 10,000× g for 3 min at 4 °C. Then, 300 µL of the upper layer were transferred to a 5-kDa-cutoff filter (Human Metabolome Technologies, Tsuruoka, Japan) to remove proteins. The filtrate was dried using a centrifuge concentrator and reconstituted with 40 µL of Milli-Q water containing reference compounds (200 µmol/L each of 3-aminopyrrolidine and trimesic acid) prior to CE-TOFMS analysis.

Capillary electrophoresis mass spectrometry analysis was performed at the Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan, under the supervision of T.S. Cells were washed twice with 5% mannitol solution and covered with 700 μl of methanol containing 25 μM internal standards [20 μM each of methionine sulfone, 2-(N-morpholino)-ethanesulfonic acid and d-camphor-10-sulfonic acid] for 10 min. The resulting extracts were mixed with 200 μl of Milli-Q water and 400 μl of chloroform and centrifuged at 4600g for 15 min at 4°C. Then, 400 μl of the aqueous phase of the sample solution was subjected to ultrafiltration through a 5-kDa cutoff filter (Human Metabolome Technologies, Tsuruoka, Japan) to remove proteins. The filtrate was centrifugally concentrated and dissolved in 25 μl of Milli-Q water that contained reference compounds (200 μM each of 3-aminopyrrolidine and trimesic acid) immediately before metabolome analysis. The concentrations of all the charged metabolites were measured by capillary electrophoresis time-of-flight mass spectrometry (Agilent Technologies, Palo Alto, CA, USA) using the methods described previously (58 (link), 59 (link)). Analysis was performed using MasterHands Software (v2.13.0.8h, Keio University).

Frozen CSF samples were thawed and 40 μL aliquots were placed into 360 μL of methanol containing internal standards (20 μmol/L each of methionine sulfone and D-camphor-10-sulfonic acid). The solutions were thoroughly mixed, and then both 400 μL of chloroform and 160 μL of Milli-Q water were added, followed by centrifugation at 10,000 × g for 3 min at 4 °C. The aqueous layer was transferred to a 5-kDa-cutoff filter (Human Metabolome Technologies, Tsuruoka, Japan) to remove proteins. The filtrate was dried using a centrifuge concentrator and reconstituted with 50 μL of Milli-Q water containing reference compounds (200 μmol/L each of 3-aminopyrrolidine and trimesic acid) prior to CE-TOFMS analysis.

This study was approved by the Tokushima University Hospital Ethics Committee (Approved no. 1815), and the corresponding regulatory agencies and all experiments were carried out in accordance with approved guidelines. All patients involved in the study signed an informed consent form.
Tumor and surrounding nontumor tissues were surgically resected from 13 patients with HCC and 3 patients with metastatic liver cancer. The resected tissue samples were quickly frozen at −80 °C until sample preparation.
Liver tissues were weighed (~100 mg) and homogenized in methanol (500 μL) containing internal standards (20 μM methionine sulfone and 50 μM Phe-Gly-¹³C₉-¹⁵N₁) using a Shake Master NEO instrument (Biomedical Science Co., Ltd., Tokyo, Japan). The homogenates were mixed with chloroform (500 μL) and Milli-Q water (200 μL), and the mixture was centrifuged at 4600× g for 15 min at 4 °C. The upper aqueous layer (300 μL) was centrifugally filtered through a 5-kDa cutoff filter (Human Metabolome Technologies, Tsuruoka, Japan) to remove proteins. The filtrate was centrifugally concentrated in a vacuum evaporator and dissolved in Milli-Q water (25 μL) immediately before CE-MS/MS analysis. This sample was diluted by 5-fold with Milli-Q water and subjected to LC-MS/MS analysis.