| MS Description
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The frozen tissues were homogenized in fiv … The frozen tissues were homogenized in five volumes of 80% aqueous methanol containing 0.5 mg l)1 lidocaine and D-camphor sulfonic acid (Tokyo Kasei) using a mixer mill (MM 300, Retsch, http://www.retsch.com) with a zirconia bead for 6 min at 20 Hz. Following centrifugation of 15 000 g for 10 min and filtration.<br /><br />
<LC-ESI-MS><br />
The sample extracts (2 μl) were analyzed using an LC-MS system equipped with an electrospray ionization (ESI) interface (HPLC, Waters Acquity UPLC system; MS, Waters Q-Tof Premier, http://www.waters.com). The analytical conditions were as follows. HPLC: column, Acquity bridged ethyl hybrid (BEH) C18 (pore size 1.7 μm, length 2.1 · 100 mm, Waters); solvent system, acetonitrile (0.1% formic acid):water (0.1% formic acid); gradient program,1 : 99 v/v at 0 min, 1 : 99 v/v at 0.1 min, 99.5 : 0.5 at 15.5 min, 99.5 : 0.5 at 17.0 min, 1 : 99 v/v at 17.1 min and 1 : 99 at 20 min; flow rate, 0.3 ml min-1; temperature, 38℃; MS detection: capillary voltage, +3.0 keV; cone voltage, 22.5 V; source temperature, 120℃; desolvation temperature, 450℃; cone gas flow, 50 l h-1; desolvation gas flow, 800 l h-1; collision energy, 2 V; detection mode, scan (m/z 100–2000; dwell time 0.45 sec; interscan delay 0.05 sec, centroid). The scans were repeated for 19.5 min in a single run. The data were recorded using MASSLYNX version 4.1 software (Waters).<br />
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<MS2T data acquisition by LC-Q-TOF-MS><br />
The sample extracts prepared by the method above (2 μl) were subjected to the same LC-Q-TOF-MS system operated under the same conditions mentioned above, except for the following changes: gradient program, 1 : 99 v/v at 0 min, 1 : 99 v/v at 0.2 min, 99.5 : 0.5 at 31 min, 99.5 : 0.5 at 34.0 min, 1 : 99 v/v at 34.2 min and 1 : 99 at 40 min; flow rate 0.15 ml min-1; survey detection mode for MS detection. In this mode, following acquisition of the MS spectrum (m/z 100–1000; dwell time 0.45 sec, inter-scan delay 0.05 sec), the MS/MS data of the most abundant ions were automatically obtained (m/z 50–1000; dwell time 2.5 sec; inter-scan delay 0.5 sec, data acquisition, centroid mode; collision energy ramped from 5 to 60 V). The mass/charge ratio (m/z) was calibrated using the lock-mass function with leucine enkephalin. The analyses were repeated 25 times by shifting the m/z ranges of the target ion selection window for the MS/MS analysis (m/z 100–160, 130–190, 160–220 … 880–940, 940–1000).The data were converted into ASCII format using DataBridge (Waters). The information in each MS/MS spectrum was formatted to the MS2T
libraries using in-house Perl scripts. Low-intensity signals of fewer
than 5 counts/sec were discarded in this process. The original retention time values were divided by two to compensate for the difference in peak elution conditions. the difference in peak elution conditions.
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