Micromass

Acylcarnitines were determined by electrospray–tandem mass spectrometry in plasma or dry bloodspots (Micromass, Waters, Milford, MA, USA) [7 (link)]. Organic acids in urine were determined qualitatively by gas chromatography–mass spectrometry (GC-MS) (Agilent, Santa Clara, CA, USA) [8 (link)]. Plasma amino acid analysis was performed on a Biochrom 30 amino acid analyzer according to the manufacturer’s instructions (Biochrom, Holliston, MA, USA).

The sequences of STAP1 to STAP14 were analyzed using an Applied Biosystems 494 protein sequencer (Perkin Elmer, USA) (9 (link)). The MWs of STAP1 to STAP14 were determined by employing a quadrupole time-of-flight (Q-TOF) mass spectrometric device (Micromass, Waters, USA) in the combination of an electrospray ionization (ESI) source (50 (link)).

Optical rotations were measured in CH₂Cl₂ on a PerkinElmer 241 polarimeter (Waltham, MA, USA), by using a sodium lamp operating at 589 nm. NMR spectra were recorded on a Bruker AVANCE 500 MHz or 600 MHz, as required. NMR spectra were obtained dissolving samples in CDCl₃ (99.9%) and chemical shifts are reported relative to solvent (δ_H 7.26 and δ_C 77.0 ppm) and TMS as internal pattern. Bruker AVANCE 600 MHz instrument is equipped with a 5 mm TCI inverse detection cryoprobe. Standard Bruker NMR pulse sequences were utilized. HR-ESI-MS (High-Resolution ElectroSpray Ionization Mass Spectrometry) and HR-EI-MS (High-Resolution Electron Impact Mass Spectrometry) data were obtained on a Waters LCT Premier XE Micromass (Manchester, UK) and VG-AutoSpec Micromass (Manchester, UK) spectrometers, respectively. IR spectra were recorded on a Bruker IFS66/S (Ettlingen, Germany) equipped with an ATR accessory using CH₂Cl₂ solutions. EnSpire^® Multimode Reader (Perkin Elmer, Walt, MA, USA) using absorbance values of Alamar Blue^® reagent. TLC (Thin layer chromatography) (Merck, Darmstadt, Germany) was visualized by UV light (254 nm) and spraying with cobalt chloride reagent (2% in sulfuric acid, 10%) and heating.

Ten ml of blood was collected from NC patients. WBC were separated using dextran 3%, and pellets were kept at −80°C with 150 μl of N-ethylmaleimide and 50 μl of 12% sulfosalicylic acid till assayed. Cystine was quantified by LC-MS/MS (Micromass, Waters) as previously described [6 (link)].

The AA sequences of eighteen peptides (MSP1-MSP18) from monkfish swim bladders were determined by a 494-protein sequencer of Applied Biosystems (Perkin Elmer Co., Ltd. Foster City, CA, USA). The MWs of eighteen peptides (MSP1-MSP18) were determined by a Q-TOF mass spectrometer with an ESI source (Micromass, Waters, Milford, MA, USA) [81 (link),82 (link)].

To investigate the absorption of CG-598 into intestine or systemic blood, C57BL/6 mice (n=3 per group) were orally administered with 150 mg/kg of CG-598 in 1% carboxymethyl cellulose (CMC). Colon tissues and plasma were sampled at 1, 2, 4, and 8 h following oral administration of CG-598. To analyze the excretion of CG-598, fasted male SD rats (n=3) were orally administered with 150 mg/kg of CG-598. After dosing, feces and urine were collected separately during 0 to 4 h, 4 to 8 h, 8 to 24 h, and 24 to 48 h and the content of CG-598 was quantified by measuring with Micromass/HPLC Alliance 2795 and analyzing with QuantiLynx (Waters, MA, USA). To analyze generation of reticulocytes/red blood cells from bone marrow, male ICR mice (n=5 per group) were orally fed once a day with the indicated doses of CG-598 or 100 mg/kg PN-3602. Body weights were monitored every 4 days. At 15 days after the first oral administration, blood samples were analyzed via auto hematology analyzer (BC-2800vet, Mindary).