Adrenocorticotropic hormone

Purified compound from RP-HPLC was analyzed through matrix assistant laser desorption ionization time of flight-mass spectroscopy (MALDI–TOF-MS) to find the exact molecular mass. 2 µL of sample mixed with 2-µL matrix solution (2 mg of alpha-cyano-4-hydroxycinnaminic acid per ml in acetonitrile-methanol–water (1:1:1) on the target plate). MALDI–TOF-MS spectra were recorded using a 337-nm nitrogen laser for desorption and ionization. The mass spectrometer was operated in the linear mode at an accelerating voltage of 18 kV with an ion flight path that was 0.7-m long. The delay time was 375 ns. Matrix suppression was also used, and the mass spectra were averaged over 50–100 individual laser shots. The laser intensity was set just above the threshold for ion production. External calibration was performed using the [M + H]⁺ signals of renin, adrenocorticotropic hormone, insulin oxidized B, and bovine insulin (Sigma-Aldrich Co.).

Twelve Xinhuai sows were randomly divided into two groups. 1 U/kg adrenocorticotropic hormone (Sigma, USA) or equivalent volume saline was injected via cervical vein per day for 9 days. Then the levels of serum cortisol were detected, and backfat and LD muscle samples were harvested. Isolated the IMAT with Ophthalmic forceps in the LD muscle.

Water was double distilled using quartz apparatus from Heraeus Quarzschmelze (Hanau, Germany). Acetonitrile, glycine (Gly), and leucine (Leu) were bought from PENTA (Praha, Czech Republic). Trifluoroacetic acid (TFA), α-cyano-4-hydroxycinnamic acid (CHCA), ammonium bicarbonate (ABC), 3,5 dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA), tryptophan (Trp), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (Steinheim, Germany). Black phosphorus was purchased from Chempur (Karlsruhe, Germany). Phenylalanine (Phe) was purchased from Merck (Darmstadt, Germany). Bradykinin fragment 1–7, angiotensin II, [glu¹]-fibrinopeptide, adrenocorticotropic hormone [1–17] (ACTH [1–17]), and adrenocorticotropic hormone [18–39] (ACTH [18–39]) were purchased from Sigma-Aldrich (Steinheim, Germany). Humanin peptides A (C₈₄H₁₃₈N₂₀O₂₄S, PRGFSCLLLLTGEIDLP), B (C₈₃H₁₃₆N₂₀O₂₃S₂, PRGFSCLLLLCGEIDLP), C (C₈₅H₁₄₀N₂₀O₂₅S, PRGFSCLLLLTSEIDLP), and D (C₁₁₈H₂₀₂N₃₄O₃₁S₂, MAPRGFSCLLLLTGEIDLPVKRRA) were purchased from Clonestar biotech (Brno, Czech Republic). CCTL14 line of hESC [46, XX] registered in Human Pluripotent Stem Cell Registry was used in this study. This hESC line was kindly provided by Dr. Aleš Hampl, Ph.D. from the Department of Histology and Embryology, Faculty of Medicine, Masaryk University, and used in consonance with the official permission (MSMT-14648/2016-5).