Ase in the freshly ready two-phase IKK-β Inhibitor Storage & Stability Bligh-Dyer mixture (chloroform/methanol/water, two:two:1.8 (v/v/v)). The pure lipid A preparations (B. japonicum, 36 mg; B. yuanmingense, 18 mg; Bradyrhizobium sp. (Lupinus), 12 mg) had been stored at 20 in CHCl3/MeOH (three:1, v/v). O-Deacylation of lipid A samples was performed by incubation (1? mg) in chloroform, methanol, 0.six M aqueous NaOH, two:three:1 (v/v/v), for 1.five h at room temperature, in line with Que-Gewirth and co-workers (37). Fatty Acids, Hopanoid Lipids, and Sugars Analysis–For total fatty acid and hopanoid lipids determination, lipid A preparations were subjected to hydrolysis in 4 M HCl (one hundred , four h). Liberated fatty acids and hopanoids were extracted with chloroform and converted to their methyl esters with diazomethJOURNAL OF BIOLOGICAL CHEMISTRYDECEMBER 19, 2014 ?VOLUME 289 ?NUMBERHopanoid-containing Lipid A of Bradyrhizobiumane. Just after evaporation to dryness, hydroxyl groups of fatty acids and hopanoid lipids have been derivatized with BSTFA (16 h at area temperature). Neutral and amino sugar analyses were performed as outlined by typical protocols described elsewhere (21). GC-MS analyses of fatty acids and sugars had been performed on a Hewlett Packard gas chromatograph 5890 series II and Agilent Technologies GC Program 7890A connected to a mass selective detector EI/CI MSD 5975C, equipped with a HP-5MS column (30 m 0.25 mm) with helium as a carrier gas (flow rate: 0.7 ml min 1). The temperature plan was as follows: 150 for 3 min, then raised to 250 at 3 min 1, then to 320 , 25 min 1. The final temperature was kept for 10 min for sugar analysis and 20 min for fatty acid evaluation. Mass Spectrometry–Lipid A samples obtained from B. Estrogen receptor Agonist Storage & Stability japonicum were analyzed on a higher resolution hybrid Fourier transform ion cyclotron resonance mass spectrometry (FTICR) instrument (Apex Qe Bruker Daltonics, Billerica, MA) with electrospray ionization (ESI), equipped using a 7 tesla actively shielded magnet. Samples for evaluation were ready as described earlier (21) and measured within the negative ion mode. Mass Spectra were charge deconvoluted and mass numbers offered refer to the monoisotopic peaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was performed having a Bruker-Daltonics Reflex III instrument (Bruker Daltonics, Bremen, Germany) at an acceleration voltage of 20 kV and delayed ion extraction. Lipid A preparations have been dispersed in methanol/water (1:1, v/v) at a concentration of 1 g/ l with addition of 20 mM EDTA. The matrix option was prepared from 2,5dihydroxybenzoic acid in 1 trifluoroacetic acid as well as the spectra have been recorded in optimistic or negative ion modes. NMR Spectroscopy–For NMR analysis a sample containing 18 mg of native lipid A from B. japonicum dissolved in 0.6 ml of CDCl3/CD3OD (2:1, v/v) with five l of D2O, was applied. One- and two-dimensional NMR spectra had been recorded at 700 MHz on an AVANCE III spectrometer with Cryoprobe (Bruker) using Bruker software. Spectra were recorded at 27 . The following two-dimensional NMR experiments were performed: COSY, DQF-COSY, TOCSY, ROESY, HSQCnd, HSQC-DEPT, and HMBC. The 1H and 13C resonances were measured relative to TMS ( H 0.0/ C 0.0).TABLE 1 Fatty acid, hopanoids, and sugar elements of lipid A isolated from LPS of Bradyrhizobium strainsThe symbols represent: , present; lack of element; tr, traces. Element Fatty acids 12:0(3-OH) 14:0(3-OH) 26:0(25-OH) 27:0(26-OH)a 28:0(27-OH) 29:0(28-OH)a 30:0(29-OH).
