Ns are visible on the spectrum of native lipid A. The main signals at m/z 2669.1, 3133.4, and 3660.6 were consistent with calculated masses of hexa-acylated lipid A, hepta-acylated lipid A, and hepta-acylated-hopanoid containing lipid A, respectively. The mass spectrum in the O-deacylated sample (Fig. 3A) showed a major signal at m/z 1692.3, originating from sodiated tetra-acyl lipid A, composed of two GlcpN3N units, two Manp, 1 GalpA, and four amide-linked fatty acids (two 12:0(3-OH) and two 14:0(3-OH) H2O). The calculated monoisotopic mass for the [M Na] ion was determined to m/z 1692.0. The signals identified as hexa-acyl lipid A derived from molecules containing two ester-linked VLCFAs (for instance 33:0(32-OH) and 34:0(33-OH), with a calculated [M-H2O H] m/z of 2669.02,Fig. 3B). The ion cluster at m/z 3100 ?200 was identified as hepta-acyl lipid A containing a third acyl residue (for instance 31:0(30-OH), theoretical m/z of [M-H2O H] 3133.48). The lipid A molecules with mass about 3660 Da in addition contained one particular hopanoid residue, ester-linked as in lipid A from B. japonicum to the ( -1)-OH-group of on the list of VLCFA residues. Fig. 4 shows the MALDI-TOF mass spectrum (damaging ion mode) of the lipid A from Bradyrhizobium sp. (Lupinus). Two sets of signals are visible, one around m/z 2583.0 as well as the other at m/z 3095.2, which have been assigned to hexa-acyl lipid A carrying two VLCFAs (e.g. 30:0(29-OH) and 31:0(30-OH), theoretical m/z for [M-H2O H] 2582.91) along with a hexa-acylated lipid A moiety also bearing a single hopanoid residue (calculated m/z [M-H2O H] 3095.33). NMR Spectroscopy of B. japonicum Lipid A–The native lipid A preparation isolated from B. japonicum was dissolved in CDCl3/CD3OD (two:1, v/v) with traces of D2O and completely characterized by one- and two-dimensional NMR spectroscopy. Lipid A was not substituted by phosphate residues, as confirmed by 31 P NMR spectroscopy. The HSQC-DEPT spectrum of the lipid A (Fig. 5, blue and green) contained 5 signals of anomeric IRAK4 Inhibitor Purity & Documentation carbons ( 92.25?03.14), 4 signals of nitrogen-bearVOLUME 289 ?Quantity 51 ?DECEMBER 19,35648 JOURNAL OF BIOLOGICAL CHEMISTRYHopanoid-containing Lipid A of BradyrhizobiumTABLE 2 Annotation of mass spectrometric information (major signals) obtained for the O-deacylated and native lipid A samples from B. japonicum (see Fig. two, A and B)Form of lipid A Lipid A O-deacylated Measured massDaType of molecule Hopanoid Lipid Atetra-acylCompositionCalculated monoisotopic massDa530.4312 1669.Lipid A native760.4712 1008.907 1651.[Y]-ion variety Hopanoid VLCFA Lipid Atetra-acyl –Cathepsin B Inhibitor web H2O2087.Lipid Apenta-acyl -H2O2583.Lipid Ahexa-acyl3096.Lipid Ahexa-acyl hopanoid3650.Lipid Ahexa-acyl (two hopanoid)two GlcpN3N two Manp 1 GalpA 2 14:0 (3-OH), two 12:0 (3-OH) -H2O 1 GlcpN3N 1 GalpA 1 14:0 (3-OH), 1 12:0 (3-OH) Hopanoid 530.4335 u 1 32:0 (31-OH) two GlcpN3N 2 Manp 1 GalpA two 14:0 (3-OH), 2 12:0 (3-OH) 2 H2O 2 GlcpN3N two Manp 1 GalpA 2 14:0 (3-OH), 2 12:0 (3-OH) 1 29:0 (28-OH) 2 H2O two GlcpN3N two Manp 1 GalpA 2 14:0 (3-OH), two 12:0 (3-OH) 1 29:0 (28-OH), 1 32:0 (31-OH) -H2O 2 GlcpN3N 2 Manp 1 GalpA two 14:0 (3-OH), 2 12:0 (3-OH) 1 29:0 (28-OH), 1 32:0 (31-OH) 1 hopanoid 530.4335 -H2O 2 GlcpN3N two Manp 1 GalpA 2 14:0 (3-OH), 2 12:0 (3-OH) 1 31:0 (30-OH), 1 32:0 (31-OH) 1 hopanoid 530.4335 1 hopanoid 544.4491 -H2O530.4335 1669.760.4726 1008.9084 1651.2087.2583.3096.3650.ing carbons ( 52.02?four.49), these of sugar ring carbon atoms ( 61.47?6.76) and signals for CH-OR too as for CH-OH groups from fatty acids and hopano.
