Unequivocal assignments of methylene hydrogens was achieved by 1H-1H COSY experiments.
The stereochemical assignments of methylene hydrogens shown in Table 2 [see Additional files 3, 4, 5] were made based on gradient NOESY cross-peaks and were consistent with distances determined from a computational, energy-minimized model of the molecule utilizing the MMX force-field (PCModel, version 9.2; Serena Software, Bloomington, IN).
Correcting erroneous atom assignments requires the analysis of hydrogen bonding interactions.
Given the previous assignment of the SIE to hydrogen bond rearrangement for other LOX isozymes, it appears that addition of ATP increases the dependence of the rate-limiting step on hydrogen bond rearrangement (k3 and k7) relative to hydrogen atom abstraction (k5 and k11).
Bi-dimensional H homonuclear correlation spectroscopy (COSY) was used to identify the pattern of hydrogen signal assignments [ 35].
Many standard chemistry algorithms are included, for example, determination of the smallest set of smallest rings, bond order perception, addition of hydrogens, and assignment of Gasteiger charges.
The final score was calculated by the integration of mass accuracy, isotopic ratio, product ion assignment, neutral loss assignment, bond dissociation energy, penalty of fragment linkage, penalty of hydrogen rearrangement rules, and existence of the compound in the internal MS-FINDER structure databases (see Additional file 1 for full details).
The protein molecule preparation included adding of hydrogen atoms and the assignment of the standard OPLS atom types.
In addition, several hydrogens of lactose are observed, which allow the direct assignment of individual hydroxyl groups as either hydrogen bond donors or acceptors.
