FIGURE 3.

Hydroxylamine and iodoacetamide treatment to unambiguously identify Cys-9 as the modified residue. Samples from the experiments depicted in Fig. 2, A and B were treated with iodoacetamide to unambiguously identify the palmitoylated/oleylated residue (panels A and B, respectively). As described above, 2 peaks were detected in the untreated sample (panel A; m/z = 4011.3 and 4036.0 Da). Treatment with hydroxylamine (+ NH₂OH, black arrow) resulted in the apparition of a unique peak at m/z = 3774.0 Da suggesting that post-translational modifications are present on a cysteine residue. Differences in masses of the peaks before and after treatment correspond to the mass of a palmitic acid and an oleic acid (Δm/z = 236.2 and 262.8, respectively). Iodoacetamide was added on the two samples (untreated and hydroxylamine treated, +IAM, gray arrows). As depicted in panels C and D, iodoacetamide only affected the peak corresponding to the deacylated SLN whose m/z increased from 3774.0 to 3830.5 Da: the difference should correspond to the acetamide addition to the SH side-chain of the cysteine. IAM reaction was probably not total, as suggested by the presence of some residual unmodified SLN (*).