Supplementary Materials [chapter]

Katrin Becker
2016 Choosing and Using Digital Games in the Classroom  
The purified SDP was subjected to high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to obtain a monoisotopic mass m/z 4311.209 [M+H] + (Fig. S3) , and this mass matched Cys 141 -Ser 182 of SdpC-2.016 Da, indicating the likely presence of a disulfide crosslink. The fragmentation by collision-induced dissociation (CID) and FT-ICR MS analysis confirmed the sequence with a disulfide crosslink localized to residues Cys 141 and Cys 147 (Fig. 2, Fig. S5 , Table S4
more » ... ). The N-terminal boundary of SDP is in agreement with the N-terminal Edman amino acid sequencing previously performed on SDP (1). Purified SKF was also analyzed by high-resolution mass spectrometry and found to have a mass of 2781.302 Da (Fig. S3 ). This mass could not be readily matched to the C-or N-terminal portions of the 55 amino acid protein precursor SkfA (1). When purified SKF was subjected to high-resolution tandem mass spectrometry, the sequence tag WASKSI was obtained (Fig. S8A, Fig. S9 ). To define the number of amino acids that were involved in the mature SKF metabolite, a 15 N feeding experiment was performed. The feeding experiment with 98% pure [ 15 N]ammonium chloride resulted in a 36 Da increase in mass, indicating that SKF contained 36 nitrogen atoms which matched to the number of nitrogens in the last 26 amino acids of the SkfA sequence, CMGCWASKSIAMTRVCALPHPAMRAI (Fig. S8B ). The calculated mass of this peptide is 2803.340, and therefore, the mature form of SkfA is 22.038 Da less than that expected from the intact peptide sequence. The absence of observable y ions provided an indication that SKF may be cyclic (2). We postulated that SKF was cyclic accounting for 18.011 of the 22.038 Da mass differences. The remaining 4.027 Da difference between the parent SkfA peptide and the cyclized precursor could be explained by two crosslinks, possibly a disulfide and a thioether. To confirm that SKF was indeed cyclic, all thiols were replaced with a proton using a reductive dethiolation reaction composed of NiCl 2 and NaBH 4 ( Fig. S10) (3). Subjecting SKF to this reaction resulted in an ion with a mass of 2551.444 Da. Tandem mass spectrometry by collision induced dissociation (CID) confirmed the sequence to the 26 amino acids on the C-terminal end of SkfA as well as a cyclic head-to-tail linkage between isoleucine and cysteine. The calculated mass of this molecule is 2551.450. To define the connection of the thioether linkage, the reductive dethiolation was repeated but with deuterated solvents and NiCl 2 /NaBD 4 and resulted in a species with mass of 2558.491, 7.041 Da larger compared to the product resulted from NiCl 2 /NaBH 4 reaction, suggesting 7 thiol connections. Six deuteriums were introduced from the replacement of three methionine and three cysteine side chains with deuterons while the remaining deuterium was introduced at the site of the thioether linkage (Fig. S11A ). Therefore, it became possible to map out the position of the thioether linkage by locating this extra deuterium that gave rise to the 1 Da mass shift. To map the thioether linkage, multiple stages of tandem mass spectrometry were obtained on the deuterated dethiolated 3 SKF. To find the position of the additional deuterium, a mass list with manually deconvoluted fragment ions was analyzed by an algorithm NRP-comparative dereplication (4) against the theoretical structure of SKF with a deuterium labeled on each desulfurized position (Fig. S11A) . NRP-dereplication matched the MS fragments with the structure and Met 12 had the lowest score indicated the strongest correlation of the 1 Da increase between the observed ions in the tandem mass spectrometry experiment when compared to the theoretically predicted ions of the deuterated dethiolated SKF template (Fig. S8C and Fig. S11B) . Guided by the NPR-dereplication result, manual annotation was performed, again verifying the extra deuterium on Met 12 ( Fig. S11C and Table S5 , S6). The position of the disulfide bond was determined by reduction, iodoacetamide alkylation and tandem mass spectrometry. Reduction and alkylation of SKF resulted in a mass increase of 116.061 Da, in agreement with two free thiols (Fig. S10) . Via tandem mass spectrometry, the alkylated residues were found to be Cys 1 and Cys 16 , positioning the disulfide between these two cysteines. Even though the tandem mass spectrometry of the desulfurized SKF indicated that the thioether linkage is connected to the methionine, it did not provide regiochemical information to which carbon of Met 12 that Cys 4 was connected. To determine the regiospecificity of the tetrahedral linkage, we resorted to nuclear magnetic resonance (NMR) spectroscopy (Figs. S12 and Table S7 ). To determine the relevant proton signals, the NMR signal that corresponded to the methionine involved in the thioether cross-link needed to be identified. To find the modified methionine with an absent proton, a 1 H-1 H total correlation spectroscopy (TOCSY) was obtained first. From the TOCSY, one set of methionine correlations lacking an α-proton was observed, and suggested that the linkage of Cys 4 to Met 12 is via the α-carbon of the latter residue ( Fig. S12B and C ). An α-connection would result in a 13 C-chemical shift at this α-carbon of about 70 ppm while a β-connection would result in 13 C-chemical shift of 40-50 ppm (3). The 13 C-chemical shift information was obtained indirectly by heteronuclear multiple bond correlation (HMBC) ( Fig. S8D and E, Fig. S12D ), because of the small quantities of pure SKF available. The same methionine that was missing the proton in the TOCSY was scrutinized in the HMBC spectrum. In the HMBC, in agreement with the findings by TOCSY, there was no evidence of an α-proton in Met 12 ; however, the β-proton possessed long range correlations between the β-protons and two quaternary carbons, located at δ 67.9 and 180.7 ( Fig. S8D and E) , and thus consistent with a thioether bridge connecting to the α-carbon of Met 12 . A post-translational modification of a cysteine to the α-carbon of methionine has not been previously reported (5). The functional annotation of the skf biosynthetic operon.
doi:10.1007/978-3-319-12223-6_12 fatcat:s5jo3ddw2zfqvb42xq3m3cfb64