Reaction SMILES: [CH3:1][CH2:2][C@@H:3]([C@H:5]1[NH:36][C:34](=[O:35])[C@H:33]([CH2:37][CH2:38][CH2:39][NH2:40])[NH:32][C:30](=[O:31])[C@H:29]([NH:41][C:42]([C@H:44]([NH:49][C:50]([C@@H:52]([NH:58][C:59]([C@H:61]([NH:66][C:67]([C@@H:69]2[N:73]=[C:72]([C@H:74]([NH2:79])[C@H:75]([CH2:77][CH3:78])[CH3:76])[S:71][CH2:70]2)=[O:68])[CH2:62][CH:63]([CH3:65])[CH3:64])=[O:60])[CH2:53][CH2:54][C:55]([OH:57])=[O:56])=[O:51])[C@H:45]([CH2:47][CH3:48])[CH3:46])=[O:43])[CH2:28][CH2:27][CH2:26][CH2:25][NH:24][C:22](=[O:23])[C@@H:21]([CH2:80][C:81]([NH2:83])=[O:82])[NH:20][C:18](=[O:19])[C@H:17]([CH2:84][C:85]([OH:87])=[O:86])[NH:16][C:14](=[O:15])[C@@H:13]([CH2:88][C:89]2[N:93]=[CH:92][NH:91][CH:90]=2)[NH:12][C:10](=[O:11])[C@H:9]([CH2:94][C:95]2[CH:96]=[CH:97][CH:98]=[CH:99][CH:100]=2)[NH:8][C:6]1=[O:7])[CH3:4].N[C@H](C(O)=O)[C@H](CC)C.N[C@H](C(N1CSC[C@H]1C(O)=O)=O)[C@H](CC)C.N[C@H](C(N1CSC[C@H]1C(N[C@H](C(O)=O)CC(C)C)=O)=O)[C@H](CC)C.N[C@H](C(N1CSC[C@H]1C(N[C@H](C(N[C@H](C(O)=O)CCC(=O)O)=O)CC(C)C)=O)=O)[C@H](CC)C.N1CSC[C@H]1C(N[C@H](C(O)=O)CC(C)C)=O.N1CSC[C@H]1C(N[C@H](C(N[C@H](C(O)=O)CCC(=O)O)=O)CC(C)C)=O>>[CH3:1][CH2:2][C@@H:3]([C@@H:5]1[NH:36][C:34](=[O:35])[C@@H:33]([CH2:37][CH2:38][CH2:39][NH2:40])[NH:32][C:30](=[O:31])[C@@H:29]([NH:41][C:42]([C@@H:44]([NH:49][C:50]([C@H:52]([NH:58][C:59]([C@@H:61]([NH:66][C:67]([C@H:69]2[N:73]=[C:72]([C@@H:74]([NH2:79])[C@H:75]([CH2:77][CH3:78])[CH3:76])[S:71][CH2:70]2)=[O:68])[CH2:62][CH:63]([CH3:64])[CH3:65])=[O:60])[CH2:53][CH2:54][C:55]([OH:57])=[O:56])=[O:51])[C@H:45]([CH2:47][CH3:48])[CH3:46])=[O:43])[CH2:28][CH2:27][CH2:26][CH2:25][NH:24][C:22](=[O:23])[C@H:21]([CH2:80][C:81]([NH2:83])=[O:82])[NH:20][C:18](=[O:19])[C@@H:17]([CH2:84][C:85]([OH:87])=[O:86])[NH:16][C:14](=[O:15])[C@H:13]([CH2:88][C:89]2[NH:93][CH:92]=[N:91][CH:90]=2)[NH:12][C:10](=[O:11])[C@@H:9]([CH2:94][C:95]2[CH:96]=[CH:97][CH:98]=[CH:99][CH:100]=2)[NH:8][C:6]1=[O:7])[CH3:4]
Reactant: CC[C@H](C)[C@@H](N)C1=N[C@@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](C(=O)N[C@@H]2CCCCNC(=O)[C@@H](CC(N)=O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H](Cc3c[nH]cn3)NC(=O)[C@H](Cc3ccccc3)NC(=O)[C@@H]([C@@H](C)CC)NC(=O)[C@H](CCCN)NC2=O)[C@@H](C)CC)CS1
Agent: CC(C)C[C@H](NC(=O)[C@@H]1CSCN1)C(=O)N[C@@H](CCC(=O)O)C(=O)O
Agent: CC(C)C[C@H](NC(=O)[C@@H]1CSCN1)C(=O)O
Agent: CC[C@H](C)[C@H](N)C(=O)N1CSC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O
Product: CC[C@H](C)[C@H](N)C1=N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(=O)O)C(=O)N[C@H](C(=O)N[C@H]2CCCCNC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](CC(=O)O)NC(=O)[C@H](Cc3cnc[nH]3)NC(=O)[C@@H](Cc3ccccc3)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](CCCN)NC2=O)[C@@H](C)CC)CS1

Procedure:
Using the same methodology as for Bacitracin A, the location of a modification responsible for the +12 Da difference from Bacitracin A, could be determined. The doubly protonated molecular ion ([M+2H]2+, m/z 718.0) was isolated and fragmented and the resulting product ion (MS/MS) spectrum is shown in FIG. 4B. The spectrum contains a full set of y″ ions with m/z 1349.7/675.82+ (loss of Ile), m/z 1236.7/619.12+ (loss of IleThz), m/z 1123.7 (loss of IleThzLeu), m/z 994.6/498.12+ (loss of IleThzLeuGlu) and m/z 869.6 (loss of IleThzLeuGluMil). These fragment assignments are visualized in FIG. 4A. In addition to the b ions with m/z 566.4 (IleThzLeuGluMil) and m/z 441.3 (IleThzLeuGlu), fragment ions resulting from pairs of bond cleavages can be seen at m/z 227.1 (ThzLeu), m/z 356.1 (ThzLeuGlu) and at m/z 481.2 (ThzLeuGluMil).