Tandem MS analysis of the different trypsin-treated, dehydrated species of His6-SgbA. If no mass change was observed for a specific b or y fragment after a single dehydration event, they are highlighted in blue or red, respectively. If a shift of −18 Da is observed for a specific b or y fragment, compared to the preceding +1 H2O species, they are colored in green or orange. It is apparent that the His6-SgbL(lyase-kinase 1-548) catalyzed dehydration starts at Thr2, proceeds to Thr10, continues to Thr14 and ends with modification of Ser20. The absence of some of the last b and first y fragments for the four-fold dehydrated peptide suggests that during the overnight incubation to obtain this species non-enzymatic cyclization of Cys16 and Dha20 occurred, which is in line with the NEM labeling results (Supporting Information Figure S11). For calculated and observed m/z values see Supporting Information Table S5.