Novel in-source fragments used to identify linear microcystins with C-terminal arginine by UHPLC-ESI-MS/MS

Degradation of cyclic cyanotoxins microcystins (MCs) leads to the formation of linear MC heptapeptides. Herein, several linear MCs were identified by UHPLC-ESI-Orbitrap-MS/MS in water samples collected from Lake Erie shore during the cyanobacterial harmful algal bloom (cyanoHAB) 2022. Consequently, novel in-source fragments of linear MCs, which contain a modified Adda moiety and C-terminal arginine (Arg), were discovered and their structures were putatively assigned using high-resolution MS and MS/MS data. The fragment ion at m/z 175.11 was used as the major diagnostic ion to identify whether a MC is linear with Arg at the C-terminus. The distinguishing feature of the novel linear MC in-source fragments is the absence of the characteristic Adda fragment ion m/z 135.08. However, these contain other fragment ions formed by the remaining part of the Adda, such as m/z 135.11 and m/z 163.11. In-source fragments that were formed when C-terminal Arg-containing linear MC precursor ions lost C₆H₅-CH=CH(OCH₃) and NH₃ from the Adda moiety confirmed the presence of linear MC-RR, MC-LR, [Asp³]MC-LR, MC-YR, MC-HilR, MC-HtyR, MC-FR, and MC-HphR in Lake Erie water. The loss of specific neutral molecules during ESI assisted the identification of linear MCs and helps avoiding the missannotation of in-source fragments as precursor ions resulting from MC degradation products or other precursor molecules.