Laser irradiation desorption of microcystins from protein complex in fish tissue and liquid chromatography‐tandem mass spectrometry analysis

Abstract

Microcystins are a group of cyanotoxins which interact with the C‐terminal region of PP1 and PP2A proteins, so denaturation and inactivation are necessary for breaking covalent binding to release microcystins. In this study, a novel extraction method was developed by laser irradiation desorption of microcystins from fish protein. The sample was mixed with aqueous methanol and irradiated by a 450 nm laser, with an optimized value of laser power density at 8 W and exposure time at 5 min. ThenLC–MS/MS was applied for the determination of microcystins in fish extracts. The ionization behaviors of microcystins were investigated firstly, and doubly charged microcystins were selected as precursor ions in multiple reaction monitoring scan for quantification. This proposed quantitative method was well validated in terms of selectivity, linearity, sensitivity, accuracy, recovery, and stability. The successful application of this LC–MS/MS method showed its ability for the analysis of microcystins in low concentration, and it would be of significant interest for environmental and food safety applications to ensure the safety of fish and related products.

Analysis of Total-Forms of Cyanotoxins Microcystins in Biological Matrices: A Methodological Review

“…A recent method developed by Shen et al [120] used laser irradiation desorption (LID) as a means to break the covalent bond between MCs and proteins, including PP1 and PP2A. The tests were conducted during the development of the LID protocol to study the relationship between extraction efficiency and laser power density.…”

Regulation Effectiveness and Mechanism of Biotransformation Pathway on the Toxicity of Microcystin-LR Target to Protein Phosphatase 2A

“…Protein phosphatases (PPs) play a pivotal role in regulating reversible phosphorylation in many intracellular signaling pathways [5]. After the uptake of MCs in humans, MCs mainly irreversibly inhibit the activity of PPs, resulting in hyperphosphorylation of vital cellular proteins, the disintegration of hepatocyte structures, apoptosis, intrahepatic hemorrhage, and death [6,7]. A crystal structure analysis of the complexes of MCLR (the most widespread and toxic MC) and PPs showed that MCs undergo two-step interactions with the catalytic centers of PPs [8,9].…”

“…In the first step, the side chain of Adda 5 was rapidly wrapped in the hydrophobic cage structure of the PPs. In the second step, the unsaturated carbonyl of Mdha 7 is irreversibly bound to specific cysteine residues by nucleophilic addition reaction to form covalent bonds. As a result, the key interactions between the conserved domains of PPs (especially nine strictly conserved amino acids) and the metal ions/introduced phosphate group changed accordingly, resulting in the inhibition of the PPs' activity [10].…”

“…In addition to these strategies, the biotransformation pathway is considered a valuable strategy through which to regulate the toxicity of MCs [15][16][17]. Pioneering toxicology studies found that the unsaturated Mdha 7 of MCs can covalently bind to dissociative biothiols (forming MC-biotransformation products, MC-BTPs) and block the covalent binding of MCs to PPs [18,19]. However, partial studies proposed that the first step was required for the inactivation of PPs activity, whereas the formation of covalent adducts was not [20].…”

“…Based on the same strategy, other MCLR-BTPs were identified (see TableS1). By comparing these fragment ions, it was found that the mass changes were all related to the Mdha7…”