Structural Characterization and Absolute Quantification of Microcystin Peptides Using Collision-Induced and Ultraviolet Photo-Dissociation Tandem Mass Spectrometry
Microcystin (MC) peptides produced by cyanobacteria pose a hepatotoxic threat to human health upon ingestion from contaminated drinking water. While rapid MC identification and quantification in contaminated body fluids or tissue samples is important for patient treatment and outcomes, conventional immunoassay-based measurement strategies typically lack the specificity required for unambiguous determination of specific MC variants, whose toxicity can significantly vary depending on their structures. Furthermore, the unambiguous identification and accurate quantitation of MC variants using tandem mass spectrometry (MS/MS)-based methods can be limited due to a current lack of appropriate stable isotope-labeled internal standards. To address these limitations, we have systematically examined here the sequence and charge state dependence to the formation and absolute abundance of both “global” and “variant-specific” product ions from representative MC-LR, MC-YR, MC-RR, and MC-LA peptides, using higher-energy collisional dissociation (HCD)-MS/MS, ion-trap collision-induced dissociation (CID)-MS/MS and CID-MS3, and 193 nm ultraviolet photodissociation (UPVD)-MS/MS. HCD-MS/MS was found to provide the greatest detection sensitivity for both global and variant-specific product ions in each of the MC variants, except for MC-YR where a variant-specific product uniquely formed via UPVD-MS/MS was observed with the greatest absolute abundance. A simple methodology for the preparation and characterization of 18O-stable isotope-labeled MC reference materials for use as internal standards was also developed. Finally, we have demonstrated the applicability of the methods developed herein for absolute quantification of MC-LR present in human urine samples, using capillary scale liquid chromatography coupled with ultra-high resolution / accurate mass spectrometry and HCD-MS/MS.
KeywordsMicrocystin Tandem mass spectrometry Ultraviolet photodissociation Absolute quantitation
The findings and conclusions in this study are those of the authors and do not necessarily represent the views of the US Department of Health and Human Services or the US Centers for Disease Control and Prevention. Use of trade names and commercial sources is for identification only and does not constitute endorsement by the US Department of Health and Human Services or the US Centers for Disease Control and Prevention.
Financial support for this work was received under contract 200-2014-59255 from The Centers for Disease Control and Prevention (CDC), National Center for Environmental Health (NCEH), Division of Laboratory Sciences (DLS), Emergency Response Branch (ERB), Atlanta, Georgia, USA.
- 11.Ueno, Y., Nagata, S., Tsutsumi, T., Hasegawa, A., Watanabe, M.-F., Park, H.-D., Chen, G.-C., Chen, G., Yus, S.-Z.: Detection of microcystins, a blue-green algal hepatotoxin, in drinking water sampled in Haimen and Fusui, endemic areas of primary liver cancer in China, by highly sensitive immunoassay. Carcinogenesis. 17, 1317–1321 (1996)CrossRefGoogle Scholar
- 13.Diehnelt, C.W., Peterman, S.M., Budde, W.L.: Liquid chromatography–tandem mass spectrometry and accurate m/z measurements of cyclic peptide cyanobacteria toxins. Trends Anal. Chem. 24, 622–634 (2005)Google Scholar
- 18.Namikoshi, M., Rinehart, K.L., Sakai, R.: Structures of three new cyclic heptapeptide hepatotoxins produced by the cyanobacterium (blue-green alga) Nostoc sp. strain 152. J. Org. Chem. 55, 6135–6139, 1990Google Scholar
- 22.Namikoshi, M., Sun, F., Choi, B.W., Rinehart, K.L., Carmichael, W.W., Evans, W.R.: Seven more microcystins from Homer Lake cells: application of the general method for structure assignment of peptides containing alpha-, beta-dehydroamino acid unit(s). J. Org. Chem. 60, 3671–3679 (1995)CrossRefGoogle Scholar
- 27.Yuan, M., Namikoshi, M., Otsuki, A., Rinehart, K.L., Sivonen, K., Watanabe, M.F.: Low-energy collisionally activated decomposition and structural characterization of cyclic heptapeptide microcystins by electrospray ionization mass spectrometry. J. Mass Spectrom. 34, 33–34 (1999)CrossRefGoogle Scholar
- 35.Fort, K.L., Dyachenko, A., Potel, C.M., Corradini, E., Marino, F., Barendregt, A., Makarov, A.A., Scheltema, R.A., Heck, A.J.R.: Implementation of ultraviolet photodissociation on a benchtop Q exactive mass spectrometer and its application to phosphoproteomics. Anal. Chem. 88, 2303–2310 (2016)CrossRefGoogle Scholar
- 36.Cleland, T.P., DeHart, C.J., Fellers, R.T., VanNispen, A.J., Greer, J.B., LeDuc, R.D., Parker, W.R., Thomas, P.M., Kelleher, N.L., Brodbelt, J.S.: High-throughput analysis of intact human proteins using UVPD and HCD on an Orbitrap mass spectrometer. J. Prot. Res. 16, 2072–2079 (2017)CrossRefGoogle Scholar
- 40.Ozawa, K.; Fujioka, H.; Muranaka, M.; Yokoyama, A.; Katagami, Y.; Homma, T.; Ishikawa, K.; Tsujimura, S.; Kumagai, M.; Watanabe, M.F.; Park, H-D. Spatial distribution and temporal variation of Microcystis species composition and microcystin concentration in Lake Biwa. Environ. Toxicol. 20, 270–276 (2005)Google Scholar
- 42.Flores, C., Caixach, J.: An integrated strategy for rapid and accurate determination of free and cell-bound microcystins and related peptides in natural blooms by liquid chromatography-electrospray-high resolution mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry using both positive and negative ionization modes. J. Chromatogr. A. 1407, 76–89 (2015)CrossRefGoogle Scholar
- 55.Robinson, N.A., Pace, J.G., Matson, C.F., Miura, G.A., Lawrence, W.B.: Tissue distribution, excretion and hepatic biotransformation of microcystin-LR in mice. J. Pharmacol. Exp. Therapeutics. 256, 176–182 (1991)Google Scholar