LC/QTOF Identification of Microcystin Variants in Cell- Bound Algae from the San Francisco Estuary

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1 LC/QTOF Identification of Microcystin Variants in Cell- Bound Algae from the San Francisco Estuary Ralph Hindle Vogon Laboratory Services Ltd. Cochrane, Alberta, Canada Max Mizel Dept. of Civil and Environmental Engineering UC Davis Davis, California 1

2 Investigate impact of Microcystis aeruginosa in San Francisco Estuary Delta in summer/fall of 2014 Magnitude Timing Distribution Toxin content (cell-bound) Identify and quantitate MC-LR Identify other MC s that may be present Nitrogen source Food web impact Project Outline

3 Sampling Sites 3

4 General Microcystin Structure > 70 microcystins have been identified in the literature Microcystin Examples X Z Formula Neutral Mass LR Leucine Arginine C 49 H 74 N 10 O Desmethyl LR Leucine Arginine C 48 H 72 N 10 O RR Arginine Arginine C 49 H 75 N 13 O YR Tyrosine Arginine C 52 H 72 N 10 O LW Leucine Phenylalanine C 54 H 72 N 8 O LF Leucine Tryptophan C 52 H 71 N 7 O HtyR Homotyrosine Arginine C 53 H 74 N 10 O

5 QQQ MRM Parameters a Microcystin Precursor b (m/z) Product Ion (m/z) LR Desmethyl-LR RR YR LW LF HtyR Collision Energy (V) a These patterns are useful, as they show that all supplied MCs fragment to the same 2 diagnostic ions b RR readily shows a doubly-charged precursors. Depending on source conditions, other MCs can show either singly- or doubly-charged ions. 5

6 Common Microcystin Fragments m/z = and Glu + Mdha = C 9 H 13 N 2 O 4 + m/z = C 9 H 11 O + m/z =

7 Zoom in on common MC fragments De-methylation is common in microcystins Two 135 ions are present from 2 different fragments Indicates that Adda group is intact and not de-methylated C 9 H 11 O = C 9 H 11 O + C 10 H = C 10 H 15 +

8 LC-QTOF Instrumentation HPLC Mass Spectrometer 1290 Binary Pump 6520 or 6540 QTOF 1200 Degasser Electrospray Source 1290 Autosampler 1290 TCC 8

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10 Database Score Monoisotopic mass (varies in ppm) Isotope distribution (varies in %) Isotope spacing (varies in ppm) Score based on: Mass match measured versus given. Abundance score abundance pattern of measured isotope cluster compared with values predicted from the proposed formula. Spacing match how the m/z spacing between the lowest m/z ion and the A+1 and A+2 ions compared with the values predicted from the proposed formula.

11 Chromatography Mass tolerance 5 ppm; EIC extraction 50 ppm; spectral average < 10% saturation

12 Product Ion Spectrum for MC-LR Score = 97.7 Acc = 1.9 ppm 12

13 Product Ion Spectrum for MC-RR Score = 96.6 Acc = 2.3 ppm 13

14 Product Ion Spectrum for MC-LA Score = 95.0 Acc = 1.0 ppm 14

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16 MC-LR Desmethylation Sites C 49 H 74 N 10 O 12 Mass = CH C 48 H 72 N 10 O 12 Mass = Adda5 Glu6 Mdha7 Ala1 Leu2 Arg4 MeAsp3

17 Comparison of IC 50 for MC Variants MC Variant IC 50 (µg/ml) [Asp 3, Z-Dhb 7 ] MC-LR 0.05 [Asp 3, Z-Dhb 7 ] MC-HtyR 0.12 [Asp 3, E-Dhb 7 ] MC-LR 0.13 [Asp 3, Dha 7 ] MC-LR 0.22 [Asp 3 ] MC-LR 0.22 [Dha 7 ] MC-LR 0.22 [Asp 3, E-Dhb 7 ] MC-HtyR 0.33 [Asp 3 ] MC-HtyR 0.35 [Dha 7 ] MC-YR 0.42 MC-LR 0.80 MC-YR 1.48 [Asp 3, Dha 7 ] MC-RR 4.11 [Asp 3, E-Dhb 7 ] MC-RR 4.95 [Dha 7 ] MC-RR 5.33 [Asp 3 ] MC-YR > 10 MC-RR > 10 IC 50 = Inhibitory Concentration for cytotoxicity, from Shimizu et al, Toxins 2014, 6,

18 Arg214 Trp200 His191 3 Cys269 Glu 5 Ile123 Gln122 Tyr265 Arg89 Tyr127 Val126 X-ray crystal structures of MC-LR bound to the catalytic domain of PP2A. The data is from the protein database on the net, provided as part of Xing, Y., Xu, Y., Chen, Y., Jeffrey, P. D., Chao, Y., Lin, Z., Li, Z., Strack, S., Stock, J. B., and Shi, Y. (2006) Structure of protein phosphatase 2A core enzyme bound to tumor-inducing toxins. Cell 127, The picture was generated by Christopher Miles (Oslo, Norway) using Pymol.

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20 Fragment Without Desmethylation 20

21 ASP 3 or dm-adda 5 21

22 Desmethylation of Adda 5 22

23 dm-adda 5 23

24 40 Microcystins Found in Trial Sample Name Formula Mass Score MC-LA C46 H67 N7 O MCYST-AR C46 H68 N10 O MCYST-AR C46 H68 N10 O [D-Asp3,Glu(OMe)]MCYST-LAba C47 H69 N7 O MCYST-LAba C47 H69 N7 O [D-Asp3, Dha7]MCYST-LR C47 H70 N10 O MCYST-ER C48 H70 N10 O14 1, [D-Asp3]MCYST-LL C48 H71 N7 O [D-Asp3]MCYST-LL C48 H71 N7 O MCYST-LM C48 H71 N7 O12 S [DMAdda5]MCYST-LR C48 H72 N10 O [DMAdda5]MCYST-LR C48 H72 N10 O MCYST-MR C48 H72 N10 O12 S 1, MCYST-M(O)R C48 H72 N10 O13 S 1, [D-Asp3]MCYST-RR C48 H73 N13 O12 1, MCYST-FA C49 H65 N7 O MCYST-YAba C49 H65 N7 O MCYST-LL C49 H73 N7 O LR C49 H74 N10 O RR C49 H75 N13 O12 1, Name Formula Mass Score [L-Mser7]MCYST-LR C49 H76 N10 O13 1, MCYST-HilR C50 H76 N10 O12 1, MCYST-WA C51 H66 N8 O MCYST-NfkA C51 H66 N8 O14 1, [Asp3]MCYST-LF C51 H69 N7 O [D-Asp3]MC-FR C51 H70 N10 O12 1, [D-Asp3]MCYST-(H4)YR C51 H74 N10 O13 1, [D-Asp3]MCYST-(H4)YR C51 H74 N10 O13 1, MCYST-WAba C52 H68 N8 O LF C52 H71 N7 O MCYST-FR C52 H72 N10 O12 1, [D-Asp3]MCYST-HtyR C52 H72 N10 O13 1, MCYST-(H4)YR C52 H76 N10 O13 1, [D-Asp3]MCYST-LW C53 H70 N8 O12 1, [DMAdda5]MC-Y(OMe)R C53 H74 N10 O14 1, LW C54 H72 N8 O12 1, LW C54 H72 N8 O12 1, MCYST-WR C54 H73 N11 O12 1, LR-GSH C59 H91 N13 O18 S 1, RR-GSH C59 H92 N16 O18 S 1,

25 MC Variants All Sites 25

26 Putative MC-WR MS/MS Spectrum C 9 H 11 O + Glu + Mdha = C 9 H 13 N 2 O 4 + m/z = C 10 H

27 Microcystins Reviewed dm-rr FA FR LR MC-LA RR WA WR

28 40 microcystin variants were detected in a single San Francisco Delta algae sample immediately preceding study Accurate mass search of database with 123 unique chemical formulas Mass identification scores > 90 Mass accuracy within 2 ppm Summary MS/MS spectra for putative compounds without standards are being reviewed Semi-quantitation will be done using response factors for known standards

29 Acknowledgements California Department of Water Resources Tomo Kurobe (Project Manager) UC Davis, Davis, CA Swee Teh (data release) UC Davis, Davis, CA Christopher Miles (MC database) Norwegian Veterinary Institute (Oslo) Joe Weitzel (Travel to UC Davis) Global Marketing Manager (Agilent - USA)