First identification of PLTX-like molecules in the Atlantic coral species. Palythoa canariensis

Transcription

1 Supporting information First identification of PLTX-like molecules in the Atlantic coral species Palythoa canariensis M Fraga 1, N Vilariño 1, MC Louzao 1, L Molina 2, Y López 2, M Poli 3 and LM Botana 1 1 Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain 2 Grupo de Investigación en Acuicultura (GIA) Instituto Canario de Ciencias Marinas P.O. Box Telde, Las Palmas, Canary Islands, Spain 3 Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA Table of contents: Figure S ps3 Figure S ps4 Table S ps5 Table S ps7 Table S ps8 Figure S ps9 Table S ps10 Table S ps12 Figure S ps13 Table S ps14 Table S-7 ps16 Figure S ps18 Table S-8 ps19 S1

2 The present Supporting information contains data obtained along the experimental process. The first figure displays the chemical structure of the palytoxin (PLTX). The second figure presents the ionization (MS 1 ) and fragmentation (MS 2 ) spectra of PLTX found in the commercial standard (Palythoa tuberculosa origin) obtained from Wako. The first and second tables show, respectively, theoretical formulae and m/z expected for the ionization pattern of PLTX and some of its cleavage ions and relate them to those found in the PLTX standard and in the Palythoa canariensis sample. The third table compares the similarity of the spectra of the main peak (m/z ) from PLTX and the spectra of molecules with the same main ion eluting a different RT found in the PLTX standard and Palythoa canariensis sample. The third figure reflects the ions (MS 1 ) and fragments (MS 2 ) found for OH-PLTX in the Palythoa tuberculosa extract. The fourth and fifth tables display the respective theoretical ionization (MS 1 ) and some fragments (MS 2 ), including theoretical formulae and m/z, found for OH-PLTX (from the Palythoa tuberculosa extract) and relate them to those in the Palythoa canariensis sample. The fourth figure shows the ionization and the fragmentation spectra of m/z found in the commercial standard from Wako. The sixth table describes the theoretical m/z of this compound based on PLTX ionization profile and the possible correlation with the MS 1 spectrum of the molecule with m/z found in the Palythoa tuberculosa and Palythoa canariensis samples. The seventh table reflects the estimated mass pattern for the molecule D found in the Palythoa canariensis sample. The fifth figure shows the calibration curve for PLTX obtained with the microsphere-based immunoassay. The eighth table shows the quantification of PLTX and PLTX-like molecules of the Palythoa canariensis extract using the immunoassay and UPLC-IT-TOF-MS. S2

3 Figure S-1. Chemical structure of PLTX. S3

4 Figure S-2. MS 1 and m/z MS 2 obtained by UPLC-IT-TOF-MS using the PLTX commercial standard. A) MS 1 spectrum, with i) tri-; ii and iii) bi-; iv) A-moiety and v) monocharged ions, obtained for the most intense peak with RT of 7.2 (Figure 2B of main text). B) MS 2 spectra obtained after the fragmentation of m/z : i) m/z ; ii) m/z ; and iii) m/z S4

5 Table S-1. Correspondence between the theoretical mass pattern described for PLTX and the m/z found by UPLC-IT-TOF-MS in the PLTX standard and the Palythoa canariensis sample. The information in brackets specifies the closest m/z found within the indicated cluster from Figure 3A (main text) for Palythoa canariensis or Figure S-2A for the palytoxin standard. *ions with intensity lower than 3X background signal. Theoretical m/z P. tuberculosa sample from Wako Retention time 7.2 min m/z found P. canariensis sample (M+H) (M+H-H 2 O) (M+H-2H 2 O) (M+H-A moiety-h 2 O) (M+H-A moiety-2h 2 O) ( )* a (M+H-A moiety-3h 2 O) ( ) ( ) (M+H-A moiety-4h 2 O) ( ) ( ) (M+H-A moiety-5h 2 O) * ( ) (M+H-A moiety-6h 2 O) * ( ) (M+H-A moiety-7h 2 O) * (M+Ca+H 2 O) (M+H+K+H 2 O) (M+Mg+H 2 O) (M+H+Na+H 2 O) (M+Ca) (M+H+K) (M+Mg) (M+H+Na) ( ) * (M+2H) * - (M+2H-H 2 O) (M+2H-2H 2 O) (M+2H-3H 2 O) ( ) (M+2H-4H 2 O) (M+2H-5H 2 O) (M+2H-6H 2 O) (M+2H-7H 2 O) (M+2H-8H 2 O) (M+2H-9H 2 O) S5

6 Continuation of table S-1 Theoretical m/z P. tuberculosa sample from Wako Retention time 7.2 min m/z found P. canariensis sample (M+2H-A moiety) (M+2H-A moiety-h 2 O) (M+2H-A moiety-2h 2 O) (M+2H-A moiety-3h 2 O) (M+2H-A moiety-4h 2 O) * - (M+2H-A moiety-5h 2 O) ( ) (M+2H-A moiety-6h 2 O) (M+2H-A moiety-7h 2 O) ( ) (M+2H-A moiety-8h 2 O) ( ) (M+2H-A moiety-9h 2 O) ( ) (M+2H-A moiety-10h 2 O) ( ) (M+2H-A moiety-11h 2 O) ( ) ( ) (M+H+Ca+H 2 O) (M+2H+K+H 2 O) (M+H+Mg+H 2 O) (M+2H+Na+H 2 O) (M+H+Ca) (M+2H+K) (M+H+Mg) (M+2H+Na) (912.80) (907.48) (907.48) (M+3H) (894.13) (894.13) (M+3H-H 2 O) * (888.13) (M+3H-2H 2 O) * (882.13) (M+3H-3H 2 O) (M+3H-4H 2 O) (M+3H-5H 2 O) (M+3H-6H 2 O) (M+3H-7H 2 O) (M+3H-8H 2 O) (M+3H-9H 2 O) (M+3H-10H 2 O) S6

7 Table S-2. Correspondence between a partial theoretical mass pattern described for the internal and extreme fragmentation of PLTX and the m/z found by UPLC-IT-TOF-MS in the commercial PLTX standard and the Palythoa canariensis samples. In extreme cleavages italic text was used to indicate the fragments corresponding to the B-side of the PLTX molecule and those with higher intensity were underlined. *ions with intensity lower than 3X background signal. Cleavages INTERNAL CLEAVAGES Theoretical m/z found in P. Formula (charge) m/z tuberculosa (Wako) m/z found in P. canariensis C 8-9 /C C 36 H 64 O 13 Ca (+2) C 8-9 /C C 40 H 72 O 16 Ca (+2) C 8-9 /C C 43 H 78 O 18 Ca (+2) * (-H 2 O) * (-H 2 O) C /C C 26 H 45 O 5 Ca (+1) C /C C 27 H 47 O 6 Ca (+1) Cleavages C 8-9 C C C C EXTREME CLEAVAGES Formula (charge) Theoretical found m/z P. found m/z m/z tuberculosa (Wako) P. canariensis C 16 H 27 O 5 N 2 (+1) (-H 2 O) (-H 2 O)* * * C 113 H 195 O 48 NCa (+2) (-H 2 O)* (-H 2 O)* (-2H 2 O) (-2H 2 O) C 52 H 92 O 19 N 2 Ca (+2) C 77 H 125 O 32 NCa (+2) * C 54 H 96 O 20 N 2 Ca (+2) C 75 H 125 O 33 NCa (+2) * * C 56 H 100 O 22 N 2 Ca (+2) C 73 H 123 O 32 NCa (+2) * C 59 H 106 O 24 N 2 Ca (+2) C 70 H 117 O 30 NCa (+2) S7

8 Table S-3. Percentage of similarity of MS 1 and MS 2 of m/z obtained from PLTX versus the spectra of molecules with a tricharged ion of m/z and RT different than 7.2 (PLTX RT) found in the commercial PLTX standard and Palythoa canariensis using the LabSolutions-LCMS software (Shimadzu, Kyoto, Japan). Percentage of similarity with PLTX standard at RT 7.2 (%) Retention time Commercial PLTX (Wako) Palythoa canariensis sample (min) MS 1 MS 2 MS 1 MS S8

9 Figure S-3. MS 1 and m/z MS 2 obtained by UPLC-IT-TOF-MS for 42-OH-PLTX from the Palythoa tuberculosa extract. A) MS 1 spectrum, with i) tri-; ii and iii) bi-; iv) A- moiety and v) monocharged ions, obtained for the molecule eluting at RT 7.0 in the Palythoa tuberculosa extract (Figure 2C of main text). B) Detailed MS 2 spectrum from the fragmentation of m/z : i) m/z , ii) m/z and iii) m/z 1120 to S9

10 Table S-4. Correspondence between the theoretical mass pattern described for OH- PLTX and ion m/z found by UPLC-IT-TOF-MS in the Palythoa tuberculosa extract and the Palythoa canariensis sample. The information in brackets specifies the closest m/z found within the indicated cluster from Figure 4A (main text) for Palythoa canariensis or Figure S- 3A for Palythoa tuberculosa. *ions with intensity lower than 3X background signal. Retention time 7.0 min m/z found Theoretical m/z P. tuberculosa P. canariensis extract Sample (M+H) (M+H-A moiety-h 2 O) (M+H-A moiety-2h 2 O) (M+H-A moiety-3h 2 O) * - (M+H-A moiety-4h 2 O) ( ) - (M+H-A moiety-5h 2 O) * - (M+Ca+H 2 O) * ( ) (M+H+K+H 2 O) ( ) (M+Mg+H 2 O) ( ) ( ) (M+H+Na+H 2 O) (M+Ca) (M+H+K) (M+Mg) (M+H+Na) ( ) ( ) ( ) ( ) (M+2H) (M+2H-H 2 O) (M+2H-2H 2 O) (M+2H-3H 2 O) (M+2H-4H 2 O) (M+2H-5H 2 O) ( ) (M+2H-6H 2 O) * ( ) (M+2H-7H 2 O) ( ) (M+2H-A moiety-h 2 O) (M+2H-A moiety-2h 2 O) (M+2H-A moiety-3h 2 O) (M+2H-A moiety-4h 2 O) * * ( ) ( ) S10

11 Continuation of the table S-4 Theoretical m/z P. tuberculosa extract Retention time 7.0 min m/z found P. canariensis sample (M+2H-A moiety-5h 2 O) ( ) (M+2H-A moiety-6h 2 O) ( ) * ( ) (M+2H-A moiety-7h 2 O) * * ( ) (M+2H-A moiety-8h 2 O) * ( ) (M+2H-A moiety-9h 2 O) * ( ) * ( ) (M+2H-A moiety-10h 2 O) *( ) * ( ) (M+H+Ca+H 2 O) (M+2H+K+H 2 O) (M+H+Mg+H 2 O) (M+2H+Na+H 2 O) (M+H+Ca) (M+2H+K) (M+H+Mg) (M+2H+Na) (918.14) (918.15) (912.81) (912.82) (912.15) (912.16) (906.65) (906.82) (M+3H) (899.56) (899.46) (M+3H-H 2 O) *(893.47) (893.48) (M+3H-2H 2 O) (887.47) * (887.43) (M+3H-3H 2 O) (881.49) * (881.49) (M+3H-4H 2 O) (M+3H-5H 2 O) (M+3H-6H 2 O) (M+3H-7H 2 O) (M+3H-8H 2 O) * (M+3H-9H 2 O) (845.47) (M+3H-10H 2 O) * S11

12 Table S-5. Correspondence between a partial theoretical mass pattern, obtained through that described for PLTX, for the internal and extreme fragmentation of OH- PLTX and the m/z found by UPLC-IT-TOF-MS in the Palythoa tuberculosa extract and the Palythoa canariensis sample. In extreme cleavages italic text was used to indicate the fragments corresponding to the B-side of the OH-PLTX molecule and those with higher intensity were underlined.*ions with intensity lower than 3X background signal. Cleavages INTERNAL CLEAVAGES Theoretical found m/z Formula (charge) m/z P. tuberculosa extract found m/z P. Canariensis C 8-9 /C C 36 H 64 O 13 Ca (+2) C 8-9 /C C 40 H 72 O 17 Ca (+2) * (-H 2 O) C 8-9 /C C 43 H 78 O 19 Ca (+2) * * * (-H 2 O) (-2H 2 O) C /C C 26 H 45 O 5 Ca (+1) C /C C 27 H 47 O 6 Ca (+1) Cleavages C 8-9 C C C C EXTREME CLEAVAGES Formula (charge) Theoretical found m/z found m/z m/z P. tuberculosa extract P. Canariensis C 16 H 27 O 5 N 2 (+1) (-H 2 O) (-H 2 O) * * C 113 H 195 O 49 NCa (+2) * (-H 2 O)* (-2H 2 O) (-2H 2 O) C 52 H 92 O 19 N 2 Ca (+2) C 77 H 125 O 32 NCa (+2) C 54 H 96 O 21 N 2 Ca (+2) * C 75 H 125 O 33 NCa (+2) C 56 H 100 O 23 N 2 Ca (+2) C 73 H 123 O 32 NCa (+2) * * C 59 H 106 O 25 N 2 Ca (+2) * C 70 H 117 O 30 NCa (+2) * * S12

13 Figure S-4. MS 1 and m/z MS 2 of the molecule eluting at RT 7.4 min found in the PLTX standard obtained by UPLC-IT-TOF-MS. A) MS 1 spectra, with i) tri-; and ii and iii) bicharged ions, obtained for the molecule eluting at RT 7.4 min (Figure 2B of main text). B) Detailed MS 2 spectrum of m/z fragmentation: i) m/z ; ii) m/z ; and iii) m/z S13

14 Table S-6. PLTX-like mass pattern for compound with m/z calculated using PLTX mass pattern as a model and its correlation with m/z found by UPLC-IT-TOF-MS for suspected PLTX-like molecules with m/z in the palytoxin standard (Wako) and Palythoa canariensis samples at RT 7.4 min. The information in brackets specifies the closest m/z found within the indicated cluster from Figure 5A (main text) for Palythoa canariensis or Figure S-4A for Palythoa tuberculosa. *ions with intensity lower than 3X background signal. Theoretical m/z Retention time 7.4 min m/z found P. tuberculosa sample from Wako (M+H) (M+Ca+H 2 O) (M+H+K+H 2 O) (M+Mg+H 2 O) (M+H+Na+H 2 O) (M+Ca) (M+H+K) (M+Mg) (M+H+Na) P. canariensis sample ( ) ( ) (M+2H) (M+2H-H 2 O) (M+2H-2H 2 O) (M+2H-3H 2 O) * - (M+2H-4H 2 O) (M+2H-5H 2 O) (M+2H-6H 2 O) (M+2H-7H 2 O) (M+2H-8H 2 O) (M+2H-9H 2 O) ( ) (M+2H-10H 2 O) (M+2H-11H 2 O) ( ) (M+2H-12H 2 O) S14

15 Continuation of Table S-6 Theoretical m/z Retention time 7.4 min m/z found P. tuberculosa sample from Wako P. canariensis sample (M+2H-A moiety-4h 2 O) ( ) * (M+2H-A moiety-5h 2 O) ( ) ( ) (M+2H-A moiety-6h 2 O) ( ) ( ) (M+2H-A moiety-7h 2 O) h (M+2H-A moiety-8h 2 O) ( ) (M+2H-A moiety-9h 2 O) ( ) ( ) (M+2H-A moiety-10h 2 O) ( ) ( ) (M+2H-A moiety-11h 2 O) ( ) (M+2H-A moiety-12h 2 O) (M+H+Ca+H 2 O) (M+2H+K+H 2 O) (M+H+Mg+H 2 O) (M+2H+Na+H 2 O) (M+H+Ca) (M+2H+K) (M+H+Mg) (M+2H+Na) (870.79) (865.46) (865.46) (859.45) (859.44) (M+3H) (852.07) (852.08) (M+3H-H 2 O) (846.09) (846.09) (M+3H-2H 2 O) * (840.11) (M+3H-3H 2 O) (834.07) * (834.08) S15

16 Table S-7. Estimation of PLTX-like mass pattern for the Palythoa canariensis molecule eluting at RT 5.9 min (Figure 2A, molecule D) based on PLTX and OH-PLTX ion profiles and its correlation with actual m/z found by UPLC-IT-TOF-MS for this molecule (Figure 6, main text). The information in brackets specifies the closest m/z found within the indicated cluster from Figure 6A (main text). *ions with intensity lower than 3X background signal. Theoretical m/z Retention time 5.9 min m/z found in P. canariensis sample (M+H) (M+Ca+H 2 O) (M+H+K+H 2 O) (M+Mg+H 2 O) (M+H+Na+H 2 O) (M+Ca) (M+H+K) (M+Mg) (M+H+Na) * * (M+2H) ( ) (M+2H-H 2 O) ( ) (M+2H-2H 2 O) ( ) (M+2H-3H 2 O) (M+2H-4H 2 O) *( ) (M+2H-5H 2 O) (M+2H-6H 2 O) * (M+2H-7H 2 O) (M+2H-8H 2 O) ( ) (M+2H-A moiety) (M+2H-A moiety-h 2 O) * (M+2H-A moiety-2h 2 O) ( ) (M+2H-A moiety-3h 2 O) ( ) (M+2H-A moiety-4h 2 O) ( ) (M+2H-A moiety-5h 2 O) ( ) (M+2H-A moiety-6h 2 O) ( ) (M+2H-A moiety-7h 2 O) ( ) (M+2H-A moiety-8h 2 O) * ( ) S16

17 Continuation of Table S-7 Theoretical m/z (M+H+Ca) (M+2H+K) (M+H+Mg) (M+2H+Na) Retention time 5.9 min m/z found in P. canariensis sample (984.82) (979.50) (M+3H) (972.16) (M+3H-H 2 O) (966.18) (M+3H-2H 2 O) (960.18) (M+3H-3H 2 O) (954.18) (M+3H-4H 2 O) (948.17) (M+3H-5H 2 O) (942.17) (M+3H-6H 2 O) (936.17) (M+3H-7H 2 O) (930.16) (M+3H-8H 2 O) (924.17) (M+3H-9H 2 O) S17

18 Figure S-5. Calibration curve of PLTX using the microsphere-based immunoassay. A) Calibration curve performed in buffer solution used for the estimation of the content of PLTX-like molecules in the Palythoa canariensis extracts. B) IC 20, IC 50 and IC 80 of the PLTX calibration curve in nm and ng/ml (mean ± SEM, n=3). A) B) nm ng/ml IC ± ± 0.45 IC ± ± 0.32 IC ± ± 1.6 S18

19 Table S-8. Estimates of PLTX-like molecules content using the microsphere-based immunoassay and UPLC-IT-TOF-MS. Total PLTX-like molecules estimated by the immunoassay (n=3, mean ± SEM) or UPLC-IT-TOF are reported. For UPLT-IT-TOF-MS, individual estimated amount of each molecule with PLTX-like mass spectrum is also shown. Concentrations are expressed in mg of PLTX-like molecules per g of lyophilized coral. Microsphere-based immunoassay LC-MS-IT-TOF Total PLTX-like molecules m/z and RT Individual quantification of m/z Total PLTX-like molecules m/z , RT mg/g 2.32 ± 0.26 mg/g m/z , RT 7.2 m/z , RT mg/g 0.07 mg/g 0.55 mg/g m/z , RT mg/g m/z , RT mg/g S19