HPLC-ESI-QqTOF MS analysis of SAM and SAH extracted from PC-3 cells CZ.1.07/2.3/.00/09.0224 NANOTEAM Budování výzkumných týmů a rozvoj univerzitního vzdělávání výzkumných odborníků pro mikro- a nanotechnologie HPLC-ESI-QqTOF MS analysis of SAM and SAH extracted from PC-3 cells Roman Guráň 27.05.2016

Aims of the study Optimize SAM and SAH extraction from cells. Develop and apply HPLC-ESI-qTOF MS method for analysis of PC-3 extracts. Investigate the SAM and SAH concentrations and their ratio (SAM/SAH), compare PC-3 cells with normal prostate cells.

SAM and SAH S-(5’-adenosyl)-L-methionine (SAM) and S-(5’-adenosyl)-L-homocysteine (SAH) are intermediates in methionine cycle. SAH is formed by SAM demethylation by nicotinamide N-methyl-transferase. The enzyme is overexpressed in some types of tumors and therefore it can enhance cancer aggressiveness by draining methyl groups from SAM. Shlomi, T. and J. D. Rabinowitz (2013). "Metabolism: Cancer mistunes methylation." Nat Chem Biol 9(5): 293-294.

HPLC-ESI-QqTOF MS HPLC system: two chromatographic pumps ESA Model 584 and ESA autosampler Model 542 (ESA Inc., Chelmsford, MA). ESI-QqTOF MS: Bruker maxis impact (Bruker Daltonik GmbH, Germany)

Route of ions through Bruker maxis impact 1) API interface 2) Desolvation unit 3) Double stage Ion Funnel and Hexapole 4) Analytical Quadrupole and Collision Cell (Q-q-stage) 5) TOF mass analyzer

PC-3 harvesting and extraction protocol PC-3 cells were maintained monolayer culture in the Ham's F-12 containing 7% fetal bovine serum (FBS) and 100 units/ml penicillin and 100 mg/ml streptomycin. Cells were grown at 37°C with 5% CO2. Cells at ~80% confluence were harvested using Trypsin/EDTA. 1×106 cells were pelleted by centrifugation at 200× g for 10 min at 4 °C. SAM and SAH extraction*: 600 µl of MeOH/1M acetic acid (80:20), which could contain spikes of SAM and SAH at desired concentration, were added to frozen PC-3 pellets in microvial. Pellets were slowly thawed on ice and to complete cells lysis, the cells were shock-frozen in liquid nitrogen and again thawed on ice. The freeze/thaw cycle was repeated three times. Sample was vortexed between the cycles. Sample was centrifuged at 9000 x g at 4 °C for 5 minutes and supernatant was transfered into clean 1,5 ml glass microvial. Pellet was washed twice by 200 µl of MeOH/1M acetic acid (80:20) and all supernatants were combined → final volume of PC-3 extract was 1 ml. (At the end, Stevens et al. used also vacuum centrifugation to concentrace cell extract) *) Adapted and slightly changed from: Stevens, A. P., K. Dettmer, et al. (2010). "Quantification of intermediates of the methionine and polyamine metabolism by liquid chromatography–tandem mass spectrometry in cultured tumor cells and liver biopsies." Journal of Chromatography A 1217(19): 3282-3288.

HPLC conditions Column: Kinetex® 5 µm EVO C18 Mobile phase A: Water + 0.1% HCOOH (both in MS quality) Mobile phase B: Methanol + 0.1% HCOOH (both in MS quality) Injection loop volume: 100 µl Gradient: 0.01 min flow 0.5 ml/min and 3 % B → 6 min 30 % B → 10 min 30 % B → 12 min 85 % B → 15 min 3 % B → 20 min 3 % B → 20.01 min flow 0.05 ml/min and 3 % B → 999.99 min flow 0.05 ml/min and 3 % B

Calibration From regression equation LOD 0,01 nM LOQ 0,03 Standards: S-(5’-adenosyl)-L-methionine iodide, MW = 526.35, MWSAM,monisotopic = 398.14, wSAM = 0.7564 S-(5’-adenosyl)-L-homocysteine, crystalline, MWmonoisotopic = 384.12 Stock solution of mixed SAM and SAH was prepared in water with 0.1% HCOOH (both in MS quality). Concentrations of SAM and SAH in stock solution were both 1 mg/ml. Calibration samples were prepared in concentrations 0.01, 0.0001 and 0.000001 mg/ml by dilution of stock solution. Then the concentration mg/ml was recalculated into nM. All samples were measured three times (n = 3). From regression equation LOD 0,01 nM LOQ 0,03 According to the lowest used concentration 0,57 1,90 From regression equation LOD 0,05 nM LOQ 0,17 According to the lowest used concentration 0,78 2,60

First analysis of PC-3 extract Typical SAM and SAH extracted ion chromatogram of PC-3 extract 2 4 6 8 10 12 14 16 Time [min] 1 3 x10 Intens. PC-3 extract_4.d: EIC 399.14 +All MS PC-3 extract_4.d: EIC 385.12 +All MS Spectrum View 336.0390 385.1118 399.1267 407.0919 423.0649 437.0798 PC-3 extract_4.d: +MS, 3.2min #194 340 360 380 400 420 440 460 480 m/z 348.0555 385.1116 PC-3 extract_4.d: +MS, 6.0min #362 c [nM/106 cells] c [nmol/106 cells] SD [nM/106 cells] SD [nmol/106 cells] RSD [%] SAM (peak 1) 3584.23 3.58 187.81 0.19 5 SAH (peak 2 and 3 together) 9580.18 9.58 118.80 0.12 1 n = 3 c [nmol/106 cells] SD [nmol/106 cells] SAM (in HTZ-19 melanoma cells) 1162.50 86.17 SAH (in HTZ-19 melanoma cells) 108.15 11.45 SAM (in Mel Im melanoma cells) 942.50 15.33 SAH (in Mel Im melanoma cells) 82.60 2.73 SAM (in PLC hepatoma cells) 939.39 178.57 SAH (in PLC hepatoma cells) 15.19 3.38 Ratio cSAM/cSAH: 0.37 Comparison with literature (different cancer cells)*: *) Stevens, A. P., K. Dettmer, et al. (2010). "Quantification of intermediates of the methionine and polyamine metabolism by liquid chromatography–tandem mass spectrometry in cultured tumor cells and liver biopsies." Journal of Chromatography A 1217(19): 3282-3288.

Conclusion and outlooks The concentration of SAM in 1 ml extract of PC-3 cells was 3.58 ± 0.19 nmol/106 cells. The concentration of SAH in 1 ml extract of PC-3 cells was 9.58 ± 0.12 nmol/106 cells. These results are preliminary, more samples of PC-3 cells and also of normal prostate cells will be analyzed when they will be grown. Then PC-3 cells will be compared with normal prostate cells. New calibration will be made from PC-3 cell extracts spiked with internal standards of SAM and SAH and then new experiment focused on treating PC-3 cells with different compounds will be started. http://www.lgcstandards-atcc.org/products/all/CRL-1435.aspx?geo_country=cz#characteristics

Thank you for your attention! Acknowledgment To all colleagues from Department of Chemistry and Biochemistry. Thank you for your attention! https://en.wikipedia.org/wiki/File:PC3_cell.jpg