Formation and Determination of Endogenous Methylated. Nucleotides in Mammals by Chemical Labeling coupled with. Mass Spectrometry Analysis

Transcription

1 Supporting Information For Formation and Determination of Endogenous Methylated Nucleotides in Mammals by Chemical Labeling coupled with Mass Spectrometry Analysis Huan Zeng, 1, Chu-Bo Qi, 1,2, Ting Liu, 1 Hua-Ming Xiao, 1 Qing-Yun Cheng, 1 Han-Peng Jiang, 1 Bi-Feng Yuan 1, *, Yu-Qi Feng 1 1 Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan , P.R. China 2 Department of Pathology, Hubei Cancer Hospital, Wuhan, Hubei , P.R. China These authors contributed equally to this work. *To whom correspondence should be addressed. Tel.: ; fax: address: bfyuan@whu.edu.cn S1

2 The Supporting Information includes following items: Page S3-S4 Page S5 Page S6 Page S7 Page S8 Page S9 Page S10 Page S11 Page S12 Page S13 Page S14 Page S15 Page S16 Page S17 Page S18 Page S19 Optimization of the extraction conditions for nucleotides and DMPA labeled nucleotide. Table S1. The gender and age of the lymphoma patients and healthy controls. Table S2. The MRM transitions and optimal parameters for the analysis of native nucleotides and DMPA-labeled nucleotides by LC-ESI-MS/MS Table S3. The extraction efficiencies of 10 nucleotides by hydrophilic Cleanert NH2 SPE cartridge. Table S4. The labeling efficiencies of 10 nucleotides by DMPA. Table S5. The recoveries of DMPA-labeled nucleotides using Strata X SPE cartridge. Table S6. Comparison of LODs of nucleotides obtained by our method with previously established methods. Table S7. Calibration curves for the analysis of 10 nucleotides by DMPA labeling coupled with LC-ESI-MS/MS analysis. Table S8. Accuracy and precision for the determination of 10 nucleotides by DMPA labeling coupled with LC-ESI-MS/MS analysis. Table S9. The measured contents of 10 nucleotides in human renal carcinoma tissues and tumor adjacent normal tissues (pmol/mg protein). Table S10. The measured contents of 10 nucleotides in 293T cells and Hela cells (pmol/mg protein). Table S11. The measured contents of 10 nucleotides in urine samples of lymphoma patients and health control (pmol/mg creatinine). Figure S1. The structures of 8 normal nucleotides. Figure S2. In addition to the methyltransferase mediated methylation of DNA and RNA, pre-methylated nucleotides also can be potentially incorporated into (A) DNA and (B) RNA during replication and transcription. Figure S3. Optimization of the enrichment conditions for nucleotides using hydrophilic Cleanert NH2 SPE cartridge. Figure S4. Optimization of the enrichment conditions for DMPA-labeled nucleotides using Strata X SPE cartridge. S2

3 Optimization of the extraction conditions for nucleotides The percentage of ACN in the loading solution was optimized ranging from 50% to 90% using Cleanert NH 2 SPE cartridge. The results showed that the peak areas of nucleotides reach plateau when the percentage of ACN was 80% (in 0.25% NH 4 OH) (Figure S3A). Therefore, we chose 80% ACN (ACN/0.25% NH 4 OH, 80/20, v/v) as the loading solution. We then optimized the percentage of ACN in desorption solution ranging from 0% to 30%. The results showed that 10% ACN was enough to desorb the nucleotides (Figure S3B). In addition, we further optimized the desorption volume ranging from 0.5 ml to 3 ml. The results showed that 2 ml of desorption solution was enough to desorb the nucleotides (Figure S3C). So we chose 2 ml of 10% ACN as the desorption solution. The eluate was then collected and evaporated to dryness under a mild nitrogen stream. The residue was dissolved in 100 µl of imidazole buffer (ph 6) and proceeded to DMPA labeling. Extraction of DMPA-labeled nucleotides After DMPA labeling, excessive DMPA and EDC existed in the reaction solution, which may compromise the subsequent LC-ESI-MS/MS analysis. In this respect, 300 µl of water and 300 µl of a dichloromethane-hexane (2:1, v/v) solvent (4 C) were added to the 100 µl of reaction solution followed by vortexing and centrifugation at 13,000 g for 5 min to remove DMPA. Then 400 µl of the upper aqueous phase was collected and mixed with 600 µl of water. Strata X SPE cartridge (30 mg/ml, Phenomenex, Guangzhou, China) was used to remove excessive EDC. We used AMP and CMP to evaluate the recoveries. As shown in Figure S4 in Supporting Information, 1 ml of 20% ACN was sufficient to desorbed S3

4 DMPA-labeled nucleotides. Under the optimized extraction conditions, the extraction efficiencies of 10 DMPA-labeled nucleotides ranged from 90.3% to 103.0% (Table S5 in Supporting Information), demonstrating good extraction efficiencies were achieved. S4

5 Table S1. The gender and age of the lymphoma patients and healthy controls. Number Gender Age Diagnosis 1 Male 48 Lymphoma 2 Male 55 Lymphoma 3 Male 53 Lymphoma 4 Male 60 Lymphoma 5 Male 42 Lymphoma 6 Female 45 Lymphoma 7 Female 41 Lymphoma 8 Female 50 Lymphoma 9 Female 49 Lymphoma 10 Female 52 Lymphoma 11 Male 42 Healthy control 12 Male 48 Healthy control 13 Male 57 Healthy control 14 Male 60 Healthy control 15 Male 53 Healthy control 16 Female 45 Healthy control 17 Female 47 Healthy control 18 Female 53 Healthy control 19 Female 58 Healthy control 20 Female 48 Healthy control S5

6 Table S2. The MRM transitions and optimal parameters for the analysis of native nucleotides and DMPA-labeled nucleotides by LC-ESI-MS/MS. Analytes Precursor ion Product ion DP/V EP/V CEP/V CE/V CXP/V damp TMP dcmp dgmp AMP UMP CMP GMP Me-dCMP Me-CMP damp-dmpa TMP-DMPA dcmp-dmpa dgmp-dmpa AMP-DMPA UMP-DMPA CMP-DMPA GMP-DMPA Me-dCMP-DMPA Me-CMP-DMPA S6

7 Table S3. The extraction efficiencies of 10 nucleotides by hydrophilic Cleanert NH 2 SPE cartridge. Analytes damp TMP dcmp dgmp AMP UMP CMP GMP 5-Me-dCMP 5-Me-CMP Recovery of nucleotides in water (%) Recovery of nucleotides spiked in urine (%) S7

8 Table S4. The labeling efficiencies of 10 nucleotides by DMPA. Analytes damp TMP dcmp dgmp AMP UMP CMP GMP 5-Me-dCMP 5-Me-CMP Labeling efficiency (%) S8

9 Table S5. The recoveries of DMPA-labeled nucleotides using Strata X SPE cartridge. Analytes damp TMP dcmp dgmp AMP UMP CMP GMP 5-Me-dCMP 5-Me-CMP Recovery (%) S9

10 Table S6. Comparison of LODs of nucleotides obtained by our method with previously established methods. LODs (pmol) Nucleotides The current work Ref 1 Ref 2 Ref 3 Ref 4 Ref 5 Ref 6 Ref 7 damp / / / / / / TMP / / / / / / dcmp / / / / / 0.03 / dgmp / / / / / 0.03 / AMP UMP / / / CMP / 34 / / GMP / Me-dCMP / / / / / / / 5-Me-CMP / / / / / / / S10

11 Table S7. Calibration curves for the analysis of 10 nucleotides by DMPA labeling coupled with LC-ESI-MS/MS analysis. Analytes Regression equation R 2 Concentration range (nm) damp y= x TMP y= x dcmp y= x dgmp y= x AMP y= x y= x UMP y= x y= x CMP y= x y= x GMP y= x y= x Me-dCMP y= x Me-CMP y= x S11

12 Table S8. Accuracy and precision for the determination of 10 nucleotides by DMPA labeling coupled with LC-ESI-MS/MS analysis. Nucleotides Spiked (pg/ul) Found (pg/ul) Relative errors (%) Intra-day Inter-day (RSD%, n=3) (RSD%, n=3) damp TMP dcmp dgmp AMP UMP CMP GMP Me-dCMP Me-CMP S12

13 Table S9. The measured contents of 10 nucleotides in human renal carcinoma tissues and tumor adjacent normal tissues (pmol/mg protein). No. Tissue damp TMP dcmp dgmp AMP UMP CMP GMP 5-Me-dCMP 5-Me-CMP Adjacent 0.6 ± ± ± ± ± ± ± ± ± ± Tumor 0.5 ± ± ± ± ± ± ± ± ± ± Adjacent 1.3 ± ± ± ± ± ± ± ± ± ± Tumor 1.1 ± ± ± ± ± ± ± ± ± ± Adjacent 0.3 ± ± ± ± ± ± ± ± ± ± Tumor 0.3 ± ± ± ± ± ± ± ± ± ± Adjacent 1.2 ± ± ± ± ± ± ± ± ± ± Tumor 1.0 ± ± ± ± ± ± ± ± ± ± Adjacent 0.5 ± ± ± ± ± ± ± ± ± ± Tumor 0.2 ± ± ± ± ± ± ± ± ± ± Adjacent 1.0 ± ± ± ± ± ± ± ± ± ± Tumor 0.3 ± ± ± ± ± ± ± ± ± ± Adjacent 0.3 ± ± ± ± ± ± ± ± ± ± Tumor 0.4 ± ± ± ± ± ± ± ± ± ± Adjacent 1.0 ± ± ± ± ± ± ± ± ± ± Tumor 0.2 ± ± ± ± ± ± ± ± ± ± Adjacent 0.6 ± ± ± ± ± ± ± ± ± ± Tumor 0.4 ± ± ± ± ± ± ± ± ± ± S13

14 Table S10. The measured contents of 10 nucleotides in 293T cells and HeLa cells (pmol/mg protein). Cells damp TMP dcmp dgmp AMP UMP CMP GMP 5-Me-dCMP 5-Me-CMP 293T 0.17± ± ± ± ± ± ± ± ± ±0.001 HeLa 0.06± ± ± ± ± ± ± ± ± ±0.002 S14

15 Table S11. The measured contents of 10 nucleotides in urine samples of lymphoma patients and healthy controls (pmol/mg creatinine). No Urine damp TMP dcmp dgmp AMP UMP CMP GMP 5-Me-dCMP 5-Me-CMP 1 Control 0.53± ± ± ± ± ± ± ± ± ± Control 1.23± ± ± ± ± ± ± ± ± ± Control 2.89± ± ± ± ± ± ± ± ± ± Control 0.71± ± ± ± ± ± ± ± ± ± Control 0.76± ± ± ± ± ± ± ± ± ± Control 4.04± ± ± ± ± ± ± ± ± ± Control 6.52± ± ± ± ± ± ± ± ± ± Control 3.29± ± ± ± ± ± ± ± ± ± Control 0.79± ± ± ± ± ± ± ± ± ± Control 0.64± ± ± ± ± ± ± ± ± ± Lymphoma 0.98± ± ± ± ± ± ± ± ± ± Lymphoma 1.34± ± ± ± ± ± ± ± ± ± Lymphoma 0.76± ± ± ± ± ± ± ± ± ± Lymphoma 0.60± ± ± ± ± ± ± ± ± ± Lymphoma 0.49± ± ± ± ± ± ± ± ± ± Lymphoma 0.38± ± ± ± ± ± ± ± ± ± Lymphoma 0.64± ± ± ± ± ± ± ± ± ± Lymphoma 0.58± ± ± ± ± ± ± ± ± ± Lymphoma 0.27± ± ± ± ± ± ± ± ± ± Lymphoma 0.29± ± ± ± ± ± ± ± ± ±0.002 S15

16 Figure S1. The structures of 8 normal nucleotides. S16

17 Figure S2. In addition to the methyltransferase mediated methylation of DNA and RNA, pre-methylated nucleotides also can be potentially incorporated into (A) DNA and (B) RNA during replication and transcription. S17

18 Figure S3. Optimization of the enrichment conditions for nucleotides using hydrophilic Cleanert NH2 SPE cartridge. (A) Optimization of the percentage of ACN in the loading solution. (B) Optimization of the percentage of ACN in the desorption solution. (C) Optimization of the desorption volume in the desorption solution. (D) Recoveries of 10 nucleotides in standard solution or spiked in human urine. S18

19 Figure S4. Optimization of the enrichment conditions for DMPA-labeled nucleotides using Strata X SPE cartridge. (A) Optimization of the percentage of ACN in desorption solution. (B) Optimization of volume of desorption solution. S19

20 References (1) Yeh, C.-F.; Jiang, S.-J. Analyst 2002, 127, (2) Cichna, M.; Raab, M.; Daxecker, H.; Griesmacher, A.; Müller, M. M.; Markl, P. J Chromatogr B 2003, 787, (3) Czarnecka, J.; Cieslak, M.; Michal, K. J Chromatogr B 2005, 822, (4) Soga, T.; Ishikawa, T.; Igarashi, S.; Sugawara, K.; Kakazu, Y.; Tomita, M. J Chromatogr A 2007, 1159, (5) Cordell, R. L.; Hill, S. J.; Ortori, C. A.; Barrett, D. A. J Chromatogr B 2008, 871, (6) Zhang, W.; Tan, S.; Paintsil, E.; Dutschman, G. E.; Gullen, E. A.; Chu, E.; Cheng, Y. C. Biochem Pharmacol 2011, 82, (7) Fukuuchi T; Yamaoka N; K., K. Anal Sci 2015, 31, S20