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 430072, P.R. China 2 Department of Pathology, Hubei Cancer Hospital, Wuhan, Hubei 430079, P.R. China These authors contributed equally to this work. *To whom correspondence should be addressed. Tel.: +86-27-68755595; fax: +86-27-68755595. E-mail address: bfyuan@whu.edu.cn S1
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
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
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
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
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 332.2 136.1 20 8 15 22 3.0 TMP 323.2 127.1 34 7 15 28 3.0 dcmp 308.2 112.2 22 8 11 23 2.5 dgmp 348.2 152.1 38 7 10 25 4.2 AMP 348.2 136.2 18 7 16 20 3.2 UMP 325.1 113.2 24 6 14 15 3.5 CMP 324.2 112.2 25 7 17 28 2.4 GMP 364.1 152.2 42 6 11 25 4.5 5-Me-dCMP 322.1 126.2 32 6 16 19 2.7 5-Me-CMP 338.2 126.1 30 5 17 21 3.0 damp-dmpa 450.2 216.2 32 7 16 34 3.0 TMP-DMPA 441.2 216.2 33 7 19 29 3.2 dcmp-dmpa 426.2 216.2 26 7 19 30 2.6 dgmp-dmpa 466.2 216.1 35 8 16 34 3.5 AMP-DMPA 466.2 136.1 42 8 16 47 2.6 UMP-DMPA 443.1 216.2 40 7 20 33 3.6 CMP-DMPA 442.1 216.2 32 6 21 32 2.6 GMP-DMPA 482.2 216.2 35 8 16 36 3.5 5-Me-dCMP-DMPA 440.2 126.1 23 8 18 27 3.6 5-Me-CMP-DMPA 456.2 126.1 35 7 16 38 2.0 S6
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 (%) 90.3 90.3 100.8 101.2 96.6 95.1 90.4 92.4 92.7 91.0 Recovery of nucleotides spiked in urine (%) 90.5 92.3 99.7 103.3 97.0 95.2 88.3 93.6 91.4 89.2 S7
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 (%) 92.6 100.3 94.2 92.7 92.3 84.4 85.7 85.2 90.4 86.8 S8
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 (%) 100.1 103.0 90.6 95.5 100.2 94.3 90.3 90.8 94.9 92.6 S9
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 1.3 10-4 / / / / / 0.003 / TMP 1.3 10-4 / / / / / 0.006 / dcmp 4.2 10-4 / / / / / 0.03 / dgmp 3.8 10-4 / / / / / 0.03 / AMP 1.2 10-4 37.3 63 0.1 0.04 2.5 2.50 3.9 UMP 2.6 10-4 48.7 49 / / 5.0 0.47 / CMP 4.7 10-4 / 34 / 0.02 5.0 0.83 / GMP 4.3 10-4 58.7 63 / 0.03 5.0 0.40 4.0 5-Me-dCMP 3.0 10-4 / / / / / / / 5-Me-CMP 3.6 10-4 / / / / / / / S10
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=1.26397x+0.00441 0.9992 0.3-300 TMP y=1.24157x-0.02381 0.9981 0.3-300 dcmp y=6.24658x+0.20742 0.9998 0.3-300 dgmp y=3.10966x+0.00813 0.9943 0.3-300 AMP y=0.09878x+0.001587 0.9983 0.3-300 y=0.08642x+0.010812 0.9990 300-3000 UMP y=0.25545x+0.001197 0.9990 0.3-300 y=0.22315x+0.00825 1.0000 300-3000 CMP y=0.12577x+0.06352 0.9992 0.3-300 y=0.10347x+0.09194 0.9999 300-3000 GMP y=1.80641x+0.03011 1.0000 0.3-300 y=1.68628x+0.09154 0.9998 300-3000 5-Me-dCMP y=32.11071x-0.00458 1.0000 0.015-15 5-Me-CMP y=3.55975x-0.05666 0.9939 0.018-15 S11
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 0.1 0.09-10.0 8.2 5.3 2.0 1.84-8.0 3.7 8.5 20.0 18.66-6.7 6.9 10.2 TMP 0.1 0.09-10.0 2.5 8.0 2.0 1.95-2.5 4.9 4.4 20.0 18.4-8.0 1.1 5.6 dcmp 0.05 0.047-6.0 8.3 13.3 0.3 0.29-3.3 8.8 6.2 2.5 2.45-2.0 6.3 8.8 dgmp 0.1 0.09-10.0 7.3 12.5 2.0 1.97-1.5 4.2 8.4 20.0 19.7-1.5 6.9 5.3 AMP 2.0 1.9-5.0 3.7 2.1 20.0 20.2 1.0 2.9 3.9 100.0 98.0-2.0 1.2 1.8 UMP 2.0 1.88-6.0 8.2 6.3 20.0 17.8-11.0 9.4 12.7 100.0 99.4-0.6 5.2 8.6 CMP 2.0 1.7-15.0 4.6 7.9 20.0 18.1-9.5 3.4 3.1 100.0 90.2-9.8 2.1 4.6 GMP 0.1 0.086-14.0 7.8 11.2 2.0 1.79-10.5 5.3 8.3 20.0 19.3-3.5 6.5 12.1 5-Me-dCMP 0.01 0.0086-14.0 9.7 15.3 0.05 0.0447-10.6 8.1 12.5 1.0 0.952-4.8 9.4 8.9 5-Me-CMP 0.05 0.043-14.0 9.1 11.5 0.3 0.27-9.7 7.3 13.8 2.5 2.39-4.5 5.5 8.5 S12
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 01 02 03 04 05 06 07 08 09 Adjacent 0.6 ±0.04 1.8 ±0.08 0.96 ±0.034 2.2 ±0.13 29.5 ±1.3 9.5 ±0.5 17.7 ±0.9 6.4 ±0.1 0.0079 ±0.0000 0.013 ±0.0016 Tumor 0.5 ±0.01 1.3 ±0.09 0.37 ±0.000 1.1 ±0.08 26.6 ±0.5 13.1 ±0.6 32.5 ±0.2 9.4 ±0.4 0.0026 ±0.0002 0.011 ±0.0012 Adjacent 1.3 ±0.02 0.7 ±0.03 0.97 ±0.083 0.6 ±0.07 35.4 ±1.0 4.6 ±0.5 9.6 ±0.3 4.6 ±0.5 0.0103 ±0.0005 0.008 ±0.0007 Tumor 1.1 ±0.00 1.0 ±0.06 0.21 ±0.012 0.9 ±0.05 32.9 ±0.9 7.0 ±0.6 35.9 ±1.5 9.8 ±0.6 0.0045 ±0.0002 0.006 ±0.0000 Adjacent 0.3 ±0.02 1.1 ±0.03 1.64 ±0.065 2.0 ±0.15 49.9 ±0.7 4.4 ±0.1 11.2 ±0.9 3.1 ±0.2 0.0037 ±0.0001 0.005 ±0.0003 Tumor 0.3 ±0.01 2.1 ±0.13 0.26 ±0.029 0.6 ±0.03 16.7 ±0.8 10.8 ±0.2 14.5 ±0.5 5.9 ±0.2 0.0028 ±0.0003 0.010 ±0.0008 Adjacent 1.2 ±0.01 0.7 ±0.08 1.16 ±0.044 0.5 ±0.06 30.4 ±1.4 4.3 ±0.3 11.1 ±0.4 3.3 ±0.0 0.0035 ±0.0002 0.005 ±0.0005 Tumor 1.0 ±0.07 1.1 ±0.06 0.28 ±0.031 0.3 ±0.00 10.4 ±0.7 4.9 ±0.1 18.6 ±0.5 3.8 ±0.1 0.0026 ±0.0001 0.006 ±0.0007 Adjacent 0.5 ±0.04 0.8 ±0.09 1.41 ±0.091 0.9 ±0.03 18.4 ±1.2 3.8 ±0.4 10.3 ±0.9 2.6 ±0.3 0.0070 ±0.0008 0.004 ±0.0001 Tumor 0.2 ±0.02 1.0 ±0.09 0.49 ±0.000 0.7 ±0.05 18.7 ±1.0 4.0 ±0.5 21.6 ±0.1 6.8 ±0.5 0.0037 ±0.0004 0.006 ±0.0007 Adjacent 1.0 ±0.08 0.8 ±0.05 1.17 ±0.059 0.5 ±0.01 16.2 ±0.9 2.9 ±0.2 13.6 ±0.3 4.8 ±0.3 0.0100 ±0.0009 0.008 ±0.0009 Tumor 0.3 ±0.02 1.1 ±0.01 0.27 ±0.030 1.3 ±0.05 14.4 ±0.8 4.5 ±0.1 23.9 ±0.5 5.3 ±0.4 0.0074 ±0.0006 0.004 ±0.0000 Adjacent 0.3 ±0.01 1.1 ±0.05 0.43 ±0.032 0.9 ±0.08 27.2 ±1.2 3.7 ±0.2 9.2 ±0.5 4.4 ±0.3 0.0100 ±0.0008 0.006 ±0.0002 Tumor 0.4 ±0.01 3.4 ±0.04 0.87 ±0.052 0.5 ±0.05 23.6 ±1.0 5.2 ±0.6 22.8 ±1.3 6.7 ±0.3 0.0073 ±0.0007 0.005 ±0.0004 Adjacent 1.0 ±0.00 0.7 ±0.07 0.89 ±0.065 0.5 ±0.03 17.6 ±0.9 5.5 ±0.2 6.9 ±0.6 2.1 ±0.1 0.0053 ±0.0006 0.010 ±0.0012 Tumor 0.2 ±0.01 0.8 ±0.00 0.20 ±0.021 0.4 ±0.03 13.4 ±0.2 3.3 ±0.3 13.6±1.2 3.3 ±0.1 0.0021 ±0.0000 0.006 ±0.0008 Adjacent 0.6 ±0.04 0.7 ±0.08 0.41 ±0.030 7.3 ±0.81 31.9 ±0.9 5.6 ±0.2 6.7 ±0.8 5.9 ±0.6 0.0037 ±0.0003 0.008 ±0.0005 Tumor 0.4 ±0.03 1.7 ±0.09 0.36 ±0.040 6.2 ±0.00 25.2 ±1.1 9.1 ±0.2 22.2 ±1.0 9.5 ±0.7 0.0024 ±0.0003 0.007 ±0.0004 S13
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±0.007 8.9±0.4 0.19±0.01 2.7±0.2 273.0±2.3 256.9±2.1 72.6±1.4 45.4±0.9 0.0025±0.0003 0.010±0.001 HeLa 0.06±0.007 9.9±0.5 0.15±0.01 11.5±0.6 112.2±1.8 143.8±1.9 27.0±0.7 53.6±1.0 0.0030±0.0004 0.020±0.002 S14
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±0.03 1.67±0.52 0.41±0.03 1.03±0.08 17.5±0.9 5.1±0.3 18.1±0.8 1.5±0.1 0.03±0.000 0.17±0.008 2 Control 1.23±0.07 0.86±0.10 0.66±0.04 0.23±0.00 52.6±2.8 10.9±0.6 25.8±1.5 6.1±0.5 0.04±0.003 0.20±0.006 3 Control 2.89±0.05 1.83±0.09 0.40±0.02 0.27±0.03 30.1±1.2 14.9±0.6 19.5±1.2 2.3±0.7 0.08±0.003 0.21±0.012 4 Control 0.71±0.00 0.59±0.03 0.74±0.05 1.04±0.07 32.3±0.0 12.7±0.6 19.6±1.0 3.0±0.3 0.04±0.005 0.23±0.007 5 Control 0.76±0.04 0.22±0.03 0.24±0.02 0.21±0.01 44.7±1.9 10.6±0.7 23.4±1.5 5.4±0.2 0.02±0.000 0.05±0.000 6 Control 4.04±0.09 1.14±0.00 1.50±0.05 1.03±0.03 37.9±1.8 19.3±0.8 25.2±1.1 8.1±0.5 0.06±0.005 0.07±0.004 7 Control 6.52±0.22 0.58±0.04 0.23±0.02 0.56±0.02 58.2±2.4 17.3±0.5 23.8±1.3 3.8±0.2 0.07±0.004 0.17±0.021 8 Control 3.29±0.25 0.88±0.05 0.31±0.01 0.70±0.03 21.4±1.0 8.1±0.3 12.0±0.7 5.1±0.4 0.04±0.004 0.09±0.006 9 Control 0.79±0.32 0.56±0.03 1.15±0.00 0.33±0.01 59.0±2.3 19.3±0.6 26.7±1.3 5.0±0.2 0.07±0.003 0.27±0.000 10 Control 0.64±0.52 0.31±0.03 0.35±0.02 0.43±0.02 18.8±1.2 7.9±0.4 16.2±0.9 4.8±0.2 0.03±0.002 0.29±0.006 11 Lymphoma 0.98±0.41 0.74±0.05 0.57±0.01 0.44±0.05 50.7±2.3 12.8±0.5 36.4±1.7 4.3±0.1 0.05±0.001 0.07±0.005 12 Lymphoma 1.34±0.10 1.11±0.08 0.40±0.04 0.71±0.04 64.1±0.0 19.8±0.9 34.7±1.8 9.9±0.5 0.04±0.002 0.22±0.013 13 Lymphoma 0.76±0.02 0.22±0.01 0.24±0.02 0.21±0.01 44.7±2.4 10.9±0.5 23.3±1.2 5.4±0.3 0.02±0.001 0.05±0.006 14 Lymphoma 0.60±0.32 1.42±0.07 0.20±0.01 0.37±0.04 21.6±1.4 10.6±0.3 38.9±1.5 1.7±0.1 0.02±0.003 0.08±0.005 15 Lymphoma 0.49±0.00 0.24±0.02 0.27±0.03 0.31±0.00 35.8±1.5 8.4±0.7 24.8±1.2 4.6±0.3 0.03±0.001 0.09±0.007 16 Lymphoma 0.38±0.03 0.37±0.04 0.19±0.01 0.21±0.02 16.4±0.9 13.4±0.9 24.3±1.0 2.4±0.1 0.03±0.004 0.05±0.004 17 Lymphoma 0.64±0.04 0.28±0.01 0.58±0.04 0.28±0.03 26.2±1.7 6.1±0.6 28.0±1.3 7.0±0.5 0.03±0.002 0.08±0.005 18 Lymphoma 0.58±0.03 0.37±0.02 0.12±0.02 0.36±0.03 18.4±0.9 16.6±0.6 20.0±1.0 2.9±0.2 0.02±0.003 0.06±0.004 19 Lymphoma 0.27±0.01 0.70±0.05 0.19±0.02 0.11±0.02 19.5±0.9 10.7±0.5 28.9±1.8 1.1±0.1 0.01±0.000 0.04±0.005 20 Lymphoma 0.29±0.02 0.59±0.04 0.42±0.05 0.41±0.03 14.2±0.8 6.3±0.5 21.23±1.4 2.6±0.1 0.01±0.002 0.03±0.002 S15
Figure S1. The structures of 8 normal nucleotides. S16
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
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
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
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