Supporting Information. Mass Spectrometry Based Ultrasensitive DNA Methylation. Profiling Using Target Fragmentation Assay

Transcription

1 Supporting Information Mass Spectrometry Based Ultrasensitive DNA Methylation Profiling Using Target Fragmentation Assay Xiang-Cheng Lin, Ting Zhang, Lan Liu, Hao Tang*, Ru-Qin Yu, Jian-Hui Jiang* State Key Laboratory for Chemo/biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, , P. R. China * Corresponding author. Tel.: ; Fax: address: haotang@hnu.edu.cn, jianhuijiang@hnu.edu.cn S-1

2 EXPERIMENTAL SECTION Materials. Streptavidin-modified beads (1 µm, 10 mg/ml) were obtained Thermo Fisher Scientific Inc.. Restriction endonuclease BbvI (recognition site GCAGC) and Dam MTase (Dnmt1) were purchased from New England Biolabs (NEB). All the oligonucleotides were synthesized from Sangon Inc. (Shanghai, China). The sequences of synthetic oligonucleotides are listed in Table S1. All other chemicals were of analytical grade and obtained from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). The solutions used in all experiments were prepared using ultrapure water which was obtained through a Millipore Milli-Q water purification system (Billerica, MA) and had an electric resistance >18.2 MΩ. Magnetic beads immobilized with the capture probes were prepared by incubating the biotinylated probes (5 µl, 1 µm) with the magnetic beads (20 µl) in a Bind and Wash (B&W) buffer (10 mm Tris-HCl, 1 mm EDTA, 2M NaCl, ph 7.5) for 15 min. After the supernatant discarded, the capture magnetic beads were obtained and stored in 4 o C for futher use. Prostate tissue samples were friendly provided by the Third XiangYa Hospital (Changsha, China), which was approved by the local institutional review board. All samples are biopsies from 20 patients, one diagnosed to be normal, three were diagnosed with benign hyperplasia, and 16 were diagnosed with prostate cancer. Genomic DNA from formalin-fixed and paraffin-embedded tissue sections were isolated and extracted using QIAamp DNA mini kit (QIAGEN) according to the manufacturer s instructions. Briefly, the tissue samples were lysed thoroughly with lysis buffer containing proteinase K, followed by incubating at 56 o C for 3 h. In order to avoid RNA interference, RNase A was added to the tissue lysate. The lysate was then passed through the QIAamp spin mini column to allow the genomic DNA to be adsorbed in the column. The genomic DNA was purified by washing with water twice and collecting the elution products. DNA concentration and quality were evaluated by absorbance at 260 and 280 nm, respectively, S-2

3 using a Lambda 850 UV-vis spectrophotometer (PerkinElmer). The concentration of genomic DNA was estimated to be ~25 µg/ml. Target capture and fragmentation. Genomic DNA of 100 µl was digested with restriction endonuclease BbvI (10 µl, 2000 U/mL, 37 o C) in NEB CutSmart buffer (50 mm KAc, 20 mm Tris-Ac, 10 mm MgAc 2, 100 µg/ml BSA, ph 7.9) for 1 h. After denatured at 95 o C for 5 min and quickly cooled in ice water for 5 min, the capture magnetic beads (20 µl) was added and incubated with the digested DNA in phosphate buffered saline (PBS, 10 mm Na 2 HPO 4, mm NaCl, 2.7 mm KCl, ph 7.4) containing 0.5 M NaCl for 1 h. After magnetic separation, the collected beads were mixed with 0.2 ml of aqueous formic acid (88% w/w) and hydrolyzed at 160 o C for 30 min. The hydrolysates was evaporated under nitrogen and then dissolved in 10 µl methanol-water (1:1) followed by MS analysis. DNA methylation using Dnmt1 and study of its inhibitor. A DNA hairpin probe with one 5mC (Table S1) was designed as substrate for Dnmt1 to study DNA methylation and screening of Dnmt1 inhibitor. In the presence of Dnmt1, methylation occurred at another C of the CpG site in the hairpin probe. The methylation experiment was performed in 50 µl of methylase buffer (50 mm of Tris-HCl, 1 mm EDTA, 1 mm DTT,5% glycerol) containing 0.1µM DNA hairpin probe,100 µg/ml BSA, 100 µm S-adenosylmethionine, with different amounts of Dnmt1 and inhibitor(5-azacytidine). The reaction mixture was incubated at 37 C for 4 h. DNA hydrolytes were obtained using the same procedure and subjected for MS measurement. To investigate the effect of inhibitor on DNA methylation, a group of different concentrations of inhibitors and the fixed concentration of Dnmt1 (40 U/mL) were used. Mass spectrometry analysis. MS analysis was performed using an LCQ Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific). The sample was directly delivered using a syringe pump at flow rate of 500 nl/min into the mass spectrometer furnished with a nano electrospray ionization (nano-esi) source under the positive-ion S-3

4 mode. The spray voltage was 2.0 kv, and the source temperature was at 270 o C. For primary mass spectrometry, data was acquired in full scan mode. MS/MS mode was employed for measurement and higher energy collisionally activated dissociation (HCD) was chosen for post-source fragmentation with 90 ev collision energy. Mass spectrometry was operated in selective ion scan mode for MS/MS by monitoring a transition pair of m/z (molecular ion)/ (fragment ion) for 5-methylcytosine and m/z / for adenine, with a scan time of 50 ms for each pair. Quantification was accomplished by averaging response signals within 1 min. Methylation-specific PCR for tissue samples. Methylation-specific PCR of genomic DNA was used for qualitative analysis of DNA methylation according to a procedure described previously. 1 Briefly, target DNA that was captured using magnetic beads from 1 µg genomic DNA was treated using the EpiTect Bisulfite Kit (QIAGEN) according to the manufacturer s instructions. PCR amplification of the bisulfite-treated DNA was performed in two separate reactions with primer pairs specific for the non-methylated (U) or the methylated (M) sequences. The sequences for methylation-specific PCR primers are given in Table S3. PCR reaction mixture (25 µl) contained 0.3 µm primers, 100 µm dntps, 2.0 U JumpStart Taq polymerase (Sigma) and 1 µl of bisulfite treated DNA. The PCR conditions were as follows: 94 o C for 1 min, 35 cycles of (94 o C for 30 s, 65 o C for 25 s and 72 o C for 30 s), and 72 C for 5 min. The PCR products were separated on 2.5% agarose gels and visualized by ethidium bromide staining. REFERENCES (1) Claus, R.; Wilop, S.; Hielscher, T.; Sonnet, M.; Dahl, E.; Galm, O.; Jost, E.; Plass, C. Epigenetics 2012, 7, S-4

5 Table S1. Sequences of the synthesized oligonucleotides Probes Sequence (5 to 3 ) DNA Target 1 GGG GCG GAG CGG GGC GGG ACC ACC C DNA Target 2 GGG G(5mC)G GAG (5mC)GG GG(5mC) GGG ACC ACC C DNA Target 3 GCG G(5mC)C CAG (5mC)GC CG(5mC) GCG ACC ACC C DNA Target 4 GGG G(5mC)G GAG CGG GGC GGG ACC ACC C DNA Target 5 GGG G(5mC)G GAG (5mC)GG GGC GGG ACC ACC C Capture Probe 1 (GSTP 1) Capture Probe 2 (BCL 2) Biotin-TTTTTTTTTTGGGTGGTCCCGCCCCGCTCCGCCCC Biotin-TTTTTTTTTTCTCCGCCCCGCTCCCTGGCCCGGGT Capture Probe 3 (ESR 1) Capture Probe 4 (HIC 1) Biotin-TTTTTTTTTTGCTCTGGCCCCGGCCCTGCCCCGGG Biotin-TTTTTTTTTTCCCGTCCCTCCCGGTCCCCGGCTCG DNA hairpin probe ATGTCGGCG (5mC)ATCAGTTTTCTGATGCGCCGACAT S-5

6 Table S2. Sequences of promoters for select genes a GSTP1 gtgcagcggccgccg//gggctggggccggcgggagtccgcgggaccctccagaagagcggc CGGCGCCGTGACTCAGCACTGGGGCGGAGCGGGGCGGGACCACCCTTATAAGGCTCG GAGGCCGCGAGGCCTTCGCTGGAGTTTCGCCGCCGCAGTCTTCGCCACCAGTGAGTAC GCGCGGCCCGCGTCCCCGGGGATGGGGCTCAGAGCTCCCAGCATGGGGCCAACCCGC AGCATCAGGCC//cgggctccc BCL2 gcgcagcggagcggg//cgggtggccggcccggacgcgccctccccggccgcggccccgcgcg CCATGTGCCCCCGGCGGGACGCGCCACTCCCGGGCCTGCCGCGGCGCCTTTAACCCGG GCCAGGGAGCGGGGCGGAGGGGGCGGTCGGGTGGCTCAGAGGAGGGCTCTTTCTTTC TTCTTTTTTTGAATGAACCGTGTGACGTTACGCACAGGAAACCGGTCGGGCTGTGCAG AGAATGAAGTAAGAGGACAGGCACCACAGCCCCGCTCCCGCCCCCTTCCTCCCGCGC CCGCCCCTCCGCGCCGCCTGCCCGCCCGCCCGCCGCGCTCCCGCCCGCCGCTCTCCGT GGCCCCGCCGCGCTGCCGCCGCCGCCGCTGCCAGCGAAGGTGCCGGGGCTCCGGGCC CTCCCTGCCGGCGGCCGTCAGCGCTCGGAGCGGGCTGCGCGGCGGGAGCTCCGGGAG GCGGCCGTAGCCAGCGCCGCCGCGCAGGACCAGGAGGAGGAGAAAGGGTGCGCAGC CCGGAGGC//ggggtgcgcc ESR1 cagcagcgacgacaa//gtaaagtaaagttcagggaagctgctctttgggatcgctccaaatcg AGTTGTGCCTGGAGTGATGTTTAAGCCAATGTCAGGGCAAGGCAACAGTCCCTGGCCG TCCTCCAGCACCTTTGTAATGCATATGAGCTCGGGAGACCAGTACTTAAAGTTGGAGGC CCGGGAGCCCAGGAGCTGGCGGAGGGCGTTCGTCCTGGGACTGCACTTGCTCCCGTC GGGTCGCCCGGCTTCACCGGACCCGCAGGCTCCCGGGGCAGGGCCGGGGCCAGAGCT CGCGTGTCGGCGGGACATGCGCTGCGTCGCCTCTAACCTCGGGCTGTGCTCTTTTTCCA S-6

7 GGTGGCCCGCCGGTTTCTGAGCCTTCTGCCCTGCGGGGACACGGTCTGCACCCTGCCC GCGGCCACGGACCATGACCATGACCCTCCACACCAAAGCATCTGGGATGGCCCTACTG CATCAGATCCAAGGGAACGAGCTGGAGCCCCTGAACCGTCCGCAGCTCAAGATC//cccct ggagc HIC1 gcgcagccacgtccc//ccctggatccgccgtcagccgggcccggggctttcgacatgcccccc AGGTGGGTCCTCGAGCCGGGGACCGGGAGGGACGGGGGACCCGGGACAGCCCGGTC CTCGTGCGTCGGCCGCCTCGGGTGCATCTTCTGGCGCGGGTGCCCCATCGCGGCTGGC GGCTGGCGTTCAGGGCTCCGGGTGTCGTCCCTTTCGGACTCAGGACCACCGGGCCGC GGCTCCGCGCCGGGTTCACGGCGGGGTCAGCGGCCCGGGGCCGGCTCTGCCCGCACA TGGGCTGGAGAGGCGAGGGGAAGGGAAGGGAAGGGGAGCTGGCGGGCGGGGCTGG CAGGGGCGCTGCCCTGGCACAGCTCGGGGCCTGGCAGCGGCGGGTG//gggcatcg a // denotes the cleavage site of restriction endonuclease BbvI, and the sequences to be captured by magnetic separation were denoted by capital letters. S-7

8 Table S3. Primers for selected sequences using in Methylation-specific PCR a GSTP1 MF TTT TAG AAGA GC GGT CG GC GTC MR UF UR TCC GCG CGTACTCACTAATAACG TTT TAG AAGA GT GGT TG GT GTTG CAAACCACACATACTCACTAATAACA BCL2 MF GTGTGACGTTACGTATAGGAAATC MR UF UR GACGACGCTAACTACGACCG GTGTGATGTTATGTATAGGAAATT ACACAACAACACTAACTACAACCA ESR1 MF ATTTGTTTTCGTCGGGTC MR UF UR TCATAATCCGTAACCGCG TGTATTTGTTTTTGTTGGGTT ATAATCCATAACCACAAACAA HIC1 MF GATAGTTCGGTTTTCGTGCGTC MR UF UR GACCCGATAATCCTAAATCCG GGATAGTTTGGTTTTTGTGTGTT ACAACCCAATAATCCTAAATCCA a MF denotes the forward primers for PCR amplification of methylated target sequences, MR is the reverse primers for PCR amplification of methylated target sequences, UF denotes the forward primers for PCR amplification of non-methylated target sequences, and UR is the reverse primers for PCR amplification of non-methylated target sequences. S-8

9 Figure S1. The full scan mass spectrum of DNA target 2 without preliminary acidic degradation. Sample was dissolved in 10 nm ammonium acetate buffer, detected in negative ion mode. S-9

10 Figure S2.. The full scan mass spectrum of standard 5mC (30 nm). S-10

11 Figure S3. (A) The full scan mass spectrum for 5-methylcytosine, (B) the product ion scan spectrum obtained using the protonated molecular ion of 5-methylcytosine as the parent ion. S-11

12 Intensity of MS/MS Y=216.45X R 2 = mc (pm) Figure S4. Plot of MS/MS intensities versus concentrations of DNA target 2 (expressed in 5mC concentration). S-12

13 Figure S5. (A) The full scan mass spectrum for adenine, (B) the product ion scan spectrum obtained using the protonated molecular ion of adenine as the parent ion. S-13

14 4 Ratio of signal intensity (5m C/A) Y=1.7142X R 2 = Ratio of Concentrations (5mC/A) Figure S6. Calibration curve for ratios of MS/MS intensities for 5mC and A versus concentration ratios [5mC]/[A]. S-14

15 Figure S7. The MS/MS spectra of five targets obtained by monitoring 5-methylcytosine (A) and adenine (B). The targets were captured and acidic hydrolyzed. S-15

16 Figure S8. Methylation analysis for samples of mixtures of methylated and non-methylated DNA with different levels of [5mC]/([5mC] + [C]). S-16

17 Figure S9. Methylation-specific PCR of promoter sequences in GSTP1 (A), BCL2 (B), ESR1 (C), and HIC1 (D) genes. M denotes the bands obtained with methylation-specific primers, U denotes the bands obtained with non-methylation specific primers, and positive denotes the bands from a positive methylated target. Sample 1 was from normal person, sample 8-10 were from persons with benign hyperplasia, and the other 16 samples were from persons diagnosed with prostate cancer. (to be continued) S-17

18 Figure S9. (continued) The DNA methylation could be inferred from the presence of bands under M. Methylation in promoter sequences for GSTP1 gene was identified for samples 2-7 and 11-20, low methylation occurred from genomic DNA in sample 9, and only genomic DNA in samples of 1, 8 and 10 were non-methylated. Methylation in promoter sequences for BCL2 gene was found to occur for samples 2-7, and 20, and only genomic DNA in samples of 1, 8-10 and 19 were non-methylated. Methylation in promoter sequences for ESR1 gene was found for samples 3-5, 7, 12-20, and only genomic DNA in samples of 1, 2, 6, and 8-11 were non-methylated. Methylation in promoter sequences for HIC1 gene was found for samples 2-7, 12-20, low methylation occurred from genomic DNA in sample 10, and only genomic DNA in samples of 1, 8, and 9 were non-methylated. S-18

19 Figure S10. The MS/MS spectra of DNA hydrolysates from Dnmt1 and its inhibitor (5-azacytidine) treated DNA hairpin probe. (A) 5-methylcytosine; (B) adenine. Concentrations of Dnmt1 (plot 1: 0 U/mL; plot 2: 20 U/mL; plot 3-5: 40 U/mL). Concentration of 5-azacytidine (plot 1-3: 0 µm; plot 4: 5 µm; plot 5: 40 µm). Adensine was used as internal standard; hence the intensities of all samples were similar. The intensity of 5-methylcytosine increases (plot 1 to plot 3) along with the increase of Dnmt1 concentration (0, 20 U/mL and 40 U/mL ). Under the same concentration of Dnmt1 (40 U/mL ), the MS intensity of 5-methylcytosine decreases (plot 3 to plot 5) along with the increases of Dnmt 1 inhibitor concentration (0, 5, and 40 µm). Hence, Dnmt1 activity assay and screening of its inhibitor could be achieved by measuring MS intensity ratio of 5mC and A. S-19