Subscriber access provided by UNIV OF NEBRASKA - LINCOLN
Letter
Mass Spectrometry Based Ultrasensitive DNA Methylation Profiling Using Target Fragmentation Assay Xiang-Cheng Lin, Ting Zhang, Lan Liu, Hao Tang, Ru-Qin Yu, and Jian-hui Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04247 • Publication Date (Web): 28 Dec 2015 Downloaded from http://pubs.acs.org on December 30, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
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 of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (P. R. China) ABSTRACT: Efficient tools for profiling DNA methylation in specific genes are essential for epigenetics and clinical diagnostics. Current DNA methylation profiling techniques have been limited by inconvenient implementation, requirements of specific reagents, and inferior accuracy in quantifying methylation degree. We develop a novel mass spectrometry method, target fragmentation assay (TFA), which enable to profile methylation in specific sequences. This method combines selective capture of DNA target from restricted cleavage of genomic DNA using magnetic separation with MS detection of the non-enzymatic hydrolysates of target DNA. This method is shown to be highly sensitive with a detection limit as low as 0.056 amol, allowing direct profiling of methylation using genome DNA without pre-amplification. Moreover, this method offers a unique advantage in accurately determining DNA methylation level. The clinical applicability was demonstrated by DNA methylation analysis using prostate tissue samples, implying the potential of this method as a useful tool for DNA methylation profiling in early detection of related diseases.
DNA methylation is a major epigenetic mechanism responsible for regulation of gene expression.1 Recent studies have revealed that the dynamic reprogramming of DNA methylation pattern by methylation versus demethylation has been closely implicated in numerous biological events and involved in many diseases such as cancers, imprinting-related diseases, and psychiatric disorders.2 In particular, local increases in DNA methylation and global decreases in genomic methylation are both associated with tumor initiation and progression.3 Hyper-methylation of CpG islands in tumor suppressor genes has been regarded as one of the earliest somatic genome alterations in carcinogenesis.4 Therefore, efficient tools for profiling DNA methylation are essential for understanding the biological roles and mechanisms of this epigenetic regulation in diseases.5 There are currently three main techniques for profiling DNA methylation, chemical transformation such as bisulfite treatment,6 enzymatic cleavage such as methylationsensitive restricted enzymes,7,8 and affinity capture using methylated DNA binding proteins.9 These methods are usually time-consuming and labor-intensive, and require specific reagents such as enzymes and binding proteins. Moreover, they do not provide accurate analysis of the degree of DNA methylation. Techniques enabling accurate and convenient profiling of DNA methylation remain elusive. Mass spectrometry (MS) has become an indispensable platform for molecular and cellular biology because of its high specificity, sensitivity and throughput as well as its ability to offer accurate molecular weight and structural information.10-12 This technology has shown great potential and prevailed applications for identification, characterization and quantification of proteome and metabolome.13-15 Despite of applications of MS-based DNA/RNA analysis to determination of PCR amplicons,16 and identification of single nucleotide polymorphism,17 the implementation of MS detection for DNA/RNA oligomers has not been widely explored. One likely reason is the size-dependent tendency to fragment for the DNA/RNA
phosphodiester backbone during the ionization processes. This tendency limited the ionization efficiency for DNA/RNA oligomers with increasing size, leading to inferior sensitivity and mass resolution for direct detection of the oligonucleotides.18 Recent years have witnessed increasing applications of MS detection to nucleobases from degraded oligonucleotides for characterization of DNA/RNA damages,19 adducts20,21 and epigenetic modifications.22 The MS assays of nucleobases instead of DNA/RNA oligomers have created the possibilities for highly sensitive detection required for routine DNA/RNA detection. Nevertheless, these approaches have typically focused on global analysis of the whole genomes rather than target DNA/RNA sequences. Because most disease-associated biomarkers are associated with specific DNA/RNA sequences, assays for DNA/RNA modifications in specific sequences are highly demanded. Herein, we report a novel MS-based method, target fragmentation assay (TFA), which enables highly sensitive and target-selective profiling of DNA methylation in specific sequences. This method is realized via combining precise restricted cleavage of genomic DNA, selective capture of DNA target sequence using magnetic separation, non-enzymatic degradation of target DNA, and MS based highly sensitive detection of the hydrolysates. This method is demonstrated using DNA methylation profiling for promoters of GSTP1, BCL2, ESR1 and HIC1 genes. Aberrantly methylation of these genes is known to important prognostic or diagnostic biomarkers for tumors of prostate.23 The results revealed that with the aid of high-resolution MS/MS analysis, the TFA method is highly sensitive with a detection limit as low as 0.056 amol, which allows direct profiling of DNA methylation using genome samples without need of pre-amplification. Moreover, this method offers a unique advantage in the ability to give accurate estimate for the level of DNA methylation, which is not available from existing techniques.6-9 These advantages imply that the TFA method creates an invaluable tool
ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
for DNA methylation profiling, thereby greatly facilitating accurate validation of DNA methylation biomarkers and early detection of the related diseases. Scheme 1. Illustration of target fragmentation assay (TFA).
The analytical principle of the TFA method is illustrated in Scheme 1. A unique characteristic of the TFA method, as compared with most of current MS analysis of DNA/RNA modifications for global genome, is that TFA aims at the profiling of DNA methylation for a specific DNA target. This first step of TFA is to obtain the specific DNA target. To this end, we choose a restricted endonuclease for cleavage of genomic DNA instead of using traditional ultrasonic treatment. The restricted cleavage results in a short target DNA fragment with precise ends, which ensure a pre-defined composition for the DNA fragment. Using magnetic beads for capture probes via the streptavidin-biotin interaction, the target DNA fragments are collected, which enables downstream assays of DNA methylation for the specific DNA target. In order to profile DNA methylation with high sensitivity and high resolution, the collected DNA fragments are completely degraded into nucleobases using formic acid.24 We have optimized reaction temperature and the use of amount of formic acid to achieve complete degradation of DNA in 30 min based on previous literature (experiment details in SI). As compared to commonly used enzymatic procedures which include the use of different enzymes and tedious treatment,19-22 this nonenzymatic degradation strategy may afford the advantages of high efficiency, low cost and improved resistance to interferents possibly inhibiting enzymatic activities. . With the degraded DNA fragments, the nucleotide compositions of target sequence are directly profiled through MS/MS analysis furnished with a nano-ESI source for ionization, which was reported to have high spray efficiency and low sample injection flow rate (
