High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry.
High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry.
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DOI：
10.1016/j.aca.2021.338880
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发表时间：
2021-10-02
影响因子：
6.2
通讯作者：
Sidoli S
中科院分区：
文献类型：
--
作者：
Sun Y;Stransky S;Aguilan J;Koul S;Garforth SJ;Brenowitz M;Sidoli S
We present a direct injection mass spectrometry (DI-MS) platform that accurately, precisely, and quickly quantitates global levels of DNA cytidine methylation (5mC) and hydroxymethylation (5hmC). Our platform combines an Advion TriVersa NanoMate coupled online to a Thermo Scientific Orbitrap Fusion Lumos. Following digestion to nucleosides, the DNA samples are analyzed at the rate of <1 min per injection with comparable detection limits of 0.63 ng/μL and 0.31 ng/μL, respectively. In contrast, the detection limits for 5mC and 5hmC in state-of-art nano liquid chromatography (LC) coupled to online mass spectrometry (nLC-MS) are notably different (0.04 ng/μL and 2.5 ng/μL, respectively). The high sensitivity of DI-MS is achieved by optimizing sample buffer composition, the source fragmentation energy, and the radio frequency of the instrument ion funnel. DI-MS accurately reports the relative abundance of 5mC and 5hmC over a range of 1% to 7% (R2 > 0.98) and 0.13% to 1.75% (R2 > 0.99), respectively. Accurate measurement of C, 5mC and 5hmC is achieved by optimizing in-source fragmentation to obtain a population of up to 93% of just the nucleoside base. This protocol minimizes base dimer formation and partial base-deoxyribose dissociation in gas phase and greatly improves modified base quantitation. We also demonstrate that DI-MS overcomes biases in differential chromatographic retention and issues of sample degradation in the autosampler due to its high throughput. Finally, we present an application of our workflow to quantify DNA modifications on a batch of 81 samples in about 1.5 hours.
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影响因子：
7
作者：
Sidoli, Simone;Kori, Yekaterina;Garcia, Benjamin A.
通讯作者：
Garcia, Benjamin A.
影响因子：
50.3
作者：
Figueroa ME;Abdel-Wahab O;Lu C;Ward PS;Patel J;Shih A;Li Y;Bhagwat N;Vasanthakumar A;Fernandez HF;Tallman MS;Sun Z;Wolniak K;Peeters JK;Liu W;Choe SE;Fantin VR;Paietta E;Löwenberg B;Licht JD;Godley LA;Delwel R;Valk PJ;Thompson CB;Levine RL;Melnick A
通讯作者：
Melnick A
影响因子：
4.1
作者：
Ontiveros, R. Jordan;Stoute, Julian;Liu, Kathy Fange
通讯作者：
Liu, Kathy Fange
影响因子：
64.5
作者：
Okano, M;Bell, DW;Li, E
通讯作者：
Li, E
影响因子：
3.5
作者：
BESTOR, TH
通讯作者：
BESTOR, TH