High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry
We present a direct injection mass spectrometry (DI-MS) platform that accurately, precisely, and quickly quantitates global levels of DNA cytidine methylation (5 mC) and hydroxymethylation (5hmC). Our platform combines an Advion TriVersa NanoMate coupled online to a Thermo Scientific Orbitrap Fusion...
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| Veröffentlicht in: | Analytica chimica acta 2021-10, Vol.1180, p.338880-338880, Article 338880 |
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| creator | Sun, Yan Stransky, Stephanie Aguilan, Jennifer Koul, Sanjay Garforth, Scott J. Brenowitz, Michael Sidoli, Simone |
| description | We present a direct injection mass spectrometry (DI-MS) platform that accurately, precisely, and quickly quantitates global levels of DNA cytidine methylation (5 mC) 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 0.98) and 0.13%–1.75% (R2 > 0.99), respectively. Accurate measurement of C, 5 mC 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 h.
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•We present an ultra-rapid ( |
| doi_str_mv | 10.1016/j.aca.2021.338880 |
| format | Article |
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[Display omitted]
•We present an ultra-rapid (<1 min) mass spectrometry method to analyze major DNA modifications.•The method overcomes issues of liquid chromatography like carryover and biased retention of nucleosides of different hydrophobicities.•We demonstrate that routinely used targeted approaches overlook the formation of nucleoside byproducts affecting quantification.•We optimized instrument settings to minimize biases in quantification.</description><identifier>ISSN: 0003-2670</identifier><identifier>EISSN: 1873-4324</identifier><identifier>DOI: 10.1016/j.aca.2021.338880</identifier><identifier>PMID: 34538324</identifier><language>eng</language><publisher>AMSTERDAM: Elsevier B.V</publisher><subject>5hmC ; 5mC ; Chemistry ; Chemistry, Analytical ; Direct injection ; DNA ; High-throughput ; Hydroxymethylation ; Mass spectrometry ; Methylation ; Physical Sciences ; Science & Technology</subject><ispartof>Analytica chimica acta, 2021-10, Vol.1180, p.338880-338880, Article 338880</ispartof><rights>2021 Elsevier B.V.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>true</woscitedreferencessubscribed><woscitedreferencescount>12</woscitedreferencescount><woscitedreferencesoriginalsourcerecordid>wos000683023000004</woscitedreferencesoriginalsourcerecordid><citedby>FETCH-LOGICAL-c428t-143e8ce56cee3742dc1c6f553cce12b3f82158e6eeb5866c97308311b93b84e03</citedby><cites>FETCH-LOGICAL-c428t-143e8ce56cee3742dc1c6f553cce12b3f82158e6eeb5866c97308311b93b84e03</cites><orcidid>0000-0002-9605-4825 ; 0000-0001-6281-4833 ; 0000-0002-5747-3306 ; 0000-0001-9073-6641 ; 0000-0002-0221-354X ; 0000-0003-0048-1716 ; 0000-0003-0844-2754</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.aca.2021.338880$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>230,315,782,786,887,3552,27931,27932,39265,46002</link.rule.ids></links><search><creatorcontrib>Sun, Yan</creatorcontrib><creatorcontrib>Stransky, Stephanie</creatorcontrib><creatorcontrib>Aguilan, Jennifer</creatorcontrib><creatorcontrib>Koul, Sanjay</creatorcontrib><creatorcontrib>Garforth, Scott J.</creatorcontrib><creatorcontrib>Brenowitz, Michael</creatorcontrib><creatorcontrib>Sidoli, Simone</creatorcontrib><title>High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry</title><title>Analytica chimica acta</title><addtitle>ANAL CHIM ACTA</addtitle><description>We present a direct injection mass spectrometry (DI-MS) platform that accurately, precisely, and quickly quantitates global levels of DNA cytidine methylation (5 mC) 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 5 mC 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 5 mC and 5hmC over a range of 1%–7% (R2 > 0.98) and 0.13%–1.75% (R2 > 0.99), respectively. Accurate measurement of C, 5 mC 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 h.
[Display omitted]
•We present an ultra-rapid (<1 min) mass spectrometry method to analyze major DNA modifications.•The method overcomes issues of liquid chromatography like carryover and biased retention of nucleosides of different hydrophobicities.•We demonstrate that routinely used targeted approaches overlook the formation of nucleoside byproducts affecting quantification.•We optimized instrument settings to minimize biases in quantification.</description><subject>5hmC</subject><subject>5mC</subject><subject>Chemistry</subject><subject>Chemistry, Analytical</subject><subject>Direct injection</subject><subject>DNA</subject><subject>High-throughput</subject><subject>Hydroxymethylation</subject><subject>Mass spectrometry</subject><subject>Methylation</subject><subject>Physical Sciences</subject><subject>Science & Technology</subject><issn>0003-2670</issn><issn>1873-4324</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>HGBXW</sourceid><recordid>eNqNkU9r3DAQxUVpSTbbfIDedCwUb_XPtpZCIWybJhDaS3sWsjxea7GtrSQn8bevHC-BXEpOw2jebzS8h9AHSjaU0OLzYaON3jDC6IZzKSV5g1ZUljwTnIm3aEUI4RkrSnKOLkI4pJZRIs7QORc5l0mzQv7G7lscW-_GfXscI9ZDjTv3gCurA_728wr3ENup09G64WnYTrV3j9PLZ91NwQZcTbi2HkzEdjikMg97HQIOx9R5lyA_vUfvGt0FuDzVNfpz_f337ia7-_Xjdnd1lxnBZMyo4CAN5IUB4KVgtaGmaPKcGwOUVbyRjOYSCoAql0VhtiUnklNabXklBRC-Rl-Xvcex6qE2MESvO3X0ttd-Uk5b9XIy2Fbt3b2SyZ3ZujX6eFrg3d8RQlS9DQa6Tg_gxqBYXoqSF9utSFK6SI13IXhonr-hRM1ZqYNKWak5K7VklRi5MA9QuSYYC4OBZy4dUEhO2HwJIWJn45PXOzcOMaGfXo8m9ZdFDcntewtenYglK1U7-58z_wGC3b5x</recordid><startdate>20211002</startdate><enddate>20211002</enddate><creator>Sun, Yan</creator><creator>Stransky, Stephanie</creator><creator>Aguilan, Jennifer</creator><creator>Koul, Sanjay</creator><creator>Garforth, Scott J.</creator><creator>Brenowitz, Michael</creator><creator>Sidoli, Simone</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>BLEPL</scope><scope>DTL</scope><scope>HGBXW</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-9605-4825</orcidid><orcidid>https://orcid.org/0000-0001-6281-4833</orcidid><orcidid>https://orcid.org/0000-0002-5747-3306</orcidid><orcidid>https://orcid.org/0000-0001-9073-6641</orcidid><orcidid>https://orcid.org/0000-0002-0221-354X</orcidid><orcidid>https://orcid.org/0000-0003-0048-1716</orcidid><orcidid>https://orcid.org/0000-0003-0844-2754</orcidid></search><sort><creationdate>20211002</creationdate><title>High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry</title><author>Sun, Yan ; Stransky, Stephanie ; Aguilan, Jennifer ; Koul, Sanjay ; Garforth, Scott J. ; Brenowitz, Michael ; Sidoli, Simone</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c428t-143e8ce56cee3742dc1c6f553cce12b3f82158e6eeb5866c97308311b93b84e03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>5hmC</topic><topic>5mC</topic><topic>Chemistry</topic><topic>Chemistry, Analytical</topic><topic>Direct injection</topic><topic>DNA</topic><topic>High-throughput</topic><topic>Hydroxymethylation</topic><topic>Mass spectrometry</topic><topic>Methylation</topic><topic>Physical Sciences</topic><topic>Science & Technology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sun, Yan</creatorcontrib><creatorcontrib>Stransky, Stephanie</creatorcontrib><creatorcontrib>Aguilan, Jennifer</creatorcontrib><creatorcontrib>Koul, Sanjay</creatorcontrib><creatorcontrib>Garforth, Scott J.</creatorcontrib><creatorcontrib>Brenowitz, Michael</creatorcontrib><creatorcontrib>Sidoli, Simone</creatorcontrib><collection>Web of Science Core Collection</collection><collection>Science Citation Index Expanded</collection><collection>Web of Science - Science Citation Index Expanded - 2021</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Analytica chimica acta</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sun, Yan</au><au>Stransky, Stephanie</au><au>Aguilan, Jennifer</au><au>Koul, Sanjay</au><au>Garforth, Scott J.</au><au>Brenowitz, Michael</au><au>Sidoli, Simone</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry</atitle><jtitle>Analytica chimica acta</jtitle><stitle>ANAL CHIM ACTA</stitle><date>2021-10-02</date><risdate>2021</risdate><volume>1180</volume><spage>338880</spage><epage>338880</epage><pages>338880-338880</pages><artnum>338880</artnum><issn>0003-2670</issn><eissn>1873-4324</eissn><abstract>We present a direct injection mass spectrometry (DI-MS) platform that accurately, precisely, and quickly quantitates global levels of DNA cytidine methylation (5 mC) 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 5 mC 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 5 mC and 5hmC over a range of 1%–7% (R2 > 0.98) and 0.13%–1.75% (R2 > 0.99), respectively. Accurate measurement of C, 5 mC 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 h.
[Display omitted]
•We present an ultra-rapid (<1 min) mass spectrometry method to analyze major DNA modifications.•The method overcomes issues of liquid chromatography like carryover and biased retention of nucleosides of different hydrophobicities.•We demonstrate that routinely used targeted approaches overlook the formation of nucleoside byproducts affecting quantification.•We optimized instrument settings to minimize biases in quantification.</abstract><cop>AMSTERDAM</cop><pub>Elsevier B.V</pub><pmid>34538324</pmid><doi>10.1016/j.aca.2021.338880</doi><tpages>11</tpages><orcidid>https://orcid.org/0000-0002-9605-4825</orcidid><orcidid>https://orcid.org/0000-0001-6281-4833</orcidid><orcidid>https://orcid.org/0000-0002-5747-3306</orcidid><orcidid>https://orcid.org/0000-0001-9073-6641</orcidid><orcidid>https://orcid.org/0000-0002-0221-354X</orcidid><orcidid>https://orcid.org/0000-0003-0048-1716</orcidid><orcidid>https://orcid.org/0000-0003-0844-2754</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | 5hmC 5mC Chemistry Chemistry, Analytical Direct injection DNA High-throughput Hydroxymethylation Mass spectrometry Methylation Physical Sciences Science & Technology |
| title | High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry |
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