Capillary Monolithic Bioreactor of Immobilized Snake Venom Phosphodiesterase for Mass Spectrometry Based Oligodeoxynucleotide Sequencing

Zhao, Chao; Yin, Ruichuan; Yin, Jie; Zhang, David; Wang, Hailin

doi:10.1021/ac2029387

Cited by 9 publications

(20 citation statements)

References 45 publications

(68 reference statements)

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“…The DNase I, SVP, and ALPaseimmobilized bioreactors were fabricated in capillary silica monolith according to our previous work. 45 In brief, three major steps were included (Scheme 1). First, the silica monolith was in situ synthesized in a fused-silica capillary (i.d.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…75 μm) using the sol−gel polymerization of TMOS. 45 Then the silylation reagent (3-aminopropyltriethoxysilane) was used to introduce amino groups to the surface of the capillary monolith. Finally, the DNase I, SVP, and ALPase were immobilized onto the monolith via Schiff-Base reaction by using glutaraldehyde to link the -NH 2 groups on monolith and enzymes.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…This result is well in accordance with obviations of our previous study. 45 Then the enzymatic efficiency of three-enzyme cascade bioreactor was evaluated. To this purpose, 12 μL of 200 ng/μL ct-DNA was pumped through the cascade bioreactor and digested for 5, 10, and 15 min, respectively, until 60 μL of products were collected.…”

Section: Analytical Chemistrymentioning

confidence: 99%

“…We thus fabricated a snake venom phosphodiesterase-based capillary bioreactor that can digest oligodeoxynucleotides for mass spectrometry-based sequencing. 45 By using such a SVP bioreactor, the sequence-specific modification of single strand oligodeoynucleotides induced by a ubiquitous pollutant acrolein was identified, demonstrating its promising applications in identification of sequence-specific damages in short oligonucleotides. In terms of the genomic DNA strands, however, there has no attempt been made in developing threeenzyme immobilized bioreactor for their completely digestion.…”

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confidence: 99%

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Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides

Yin

Zhang

et al. 2016

Anal. Chem.

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Structure-based DNA modification analysis provides accurate and important information on genomic DNA changes from epigenetic modifications to various DNA lesions. However, genomic DNA strands are often required to be efficiently digested into single nucleosides. It is an arduous task because of the involvement of multiple enzymes with different catalytic acitivities. Here we constructed a three-enzyme cascade capillary monolithic bioreactor that consists of immobilized deoxyribonuclease I (DNase I), snake venom phosphodiesterase (SVP), and alkaline phosphatase (ALPase). By the use of this cascade capillary bioreactor, genomic DNA can be efficiently digested into single nucleosides with an increasing rate of ∼20 folds. The improvement is mainly attributed to dramatically increase enzymatic capacity and activity. With a designed macro-porous structure, genomic DNA of 5−30 Kb (∼1.6−10 million Daltons) can be directly passed through the bioreactor simply by hand pushing or a low-pressure microinjection pump. By coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further developed a sensitive assay for detection of an oxidative stress biomarker 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in DNA. The proposed three-enzyme cascade bioreactor is also potentially applicable for fast identification and quantitative detection of other lesions and modifications in genomic DNA.E xposure to chemical carcinogens may result in DNA damages, a key step toward the onset of cancer. 1−4 DNA damages can occur via structural modification of the nucleosides and phosphate moieties in DNA chains. 5 It is difficult to directly search for various modifications in genomic DNA since DNA strands are long biopolymers consisting of a large number of normal nucleosides (deoxyadenosine, dA; deoxygunosine, dG; deoxycytidine, dC; and thymidine monophosphates, dT) interspersing with a small quantity of modified nucleosides (e.g., 5-methylcytosine, 5mC) or trace modifications (e.g., 5-hydroxymethylcytosine, 5-formalcytosine, and 5-carboxylcytosine). To determine these DNA modifications, genomic DNA samples were often required to be efficiently digested into mononucleotides or single nucleosides. 6−10 Liquid chromatography-mass spectrometry (LC-MS) can explore whether nucleotide is modified, and is sensitive enough to quantify these modifications on DNA chains. 9,11−15 For example, during LC-MS/MS analysis of global DNA methylation and hydroxylmethylation, genomic DNA was digested into the mixture of classical DNA nucleosides (dA, dT, dG, dC) and modified nucleosides. 16−18 Recently, we discovered a new DNA modification in drosophila, N6-methyladenine. 19 To accurately identify its structure and validate its identity, the Drosophila genomic DNA was also required to be digested into single nucleosides for high sensitivity analysis. Another example, identification and analysis of oxidative damaged DNA (e.g., 8-oxo-7,8-dihydro-2′-deoxyguanosine, 8-oxodG) completely digestion of genomic DNA to maximal release 8-oxodG w...

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Analytical Chemistrymentioning

confidence: 99%

mentioning

confidence: 99%

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Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides

Yin

Zhang

et al. 2016

Anal. Chem.

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“…In one example, fabrication of the silica monolith shown in Fig. 7 was achieved through polymerization of tetramethoxysilane [93]. The silica monolith was modified with 3-aminopropyl-trimethoxysilane to conjugate enzyme to the monolith via reductive amination.…”

Section: Enzyme Immobilization Techniquesmentioning

confidence: 99%

Advances in enzyme substrate analysis with capillary electrophoresis

Gattu

Crihfield

et al. 2018

Methods

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Capillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with various detectors makes it suitable for KM determinations ranging from nanomolar to millimolar concentrations. Capillary electrophoresis fundamentals are discussed with an emphasis on the separation mechanisms relevant to evaluate sets of substrate and product that are charged, neutral, and even chiral. The basic principles of Michaelis-Menten determinations are reviewed and the process of translating capillary electrophoresis electropherograms into a Michaelis-Menten curve is outlined. The conditions that must be optimized in order to couple off-line and on-line enzyme reactions with capillary electrophoresis separations, such as incubation time, buffer pH and ionic strength, and temperature, are examined to provide insight into how the techniques can be best utilized. The application of capillary electrophoresis to quantify enzyme inhibition, in the form of KI or IC50 is detailed. The concept and implementation of the immobilized enzyme reactor is described as a means to increase enzyme stability and reusability, as well as a powerful tool for screening enzyme substrates and inhibitors. Emerging techniques focused on applying capillary electrophoresis as a rapid assay to obtain structural identification or sequence information about a substrate and in-line digestions of peptides and proteins coupled to mass spectrometry analyses are highlighted.

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Mass spectrometry analysis of nucleosides and nucleotides

Dudley

Bond

2013

Mass Spec Rev

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Mass spectrometry has been widely utilised in the study of nucleobases, nucleosides and nucleotides as components of nucleic acids and as bioactive metabolites in their own right. In this review, the application of mass spectrometry to such analysis is overviewed in relation to various aspects regarding the analytical mass spectrometric and chromatographic techniques applied and also the various applications of such analysis.

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Capillary Monolithic Bioreactor of Immobilized Snake Venom Phosphodiesterase for Mass Spectrometry Based Oligodeoxynucleotide Sequencing

Cited by 9 publications

References 45 publications

Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides

Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides

Advances in enzyme substrate analysis with capillary electrophoresis

Mass spectrometry analysis of nucleosides and nucleotides

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