Fabrication of an on-line enzyme micro-reactor coupled to liquid chromatography–tandem mass spectrometry for the digestion of recombinant human erythropoietin

Foo, Hsiao Ching; Smith, Norman W.; Stanley, Shawn M.R.

doi:10.1016/j.talanta.2014.12.033

Cited by 23 publications

(9 citation statements)

References 30 publications

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“…Trypsin can be covalently bonded or physically adsorbed onto silica or organic materials such as styrene-, acrylamide-, and methacrylate-based copolymers [6]. This can provide immobilized trypsin on membranes [8–11], modified silica capillaries [12–14], microchips [15, 16], gel networks [17, 18], etc. [7].…”

Section: Introductionmentioning

confidence: 99%

“…[7]. Utilizing immobilized trypsin for a protein digestion provides advantages such as a high enzyme-to-substrate ratio, shorter digestion times, less trypsin autolysis, and low reagent consumption while maintaining highly efficient digestions [4–7, 12, 19]. While the high abundance of trypsin allows for less competition for catalytic sites, the decrease in trypsin autolysis provides less complex mass spectra for analysis.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of an immobilized enzyme reactor for on-line protein digestion

Moore

Hess

Jorgenson

2016

Journal of Chromatography A

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Despite the developments for faster liquid chromatographic and mass spectral detection techniques, the standard in-solution protein digestion for proteomic analyses has remained relatively unchanged. The typical in-solution trypsin protein digestion is usually the slowest part of the workflow, albeit one of the most important. The development of a highly efficient immobilized enzyme reactor (IMER) with rapid performance for on-line protein digestion would greatly decrease the analysis time involved in a proteomic workflow. Presented here is the development of a silica based IMER for on-line protein digestion, which produced rapid digestions in the presence of organic mobile phase for both model proteins and a complex sample consisting of the insoluble portion of a yeast cell lysate. Protein sequence coverage and identifications evaluated between the IMER and in-solution digestions were comparable. Overall, for a yeast cell lysate with only a 10 sec volumetric residence time on-column, the IMER identified 507 proteins while the in-solution digestion identified 490. There were no significant differences observed based on identified protein’s molecular weight or isoelectric point between the two digestion methods. Implementation of the IMER into the proteomic workflow provided similar protein identification results, automation for sample analysis, and reduced the analysis time by 15 hr.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Characterization of an immobilized enzyme reactor for on-line protein digestion

Moore

Hess

Jorgenson

2016

Journal of Chromatography A

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“…Of note, DNA digestion is very different from proteomic digestion, in which the proteins can be quickly digested into small peptides and amino acids by one enzyme (e.g., the potent enzyme trypsin), which can then be directly analyzed by LC-MS. − Compared with the most commonly studied proteins, genomic DNA strands are much larger supramacromolecules, which possess molecular weight generally more than 10 million Daltions. To characterize chemical structural change of nucleotides in DNA or to quantify the stucturally changed nucleoties, long DNA chains are often required to be digested into single nucleosides facilitating followed MS analysis.…”

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confidence: 99%

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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“…It is known that enzyme-immobilized bioreactors exhibited good performances in online fast digestion proteins (typically within 10 min) coupled with LC–MS proteomics analysis, − however, up to date, seldom studies reported as for utilizing immobilized enzymes for online digestion coupled LC–MS for DNA analysis. This is because, compared to most studies, proteins and genomic DNA strands are much larger supra-macromolecules (generally, molecular weight > 10 million Daltons) and thus are more difficult to be digested into single nucleosides facilitating the followed MS analysis.…”

Section: Introductionmentioning

confidence: 99%

Multienzyme Cascade Bioreactor for a 10 min Digestion of Genomic DNA into Single Nucleosides and Quantitative Detection of Structural DNA Modifications in Cellular Genomic DNA

Yin

Chen

Zhang

et al. 2018

ACS Appl. Mater. Interfaces

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Identification and quantification of chemical DNA modifications provide essential information on genomic DNA changes, for example, epigenetic modifications and abnormal DNA lesions. In this vein, it requires to digest genomic DNA strands into single nucleosides, facilitating the mass spectrometry analysis. However, rapid digestion of such supramacromolecule DNA of several millions Daltons (molecular weight) into single nucleosides remains very challenging. Here, we constructed an immobilized benzonase capillary bioreactor and further tandemly coupled with immobilized snake venom phosphodiesterase and alkaline phosphatase capillary bioreactor to form a novel three-enzyme cascade bioreactor (BenzoSAC bioreactor). In these constructions, the chosen enzymes were immobilized onto synthetic porous capillary silica monoliths. With the tailor-made porous structure and high immobilized capacity and high digestion rate of benzonase, genomic DNA of >99.5% can be digested into single nucleosides within only 10 min when passing through the BenzoSAC bioreactor by microinjection pump. In contrast, traditional digestion requires 8-24 h. By offline coupling this benzoSAC bioreactor with liquid chromatography-tandem mass spectrometry, we detected 5-hydroxymethylcytosine, a major oxidation product of the epigenetically crucial 5-methylcytosine, in genomic DNA isolated from ladder cancer (T24) cells. The newly synthesized BenzoSAC bioreactor and the proposed mass spectrometry detection are promising for fast identification and analysis of structural modifications in DNA.

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Fabrication of an on-line enzyme micro-reactor coupled to liquid chromatography–tandem mass spectrometry for the digestion of recombinant human erythropoietin

Cited by 23 publications

References 30 publications

Characterization of an immobilized enzyme reactor for on-line protein digestion

Characterization of an immobilized enzyme reactor for on-line protein digestion

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

Multienzyme Cascade Bioreactor for a 10 min Digestion of Genomic DNA into Single Nucleosides and Quantitative Detection of Structural DNA Modifications in Cellular Genomic DNA

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