Summary of Study ST001661
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001066. The data can be accessed directly via it's Project DOI: 10.21228/M8X12C This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST001661 |
| Study Title | Extension of Diagnostic Fragmentation Filtering for Automated Discovery in DNA Adductomics |
| Study Summary | Development of high resolution/accurate mass liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) methodology enables the characterization of covalently modified DNA induced by interaction with genotoxic agents in complex biological samples. Constant neutral loss monitoring of 2´-deoxyribose or the nucleobases using data-dependent acquisition represents a powerful approach for the unbiased detection of DNA modifications (adducts). The lack of available bioinformatics tools necessitates manual processing of acquired spectral data and hampers high throughput application of these techniques. To address this limitation, we present an automated workflow for the detection and curation of putative DNA adducts by using diagnostic fragmentation filtering of LC-MS/MS experiments within the open-source software MZmine. The workflow utilizes a new feature detection algorithm, DFBuilder, which employs diagnostic fragmentation filtering using a user-defined list of fragmentation patterns to reproducibly generate feature lists for precursor ions of interest. The DFBuilder feature detection approach readily fits into a complete small molecule discovery workflow and drastically reduces the processing time associated with analyzing DNA adductomics results. We validate our workflow using a mixture of authentic DNA adduct standards and demonstrate the effectiveness of our approach by reproducing and expanding the results of a previously published study of colibactin-induced DNA adducts. The reported workflow serves as a technique to assess the diagnostic potential of novel fragmentation pattern combinations for the unbiased detection of chemical classes of interest. |
| Institute | University of Minnesota |
| Department | School of Public Health, Division of Environmental Health Sciences |
| Laboratory | Balbo Research Group |
| Last Name | Murray |
| First Name | Kevin |
| Address | 2-210 CCRB, 2231 6th St SE, Minneapolis, MN 55455 |
| murra668@umn.edu | |
| Phone | 612-626-2182 |
| Submit Date | 2021-01-25 |
| Num Groups | 1 |
| Total Subjects | 3 |
| Study Comments | Synthetic samples of authentic standards for workflow testing and validation. |
| Publications | Murray K.J.; Carlson E.S.; Stornetta A.; Balskus E.P.; Villalta P.W.; Balbo S. Extension of Diagnostic Fragmentation Filtering for Automated Discovery in DNA Adductomics. Anal. Chem. 2021. (In Revision). |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-04-25 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001066 |
| Project DOI: | doi: 10.21228/M8X12C |
| Project Title: | Extension of Diagnostic Fragmentation Filtering for Automated Discovery in DNA Adductomics |
| Project Summary: | Development of high resolution/accurate mass liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) methodology enables the characterization of covalently modified DNA induced by interaction with genotoxic agents in complex biological samples. Constant neutral loss monitoring of 2´-deoxyribose or the nucleobases using data-dependent acquisition represents a powerful approach for the unbiased detection of DNA modifications (adducts). The lack of available bioinformatics tools necessitates manual processing of acquired spectral data and hampers high throughput application of these techniques. To address this limitation, we present an automated workflow for the detection and curation of putative DNA adducts by using diagnostic frag-mentation filtering of LC-MS/MS experiments within the open-source software MZmine. The workflow utilizes a new feature detection algorithm, DFBuilder, which employs diagnostic fragmentation filtering using a user-defined list of fragmentation pat-terns to reproducibly generate feature lists for precursor ions of interest. The DFBuilder feature detection approach readily fits into a complete small molecule discovery workflow and drastically reduces the processing time associated with analyzing DNA adductomics results. We validate our workflow using a mixture of authentic DNA adduct standards and demonstrate the effectiveness of our approach by reproducing and expanding the results of a previously published study of colibactin-induced DNA adducts. The reported workflow serves as a technique to assess the diagnostic potential of novel fragmentation pattern combinations for the unbiased detection of chemical classes of interest. |
| Institute: | University of Minnesota |
| Department: | School of Public Health, Division of Environmental Health Sciences |
| Laboratory: | Balbo Research Group |
| Last Name: | Murray |
| First Name: | Kevin |
| Address: | 2-210 CCRB, 2231 6th St SE, Minneapolis, MN 55455 |
| Email: | murra668@umn.edu |
| Phone: | 612-625-2280 |
| Project Comments: | Experimental data for the reproduction and testing of the DFBuilder workflow for the automated detection of DNA adducts using diagnostic fragmentation filtering. |
| Publications: | Murray K.J.; Carlson E.S.; Stornetta A.; Balskus E.P.; Villalta P.W.; Balbo S. Extension of Diagnostic Fragmentation Filtering for Automated Discovery in DNA Adductomics. Anal. Chem. 2021. (In Revision). |
Subject:
| Subject ID: | SU001738 |
| Subject Type: | Synthetic sample |
| Subject Species: | - |
Factors:
Subject type: Synthetic sample; Subject species: - (Factor headings shown in green)
| mb_sample_id | local_sample_id | Matrix |
|---|---|---|
| SA152080 | DNA_Adducts_Blank_1 | Blank |
| SA152081 | DNA_Adducts_Sample_3 | Standards |
| SA152082 | DNA_Adducts_Sample_2 | Standards |
| SA152083 | DNA_Adducts_Sample_1 | Standards |
| Showing results 1 to 4 of 4 |
Collection:
| Collection ID: | CO001731 |
| Collection Summary: | Synthetic standard mixture of covalently modified DNA. |
| Sample Type: | Synthetic Mixture |
Treatment:
| Treatment ID: | TR001751 |
| Treatment Summary: | No treatment. |
Sample Preparation:
| Sampleprep ID: | SP001744 |
| Sampleprep Summary: | All DNA adduct standards were purchased or prepared as previously described. The nine DNA adduct standards included: O6-Methyl-2´-deoxyguanosine (O6-me-dG), 8-oxo-7, 8-dihydro-2´-deoxyguanosine (8-oxo-dG), N6-hydroxymethyldeoxyadenosine (N6-Me-dA), 1, N6-etheno-2´-deoxyadenosine (ε-dA), N2-Ethyl-2´-deoxyguanosine (N2-ethyl-dG), (6R/S)-3-(2´-deoxyribos-1´-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]-purine-10(3H)one (OH-PdG), O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dT), D5-ethyl-2´-deoxycytidine (D5-ethyl-dC), 6-(1-Hydroxyhexanyl)-8-hydroxy-1, and N2-propano-2´-deoxyguansine (HNE-dG). The nine standards were dissolved in 20% methanol and combined at a final concentration of 10 fmol/µL, respectively. All solvents were LC-MS grade and were purchased from Sigma-Aldrich. |
Combined analysis:
| Analysis ID | AN002712 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 RS |
| Column | Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) |
| MS Type | ESI |
| MS instrument type | Ion trap |
| MS instrument name | Thermo Fusion Tribrid Orbitrap |
| Ion Mode | POSITIVE |
| Units | Abundance |
Chromatography:
| Chromatography ID: | CH002001 |
| Chromatography Summary: | All analyses were conducted using identical chromatographic conditions and MS instrument settings, unless otherwise described. An UltiMate™ 3000 RSLCnano HPLC system (Thermo Scientific, Waltham, MA) was interfaced to an Orbitrap Fusion™ Tribrid™ MS (Thermo Fisher Scientific, San Jose, CA). One microliter of the authentic DNA standard mixture and five microliters of E. Coli DNA extracts were injected onto the analytical platform equipped with a 5 µL injection loop. Solvent blanks were analyzed before and after acquisition to assess contamination and sample carryover between injections. Chromatographic separation was performed using a custom-packed capillary column (75 µm ID, 20 cm length, 10 µm orifice) using a commercially available fused-silica emitter (New Objective, Woburn MA) containing Luna C18 (Phenomenex Corp. Torrance, CA) stationary phase (5 µm, 120 Å). The LC solvents were (A) 0.05% HCO2H in H2O and (B) CH3CN solutions. The flow rate was 1000 nL/min for 5.5 min at 2% B, then decreased to 300 nL/min with a 25 min linear gradient from 2 to 50% B, an increase to 98% B in 1 min, with a 4 min hold and a 5 min equilibration at 1000 nL/min to the starting conditions. The injection valve was switched at 5.5 min to remove the sample loop from the flow path during the gradient. A Nanospray Flex ion source (Thermo Fisher Scientific) was used with a source voltage of 2.2 kV and capillary temperature of 300°C. The S-Lens RF level setting was 60%. |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) |
| Flow Gradient: | The flow rate was 1000 nL/min for 5.5 min at 2% B, then decreased to 300 nL/min with a 25 min linear gradient from 2 to 50% B, an increase to 98% B in 1 min, with a 4 min hold and a 5 min equilibration at 1000 nL/min to the starting conditions. |
| Flow Rate: | 1ml/min-0.3ml/min |
| Solvent A: | 100% water; 0.05% formic acid |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002509 |
| Analysis ID: | AN002712 |
| Instrument Name: | Thermo Fusion Tribrid Orbitrap |
| Instrument Type: | Ion trap |
| MS Type: | ESI |
| MS Comments: | Untargeted DDA-CNL-MS3 analyses were performed with full scan detection followed by MS2 acquisition and constant neutral loss triggering of MS3 fragmentation. Full scan detection was performed using the Orbitrap detection at a resolution of 60,000, automatic gain control (AGC) targeted setting of 2 × 10^5, and a maximum ion injection time setting of 118 ms. Full scan range of 150 – 1000 m/z was used for analysis of the authentic standards. MS2 spectra were acquired with quadrupole isolation of 1.5 m/z, fragmentation of the top 10 most intense full scan ions with Orbitrap detection at a resolution of 15,000, an AGC setting of 5 × 10^4, and a maximum ion injection time of 200 ms. The analysis of authentic standards utilized CID fragmentation with a constant collision energy of 30% and maximum ion injection time of 75 ms. Data-dependent parameters were as follows: a triggering threshold of 2.0 × 10^4, repeat count of 1, exclusion duration of 15 s. No masses were excluded in the analysis of the authentic standards. MS3 HCD /CID fragmentation (2.5 m/z isolation width, HCD/CID collision energy of 30%) with Orbitrap detection at a resolution of 15,000 was triggered upon observation of neutral losses of 116.0474, 151. 0494, 135.0545, 126.0429 and 111.0433 m/z. A minimal product ion signal of 1.0 × 10^4 was used. All spectra were acquired with the EASY-IC lock mass (202.0777 m/z) enabled. |
| Ion Mode: | POSITIVE |
