#METABOLOMICS WORKBENCH murra668_20210125_135623 DATATRACK_ID:2421 STUDY_ID:ST001661 ANALYSIS_ID:AN002712 PROJECT_ID:PR001066 VERSION 1 CREATED_ON January 26, 2021, 11:08 am #PROJECT PR:PROJECT_TITLE Extension of Diagnostic Fragmentation Filtering for Automated Discovery in DNA PR:PROJECT_TITLE Adductomics PR:PROJECT_SUMMARY Development of high resolution/accurate mass liquid chromatography-coupled PR:PROJECT_SUMMARY tandem mass spectrometry (LC-MS/MS) methodology enables the characterization of PR:PROJECT_SUMMARY covalently modified DNA induced by interaction with genotoxic agents in complex PR:PROJECT_SUMMARY biological samples. Constant neutral loss monitoring of 2´-deoxyribose or the PR:PROJECT_SUMMARY nucleobases using data-dependent acquisition represents a powerful approach for PR:PROJECT_SUMMARY the unbiased detection of DNA modifications (adducts). The lack of available PR:PROJECT_SUMMARY bioinformatics tools necessitates manual processing of acquired spectral data PR:PROJECT_SUMMARY and hampers high throughput application of these techniques. To address this PR:PROJECT_SUMMARY limitation, we present an automated workflow for the detection and curation of PR:PROJECT_SUMMARY putative DNA adducts by using diagnostic frag-mentation filtering of LC-MS/MS PR:PROJECT_SUMMARY experiments within the open-source software MZmine. The workflow utilizes a new PR:PROJECT_SUMMARY feature detection algorithm, DFBuilder, which employs diagnostic fragmentation PR:PROJECT_SUMMARY filtering using a user-defined list of fragmentation pat-terns to reproducibly PR:PROJECT_SUMMARY generate feature lists for precursor ions of interest. The DFBuilder feature PR:PROJECT_SUMMARY detection approach readily fits into a complete small molecule discovery PR:PROJECT_SUMMARY workflow and drastically reduces the processing time associated with analyzing PR:PROJECT_SUMMARY DNA adductomics results. We validate our workflow using a mixture of authentic PR:PROJECT_SUMMARY DNA adduct standards and demonstrate the effectiveness of our approach by PR:PROJECT_SUMMARY reproducing and expanding the results of a previously published study of PR:PROJECT_SUMMARY colibactin-induced DNA adducts. The reported workflow serves as a technique to PR:PROJECT_SUMMARY assess the diagnostic potential of novel fragmentation pattern combinations for PR:PROJECT_SUMMARY the unbiased detection of chemical classes of interest. PR:INSTITUTE University of Minnesota PR:DEPARTMENT School of Public Health, Division of Environmental Health Sciences PR:LABORATORY Balbo Research Group PR:LAST_NAME Murray PR:FIRST_NAME Kevin PR:ADDRESS 2-210 CCRB, 2231 6th St SE, Minneapolis, MN 55455 PR:EMAIL murra668@umn.edu PR:PHONE 612-625-2280 PR:PROJECT_COMMENTS Experimental data for the reproduction and testing of the DFBuilder workflow for PR:PROJECT_COMMENTS the automated detection of DNA adducts using diagnostic fragmentation filtering. PR:PUBLICATIONS Murray K.J.; Carlson E.S.; Stornetta A.; Balskus E.P.; Villalta P.W.; Balbo S. PR:PUBLICATIONS Extension of Diagnostic Fragmentation Filtering for Automated Discovery in DNA PR:PUBLICATIONS Adductomics. Anal. Chem. 2021. (In Revision). #STUDY ST:STUDY_TITLE Extension of Diagnostic Fragmentation Filtering for Automated Discovery in DNA ST:STUDY_TITLE Adductomics ST:STUDY_SUMMARY Development of high resolution/accurate mass liquid chromatography-coupled ST:STUDY_SUMMARY tandem mass spectrometry (LC-MS/MS) methodology enables the characterization of ST:STUDY_SUMMARY covalently modified DNA induced by interaction with genotoxic agents in complex ST:STUDY_SUMMARY biological samples. Constant neutral loss monitoring of 2´-deoxyribose or the ST:STUDY_SUMMARY nucleobases using data-dependent acquisition represents a powerful approach for ST:STUDY_SUMMARY the unbiased detection of DNA modifications (adducts). The lack of available ST:STUDY_SUMMARY bioinformatics tools necessitates manual processing of acquired spectral data ST:STUDY_SUMMARY and hampers high throughput application of these techniques. To address this ST:STUDY_SUMMARY limitation, we present an automated workflow for the detection and curation of ST:STUDY_SUMMARY putative DNA adducts by using diagnostic fragmentation filtering of LC-MS/MS ST:STUDY_SUMMARY experiments within the open-source software MZmine. The workflow utilizes a new ST:STUDY_SUMMARY feature detection algorithm, DFBuilder, which employs diagnostic fragmentation ST:STUDY_SUMMARY filtering using a user-defined list of fragmentation patterns to reproducibly ST:STUDY_SUMMARY generate feature lists for precursor ions of interest. The DFBuilder feature ST:STUDY_SUMMARY detection approach readily fits into a complete small molecule discovery ST:STUDY_SUMMARY workflow and drastically reduces the processing time associated with analyzing ST:STUDY_SUMMARY DNA adductomics results. We validate our workflow using a mixture of authentic ST:STUDY_SUMMARY DNA adduct standards and demonstrate the effectiveness of our approach by ST:STUDY_SUMMARY reproducing and expanding the results of a previously published study of ST:STUDY_SUMMARY colibactin-induced DNA adducts. The reported workflow serves as a technique to ST:STUDY_SUMMARY assess the diagnostic potential of novel fragmentation pattern combinations for ST:STUDY_SUMMARY the unbiased detection of chemical classes of interest. ST:INSTITUTE University of Minnesota ST:DEPARTMENT School of Public Health, Division of Environmental Health Sciences ST:LABORATORY Balbo Research Group ST:LAST_NAME Murray ST:FIRST_NAME Kevin ST:ADDRESS 2-210 CCRB, 2231 6th St SE, Minneapolis, MN 55455 ST:EMAIL murra668@umn.edu ST:PHONE 612-626-2182 ST:NUM_GROUPS 1 ST:TOTAL_SUBJECTS 3 ST:STUDY_COMMENTS Synthetic samples of authentic standards for workflow testing and validation. ST:PUBLICATIONS Murray K.J.; Carlson E.S.; Stornetta A.; Balskus E.P.; Villalta P.W.; Balbo S. ST:PUBLICATIONS Extension of Diagnostic Fragmentation Filtering for Automated Discovery in DNA ST:PUBLICATIONS Adductomics. Anal. Chem. 2021. (In Revision). #SUBJECT SU:SUBJECT_TYPE Synthetic sample #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - DNA_Adducts_Blank_1 Matrix:Blank RAW_FILE_NAME=DNA_Adducts_Blank_1 SUBJECT_SAMPLE_FACTORS - DNA_Adducts_Sample_1 Matrix:Standards RAW_FILE_NAME=DNA_Adducts_Sample_1 SUBJECT_SAMPLE_FACTORS - DNA_Adducts_Sample_2 Matrix:Standards RAW_FILE_NAME=DNA_Adducts_Sample_2 SUBJECT_SAMPLE_FACTORS - DNA_Adducts_Sample_3 Matrix:Standards RAW_FILE_NAME=DNA_Adducts_Sample_3 #COLLECTION CO:COLLECTION_SUMMARY Synthetic standard mixture of covalently modified DNA. CO:SAMPLE_TYPE Synthetic Mixture #TREATMENT TR:TREATMENT_SUMMARY No treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY All DNA adduct standards were purchased or prepared as previously described. The SP:SAMPLEPREP_SUMMARY nine DNA adduct standards included: O6-Methyl-2´-deoxyguanosine (O6-me-dG), SP:SAMPLEPREP_SUMMARY 8-oxo-7, 8-dihydro-2´-deoxyguanosine (8-oxo-dG), N6-hydroxymethyldeoxyadenosine SP:SAMPLEPREP_SUMMARY (N6-Me-dA), 1, N6-etheno-2´-deoxyadenosine (ε-dA), N2-Ethyl-2´-deoxyguanosine SP:SAMPLEPREP_SUMMARY (N2-ethyl-dG), SP:SAMPLEPREP_SUMMARY (6R/S)-3-(2´-deoxyribos-1´-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]-purine-10(3H)one SP:SAMPLEPREP_SUMMARY (OH-PdG), O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dT), SP:SAMPLEPREP_SUMMARY D5-ethyl-2´-deoxycytidine (D5-ethyl-dC), 6-(1-Hydroxyhexanyl)-8-hydroxy-1, and SP:SAMPLEPREP_SUMMARY N2-propano-2´-deoxyguansine (HNE-dG). The nine standards were dissolved in 20% SP:SAMPLEPREP_SUMMARY methanol and combined at a final concentration of 10 fmol/µL, respectively. All SP:SAMPLEPREP_SUMMARY solvents were LC-MS grade and were purchased from Sigma-Aldrich. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY All analyses were conducted using identical chromatographic conditions and MS CH:CHROMATOGRAPHY_SUMMARY instrument settings, unless otherwise described. An UltiMate™ 3000 RSLCnano CH:CHROMATOGRAPHY_SUMMARY HPLC system (Thermo Scientific, Waltham, MA) was interfaced to an Orbitrap CH:CHROMATOGRAPHY_SUMMARY Fusion™ Tribrid™ MS (Thermo Fisher Scientific, San Jose, CA). One microliter CH:CHROMATOGRAPHY_SUMMARY of the authentic DNA standard mixture and five microliters of E. Coli DNA CH:CHROMATOGRAPHY_SUMMARY extracts were injected onto the analytical platform equipped with a 5 µL CH:CHROMATOGRAPHY_SUMMARY injection loop. Solvent blanks were analyzed before and after acquisition to CH:CHROMATOGRAPHY_SUMMARY assess contamination and sample carryover between injections. Chromatographic CH:CHROMATOGRAPHY_SUMMARY separation was performed using a custom-packed capillary column (75 µm ID, 20 CH:CHROMATOGRAPHY_SUMMARY cm length, 10 µm orifice) using a commercially available fused-silica emitter CH:CHROMATOGRAPHY_SUMMARY (New Objective, Woburn MA) containing Luna C18 (Phenomenex Corp. Torrance, CA) CH:CHROMATOGRAPHY_SUMMARY stationary phase (5 µm, 120 Å). The LC solvents were (A) 0.05% HCO2H in H2O CH:CHROMATOGRAPHY_SUMMARY and (B) CH3CN solutions. The flow rate was 1000 nL/min for 5.5 min at 2% B, then CH:CHROMATOGRAPHY_SUMMARY decreased to 300 nL/min with a 25 min linear gradient from 2 to 50% B, an CH:CHROMATOGRAPHY_SUMMARY increase to 98% B in 1 min, with a 4 min hold and a 5 min equilibration at 1000 CH:CHROMATOGRAPHY_SUMMARY nL/min to the starting conditions. The injection valve was switched at 5.5 min CH:CHROMATOGRAPHY_SUMMARY to remove the sample loop from the flow path during the gradient. A Nanospray CH:CHROMATOGRAPHY_SUMMARY Flex ion source (Thermo Fisher Scientific) was used with a source voltage of 2.2 CH:CHROMATOGRAPHY_SUMMARY kV and capillary temperature of 300°C. The S-Lens RF level setting was 60%. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Fusion Tribrid Orbitrap MS:INSTRUMENT_TYPE Ion trap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Untargeted DDA-CNL-MS3 analyses were performed with full scan detection followed MS:MS_COMMENTS by MS2 acquisition and constant neutral loss triggering of MS3 fragmentation. MS:MS_COMMENTS Full scan detection was performed using the Orbitrap detection at a resolution MS:MS_COMMENTS of 60,000, automatic gain control (AGC) targeted setting of 2 × 10^5, and a MS:MS_COMMENTS maximum ion injection time setting of 118 ms. Full scan range of 150 – 1000 MS:MS_COMMENTS m/z was used for analysis of the authentic standards. MS2 spectra were acquired MS:MS_COMMENTS with quadrupole isolation of 1.5 m/z, fragmentation of the top 10 most intense MS:MS_COMMENTS full scan ions with Orbitrap detection at a resolution of 15,000, an AGC setting MS:MS_COMMENTS of 5 × 10^4, and a maximum ion injection time of 200 ms. The analysis of MS:MS_COMMENTS authentic standards utilized CID fragmentation with a constant collision energy MS:MS_COMMENTS of 30% and maximum ion injection time of 75 ms. Data-dependent parameters were MS:MS_COMMENTS as follows: a triggering threshold of 2.0 × 10^4, repeat count of 1, exclusion MS:MS_COMMENTS duration of 15 s. No masses were excluded in the analysis of the authentic MS:MS_COMMENTS standards. MS3 HCD /CID fragmentation (2.5 m/z isolation width, HCD/CID MS:MS_COMMENTS collision energy of 30%) with Orbitrap detection at a resolution of 15,000 was MS:MS_COMMENTS triggered upon observation of neutral losses of 116.0474, 151. 0494, 135.0545, MS:MS_COMMENTS 126.0429 and 111.0433 m/z. A minimal product ion signal of 1.0 × 10^4 was used. MS:MS_COMMENTS All spectra were acquired with the EASY-IC lock mass (202.0777 m/z) enabled. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Abundance MS_METABOLITE_DATA_START Samples DNA_Adducts_Sample_1 DNA_Adducts_Sample_2 DNA_Adducts_Sample_3 Factors Matrix:Standards Matrix:Standards Matrix:Standards N6-etheno-dA 59000000.00 59000000.00 54600000.00 8-Oxo-dG 3015823.99 2931501.58 2452410.17 D5-ethyl-dC 5731662.15 5237348.18 5951297.65 HNE-dG 20900000.00 20200000.00 20100000.00 N2-ethyl-dG 4781059.99 4596734.55 4010626.97 N6-Me-dA 56000000.00 53900000.00 57500000.00 O2-POB-dT 22100000.00 23900000.00 22000000.00 O6-Me-dG 2060000000.00 2040000000.00 2050000000.00 OH-PdG 13600000.00 13200000.00 11800000.00 Unknown#1 1379118.07 2819709.37 1914104.73 Unknown#2 2673573.41 2426571.76 2275456.54 Unknown#3 2449519.67 2497606.79 2344876.84 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PUBCHEM quantitated m/z retention times N6-etheno-dA 10945668 266.1247355 16.60365078 8-Oxo-dG 135440064 284.0989503 15.75213062 D5-ethyl-dC 261.1605123 9.370714411 HNE-dG 154706783 424.2189636 37.63451714 N2-ethyl-dG 135742144 296.135259 26.11177088 N6-Me-dA 102175 266.1247355 16.60365078 O2-POB-dT 390.1658796 25.62979527 O6-Me-dG 73317 282.1197001 21.49885507 OH-PdG 324.1302999 17.00734837 Unknown#1 211.132958 27.13967637 Unknown#2 296.1353353 28.53294047 Unknown#3 398.1669108 22.35523217 METABOLITES_END #END