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MB Sample ID: SA152083

Local Sample ID:DNA_Adducts_Sample_1
Subject ID:SU001738
Subject Type:Synthetic sample
Subject Species:-

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Subject:

Subject ID:SU001738
Subject Type:Synthetic sample
Subject Species:-

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
DNA_Adducts_Sample_1SA152083FL017884StandardsMatrix

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
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