Accurate Measurement of FormaldehydeâInduced DNAâProtein

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Accurate Measurement of Formaldehyde–Induced DNA–Protein Crosslinks by High-resolution Orbitrap Mass Spectrometry Chih-Wei Liu, Xu Tian, Hadley J Hartwell, Jiapeng Leng, Liang Chi, Kun Lu, and James A. Swenberg Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00040 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

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Chemical Research in Toxicology

Accurate Measurement of Formaldehyde–Induced DNA–Protein Crosslinks by High-resolution Orbitrap Mass Spectrometry Chih-Wei Liu, Xu Tian, Hadley J. Hartwell, Jiapeng Leng, Liang Chi, Kun Lu* and James A. Swenberg* Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States

Corresponding Author

* Kun Lu, Phone: 919 966 7337; e-mail: [email protected]. * James A. Swenberg, Phone: 919 966 6139; email: [email protected].

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Table of Contents (TOC) graphic


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ABSTRACT

Genomic instability caused by DNA-protein crosslink (DPCs)-induced DNA damage is implicated in disease pathogenesis, aging and cancer development. The covalent linkages between DNA and protein are induced by chemical reactions catalyzed by the endogenous metabolic intermediates and exogenous agents such as aldehydes, chemotherapeutic agents, and ionizing radiation. Formaldehyde has been classified as a genotoxic carcinogen. In addition, endogenous formaldehyde-induced DPCs may increase the risks of bone marrow toxicity and leukemia. There is a need to develop an effective detection method for DPC analysis, including structural differentiation of endogenous and exogenous formaldehyde-induced DPCs. To this end, our group previously reported a useful liquid chromatography-selected reaction monitoring (LCSRM) approach coupled with stable isotope labeling and low mass resolution-triple quadrupole mass spectrometry. In the present work, we further demonstrate an accurate quantification method using a high-resolution, accurate-mass Orbitrap mass spectrometer for measurement of covalent linkage between 2’-deoxyguanosine (dG) and cysteine (Cys), specifically termed dGMe-Cys, one kind of linkages derived from formaldehyde-induced DPCs. This quantification method with wide dynamic range of at least 3 orders generates an interference-free spectrum for unbiased and unambiguous quantification, resulting in good intra- and inter-day precisions and accuracies with less than 10% variations. The endogenous and exogenous amounts of dG-MeCys in a human cell line treated with formaldehyde are analyzed by our new methodology. The quantification strategy demonstrated in this study can be widely applied to characterize and quantify other DPC linkages induced by formaldehyde or other chemical agents.



INTRODUCTION DNA damage caused by toxic chemicals generated from either endogenous metabolic pathways or exogenous environmental stimuli is implicated in disease pathogenesis and cancer development.1-5 The types of DNA damage include DNA single- and double-strand breaks, DNA adducts, abasic sites (Ap site), Ap site-mediated interstrand DNA–DNA crosslinks, and DNAprotein crosslinks (DPCs), among others.5-8 Within the bulky lesions caused by DPCs, the covalent bond formed between DNA and protein is induced by chemical reactions catalyzed by either endogenous metabolic intermediates3, 9, 10 or exogenous agents.11-13 DPCs form obstacles that affect normal DNA-protein interactions during DNA replication and transcription, which may result in accelerated aging and cancer development.14 While it is very important to understand the molecular mechanisms involved in DPCs repair, currently very little is known. Recently, it was reported that DNA-dependent proteases, Wss1 and SPRTN, might play an important role for DPC removal, protecting cells from DPC genotoxicity.1, 15 Historically, the bottleneck for studying DPCs has been due to the nonselective methods used for isolation and detection. Traditional DNA/protein precipitation methods are incapable of selectively recognizing and quantifying a specific structural DPC linkage from a complex DPC pool. Recently, the advancement in mass spectrometry-based detection and quantification methods has allowed for the characterization of specific structural DPCs that were directly induced by exogenous crosslinking agents such as nitrogen mustards13 or diepoxybutane.16 Most importantly, formaldehyde, a well-known crosslinking agent, has been classified as a carcinogen in animals and humans.9, 17, 18 Humans are exposed to formaldehyde through a wide array of industrial and environmental sources, as well as from endogenous formation during various cellular processes.19 Until recently, it has been impossible to differentiate exogenous from


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endogenous formaldehyde-induced DPCs. Lai et al. implemented the use of stable isotope labeled-formaldehyde in animal exposure experiments, which enabled the simultaneous quantification of endogenous and exogenous DPCs.10 In that study, the covalent linkage between the 2’-deoxyguanosine (dG) and cysteine (Cys), specifically termed dG-Me-Cys (Scheme 1), represents the formaldehyde-induced DPCs. Moreover, dG-Me-Cys linkage is one of the major covalent linkages derived from formaldehyde-induced DPCs. Specifically, the linkages between amino acids (cysteine, histidine, tryptophan, and lysine) and deoxynucleosides (2’deoxyguanosine, 2’-deoxyadenosine, and 2’-deoxycytidine) induced by formaldehyde have been identified by an in vitro experiment conducted by Lu et al..20 It is of note that traditional selected reaction monitoring (SRM) for targeted quantification was utilized in those three pioneering structural DPCs studies.



Scheme 1. Proposed fragmentation patterns of dG-Me-Cys and [15N5]-dG-[13CD2]-Me-Cys after HCD fragmentation. The measured m/z and mass accuracy are shown in Table 1. The “N”, “C”, and “H” highlighted with red indicate stable-isotope labeled sites of [15N5]-dG-[13CD2]-Me-Cys.

In traditional SRM performed on triple quadrupole (QqQ) mass spectrometry,21-23 the first and the third quadrupoles act as mass filters to specifically select predefined m/z values for a precursor ion. A specific fragment ion is generated from fragmentation performed in the second 6 Environment ACS Paragon Plus

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quadrupole, which is used as collision cell. A set of transitions (precursor/fragment ion pairs) are selectively monitored over time to reveal the chromatographic peak for quantification. The main drawback of using SRM for quantification is the low mass resolution of the quadrupole mass analyzer used for ion selection, which may affect the quantification accuracy, selectivity and sensitivity due to the abundance of co-eluting interferences in biological samples.23 Parallel reaction monitoring (PRM) is an emerging quantification method executed on a quadrupole Orbitrap mass spectrometer.24, 25 PRM works by performing a similar selection of the target precursor ion on the quadrupole with an isolation window of 1-2 m/z, and then full MS/MS spectrum is acquired at a high mass resolution. Following fragmentation, the Orbitrap mass analyzer provides the mass accuracy. Post-acquisition extraction of fragment ions with tight tolerance greatly improves the selectivity and quantification by eliminating interferences from co-eluting species. Herein, we demonstrated the first study for structural detection and accurate quantification of dG-Me-Cys using Orbitrap mass spectrometry. The high mass resolution and mass accuracy provided by the Orbitrap mass analyzer showed the unbiased and accurate quantification of dG-Me-Cys. Our novel method using the advanced Orbitrap platform was further applied to detect and quantify both endogenous and exogenous formaldehyde-induced DPCs in human cell lines.



MATERIALS AND METHODS Chemicals and Materials. Unless otherwise specified, all reagents and chemicals used in this study were purchased from Sigma Aldrich (St. Louis, MO). Acetonitrile (CH3CN) solvent and formaldehyde solution (37%, w/w) were purchased from Thermo Fisher Scientific (Rockford, IL). [15N5]-2’-deoxyguanosine and [13CD2]-formaldehyde solution (20% w/w in D2O) were obtained from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). DNAzol and Turbo DNase were obtained from Lift Technologies. Proteinase K was obtained from FivePrime (San Francisco, CA). Nanosep Centrifugal Devices (MWCO 3K) were purchased from Pall Lift Sciences. All solvents used were at least HPLC-grade. Synthesis of dG-Me-Cys and [15N5]-dG-[13CD2]-Me-Cys Standards The synthesized standards were prepared by enzymatic digestion of dG-Me-GSH (glutathione) according to previous publications.10,

26

Briefly, GSH was incubated with formaldehyde in

sodium phosphate buffer at 37 °C for 3 hours. Then, 2’-deoxyguanosine (dG) was added for crosslinking reaction at 37 °C for 14 hours to produce dG-Me-GSH. The dG-Me-GSH was digested by carboxypeptidase Y and leucine aminopeptidase M in sodium phosphate buffer with the presence of MgCl2 and CaCl2 at room temperature for 15 hours in order to release dG-MeCys. The same method was applied to synthesize [15N5]-dG-[13CD2]-Me-Cys using [15N5]-dG and [13CD2]-formaldehyde as starting materials. The dG-Me-Cys and [15N5]-dG-[13CD2]-Me-Cys were further purified and quantified as described previously.10, 26 The dG-Me-Cys and [15N5]-dG[13CD2]-Me-Cys were used as the analytical and internal standard to construct calibration curve for accurate quantification of endogenous and exogenous DPCs (dG-Me-Cys).


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Cell Culture and Cell Treatment with [13CD2]-Formaldehyde. HEK293T cells were obtained from the American Type Culture Collection (Camden, NJ). The cells were maintained as exponentially growing monolayer cultures in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% PenicillinStreptomycin in a humidified incubator at 37 °C with 5% CO2. HEK293T cells were subcultured one day before [13CD2]-formaldehyde treatment. The cells were washed with phosphatebuffered saline (PBS) to remove the FBS just prior to treatment. The washed cells were treated with DMEM containing 0, 100, or 250 µM [13CD2]-formaldehyde for 2 hours at 37 °C. After treatment, the cells were washed with PBS to remove the [13CD2]-formaldehyde before further isolation of DNA-protein crosslinks. DNA-protein Crosslink Isolation and Enzymatic Digestion. The DNA-protein crosslinks (DPCs) were isolated from the harvested cells using DNAzol (Thermo Fisher Scientific). The cells were dissolved in 1 mL DNAzol reagent by pipetting before adding proteinase K for overnight digestion at room temperature. Further DPCs isolation and enzymatic digestion was previously described with some minor modifications.10 Briefly, DPCs were precipitated by 100% ethanol at -20 °C for 2 hours, and then centrifuged at 14,000 × g at 4 °C for 5 min. The pellets (DPCs) were washed with 75% ethanol before being reconstituted with 0.5 mL pre-digestion buffer (40 mmol/L ammonium acetate (pH 6.0), CaCl2 (10 mmol/L), and pronase (1.4 U/mL)) for overnight digestion at room temperature. After digestion, the supernatant was collected by centrifuged at 12,000 × g at 4 °C for 5 min. Then, DPCs were precipitated at -20 °C for 2 hours using 10% volume sodium acetate (3 M) and 2.5 fold volume of 100% ethanol. DPCs were pelleted by centrifugation at 12,000 × g at 4 °C for 5



min, and then washed with 75% ethanol. Pellets were reconstituted with 0.45 mL digestion buffer (40 mmol/L ammonium acetate (pH 6.0), MgCl2 (10 mmol/L), CaCl2 (10 mmol/L), DNase I (44.4 U/mL), alkaline phosphatase (3.3 U/mL), phosphodiesterase I (0.0067 U/mL), prolidase (3.3 U/mL), carboxypeptidase Y (0.83 U/mL), and aminopeptidase M (0.083 U/mL)) for overnight digestion at room temperature. The enzymatic reaction was terminated with the addition of 10 µL of 30% acetic acid. After adding 8 fmol of internal standard ([15N5]-dG[13CD2]-Me-Cys), the final reaction mixture was subjected to centrifugation at 12,000 × g at 4 °C for 40 minutes in a Nanosep Centrifugal Device to remove enzymes prior to HPLC purification. HPLC Purification and Fractionation of DPCs The target analytes: endogenous and exogenous dG-Me-Cys, and their internal standard, were purified from the filtrate using an Agilent 1200 Series UV HPLC System with two C18 reversephase columns connected in series (Waters Atlantis T3, 3 µm, 15 cm × 4.6 mm i.d.).10 The detection wavelength and column temperature were set at 254 nm and 15 °C, respectively. The mobile phases consisted of 0.05% acetic acid in water (A) and CH3CN (B). The flow rate was 0.45 mL/min, and elution gradient conditions were set as follows: 0 min, 2% B; 3 min, 2% B; 42 min, 4.2% B; 43 min, 4.2% B; 43.5 min, 80% B; 46 minutes, 80% B; 47.5 minutes, 2% B; 55 minutes, 2% B. The target analytes were eluted at a retention time range between 37 and 41 min. The fractions containing target compounds were combined and concentrated to approximately 20 µL using a vacuum concentrator before LC-MS/MS analysis. The amount of digested dG in each sample was quantitated by the UV peak area (λ = 254 nm) based on each freshly prepared calibration curve to estimate the dG amount in each sample loaded on column. Nano-LC/ESI/MS/MS Analysis.


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LC-MS/MS analysis was conducted using an UltiMate 3000 RSLCnano system coupled to a Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer through an EASY-Spray ion source for nano-electrospray (Thermo Fisher Scientific). DPCs were separated on a PepMap C18 analytical column (2 µm particle, 50 cm x 75 µm i.d., catalog number ES503, Thermo Fisher Scientific). At first, the DPCs were loading into a C18 trapping column (5 µm particle, 0.5 cm x 300 µm i.d., catalog number 160454, Thermo Fisher Scientific) at a flow rate of 5 µL/min for 3 minutes using 0.1% formic acid in ddH2O as a loading solvent. A binary solvent system consisting of 0.1% formic acid in ddH2O (solvent A) and 0.1% formic acid in CH3CN (solvent B) was used for LC separation at a flow rate of 250 nL/min. LC separation was performed using the following gradient setting: held at 1% B for 3 min (trapping time), from 1% to 20% B in 16 min, 20% to 90% B in 0.1 min, held at 90% B for 9.9 min, 90% to 1% B in 0.1 min, and held at 4% B for 13.9 min for re-equilibrating column. MS and MS/MS data were both acquired in profile mode. One full scan coupled with targeted PRM mode with an inclusion list comprised of m/z 401.12 (endogenous dG-Me-Cys), 404.14 (exogenous dG-[13CD2]-Me-Cys), and m/z 409.13 (internal standard [15N5]-dG-[13CD2]-Me-Cys). The Orbitrap resolution for full MS (380 to 420 m/z scan range) and MS/MS spectra was set to 240,000 and 60,000 at m/z, respectively. Automatic gain control (AGC) target for full scan and MS/MS scan was 1x106 and 5x105 with maximum fill time of 100 and 250 ms, respectively. Precursors in targeted PRM mode were isolated with a window of 1.4 m/z and fragmented with HCD fragmentation (higher-energy collisional dissociation, normalized collision energy of 25). Calibration Curve.



Standard curves were established by plotting the peak area ratios of solutions containing a fixed concentration of [15N5]-dG-[13CD2]-Me-Cys (internal standard) at 0.25 nM and increasing concentrations of dG-Me-Cys (analytical standard) from 0.00390625 nM to 4 nM. The two-fold serial dilution was performed from the highest stock solution of 4 nM of analytical standard using internal standard (0.25 nM) in 0.05% acetic acid as a diluent. Linear calibration curve was based on the loading amount ratio versus the integrated peak area ratio between the analytical and internal standards. The method validation was performed for consecutive intraday and interday evaluations. Data Analysis. The LC-MS/MS raw data generated from Q Exactive HF instrument were analyzed by Xcalibur software (Thermo Fisher Scientific). The quantification analysis was performed using Skyline v3.7.0.11317,27 and the chromatographic peak area ratios of endogenous dG-Me-Cys (m/z 401.12377 m/z 164.05668) and exogenous dG-[13CD2]-Me-Cys (m/z 404.13948 m/z 167.07238) over that of internal standard [15N5]-dG-[13CD2]-Me-Cys (m/z 409.12465 m/z 172.05756) were directly reported from Skyline after manually checked peak integration.


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RESULTS Detection of dG-Me-Cys by Q Exactive HF. In our previous study, Lai et al. demonstrated the detection and quantification of dG-Me-Cys using low mass resolution triple quadrupole mass spectrometry with SRM mode used to detect and quantify dG-Me-Cys.10 In the current study, for the first time, we have evaluated the detectability for dG-Me-Cys using a high-resolution, accurate-mass Orbitrap mass spectrometry. At first, the synthetic standards, dG-Me-Cys and stable isotope labeled [15N5]-dG-[13CD2]-MeCys, were individually analyzed by LC-MS/MS Orbitrap mass spectrometry. Figure 1 shows the extracted ion chromatograms (XIC) of dG-Me-Cys (Figure 1A) and [15N5]-dG-[13CD2]-Me-Cys (Figure 1B). Because of high mass accuracy obtained from Orbitrap, the XIC was executed using the exact mass of target product ion of m/z 164.05723 and m/z 172.05811 for dG-Me-Cys and [15N5]-dG-[13CD2]-Me-Cys, respectively, within 5 ppm mass accuracy. In addition, the precursor ions of target analytes were revealed by the high mass resolution and accuracy in full scan spectra (Figure 1, left inserts).



Figure 1. Extracted ion chromatogram (XIC) of synthetic standards for (A) dG-Me-Cys (m/z 401.12377 m/z 164.05668) and (B) [15N5]-dG-[13CD2]-Me-Cys (m/z 409.12465 m/z 172.05756). Inserts show the isotope envelope patterns and corresponding MS/MS spectra. R indicates the mass resolution.

The product ion spectrum (MS/MS) after HCD fragmentation was further used to confirm the identities and structures of target analytes (Figure 1, right inserts). The fragmentation patterns under HCD fragmentation were proposed in Scheme 1. The peaks of m/z 164.05668 and m/z 172.05756 for dG-Me-Cys and [15N5]-dG-[13CD2]-Me-Cys, respectively, were the most intense


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product ions (“product ion 5”, Table 1) after fragmentation on two chemical bonds as indicated by red dashed line. After fragmentation, all five of the proposed product ions were detected, and the measured mass accuracies for precursor and product ions was less than 3 ppm compared to their exact mass (Table 1). These data demonstrate the confident identification and detectability of synthetic dG-Me-Cys by high-resolution, accurate-mass Orbitrap mass spectrometry.

Table 1. Mass accuracy of dG-Me-Cys and [15N5]-dG-[13CD2]-Me-Cys measured in Q Exactive HF Mass Spectrometry [15N5]-dG-[13CD2]-Me-Cys

dG-Me-Cys ion typea

measured m/z

theoretical m/z

mass accuracy (ppm)

measured m/z

theoretical m/z

mass accuracy (ppm)

Precursor ion

401.12344

401.12377

-0.83

409.12460

409.12465

-0.13

Product ion 1

117.05489

117.05462

2.28

117.05486

117.05462

2.03

Product ion 2

122.02722

122.02702

1.60

122.02721

122.02702

1.52

Product ion 3

280.10388

280.10403

-0.54

288.10489

288.10491

-0.07

Product ion 4

285.07626

285.07643

-0.60

293.07727

293.07731

-0.13

Product ion 5

164.05653

164.05668

-0.94

172.05757

172.05756

0.06

a

The chemical structures were shown in Scheme 1.

Linearity of Calibration Curve for Quantification For quantification of dG-Me-Cys by Q Exactive HF, we implemented targeted PRM mode.28 Briefly, this mode sequentially isolates target precursor ions for HCD fragmentation and collects the full product ion spectrum. Quantification was based on post-acquisition processing to reconstruct the chromatographic peaks using a narrow tolerance ( m/z 164.05668), exogenous dG-[13CD2]-Me-Cys (m/z 404.14 > m/z 167.07238) and internal standard [15N5]-dG-[13CD2]-Me-Cys (m/z 409.13 > m/z 172.05756). Figure 2 shows the wide dynamic range of the calibration curve for quantification of dG-Me-Cys. Plotting amount ratio versus the integrated peak area ratio over internal standard indicates the high quantification capability (at least 3 orders) using PRM mode on Orbitrap MS. In this tested calibration curve, the lowest calibrator was 15.625 amol of dG-Me-Cys loaded onto the LC column. Intra- and inter-day precisions and accuracies for dG-Me-Cys quantification were also evaluated and variations were less than 10% when measuring known dG-Me-Cys over IS amount ratio, providing accurate and precise quantification results (Table 2).

Figure 2. Calibration curve by plotting amount ratio versus area ratio. X-axis is the added dGMe-Cys (analytical standard, AS) concentrations in fixed concentration of [15N5]-dG-[13CD2]-


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Me-Cys (internal standard, IS) at 0.25 nM and final amount ratios are from 0.015625 to 16. Yaxis is the integrated peak area ratios between AS and IS. Insert shows a zoomed in region of amount ratio from 0.015625 to 0.5.

Table 2. Intra- and inter-day accuracy and precision evaluation for dG-Me-Cys (analytical standard, AS) by spiked in fixed amount of [15N5]-dG-[13CD2]-Me-Cys (internal standard, IS) determined by LC-MS/MS (n=3) Intra/inter day

Linear regression equation

R2

Added AS/IS amount ratio

Measured AS/IS amount ratio

RSD (%)

Accuracy (%)

Day 1

y = 1.0538x-0.0027

0.9994

0.03125

0.02863

6.54

91.6

0.5

0.49927

1.09

99.9

0.03152

8.87

100.9

Day 2

y = 1.0735x-0.005

0.9999

0.03125 0.5

0.48266

0.84

96.5

Day 3

y = 1.0599x-0.0113

0.9984

0.03125

0.03403

4.17

108.9

0.5

0.49524

1.33

99.0

0.03125

0.03139

8.61

100.4

0.5

0.49239

1.76

98.5

Inter-day

Measurement of Endogenous and Exogenous dG-Me-Cys in HEK293T Cells Treated with Isotope-labeled Formaldehyde. Next, we applied this quantification strategy to measure endogenous dG-Me-Cys (m/z 401.12 > m/z 164.05668) and exogenous dG-[13CD2]-Me-Cys (m/z 404.14 > m/z 167.07238) in HEK293T cells treated with [13CD2]-formaldehyde at 0, 100, and 250 µM (Table 3). While no exogenous DPCs were detected in the control group, exogenous dG-[13CD2]-Me-Cys was 0.94 ± 0.04 and 12.41 ± 0.69 crosslinks/108 dG after treated with 100 and 250 µM, respectively. In this in vivo experiment, the cells were treated with isotope-labeled formaldehyde for only 2 hours, resulting in a rapid exogenous DPCs generation; however, the amount of endogenous DPCs was not affected under this treatment. This observation indicated that the exposure-induced stress did not alter the amount of endogenous formaldehyde-induced DPCs. The chromatographic peaks for



those endogenous and exogenous DPCs, as well as internal standards integrated by Skyline software are shown in Figure 3. Clearly, the exogenous DPCs increased with increased formaldehyde concentration (Figure 3, right panel). In addition, the mass accuracy of those product ions was less than 1 ppm.

Table 3. Formaldehyde-induced exogenous dG-Me-Cys in HEK293T cell lines treated by 0, 100, and 250 µM [13CD2]-formaldehyde Formaldehyde treated concentration (µM) 0 100 250 a ND, not detected.

dG-Me-Cys (crosslink/108 dG) Endogenous

Exogenous

0.66±0.06 (n=4) 0.51±0.11 (n=4) 0.67±0.08 (n=4)

NDa 0.93±0.04 12.41±0.69


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Figure 3. Chromatographic peak integrations for quantification of endogenous (m/z 401.12377 > m/z 164.05668, left panel) and exogenous (m/z 404.13948 > m/z 167.07238, right panel) dG-MeCys in HEK293T cells treated with 0 (A), 100 (B), and 250 µM [13CD2]-formaldehyde (C) for 2 hours. The endogenous and exogenous dG-Me-Cys measured were marked with red and orange lines, respectively. The internal standard, [15N5]-dG-[13CD2]-Me-Cys (m/z 409.12465 m/z 172.05756), was marked with blue line. Mass accuracy of the integrated peaks reported by Skyline was also provided in the unit of ppm. The ratio of peak area was calculated by the integrated peak area of dG-Me-Cys over that of the internal standard.

Chromatographic Peak Integration using High-resolution, Accurate-mass Orbitrap Mass Spectrometry Orbitrap mass analyzer provides the benefits of high mass accuracy (10.


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Figure 4. XIC of exogenous dG-Me-Cys in HEK293T cells treated with 250 µM [13CD2]formaldehyde for 2 hours in mass tolerance of 5 ppm (A) and 2095 ppm (B) using Orbitrap MS. (C) LC-SRM spectrum for exogenous dG-Me-Cys in rat nasal tissue exposed to 15 ppm [13CD2]formaldehyde for 6 hours per day for 4 days using triple quadruple MS, and corresponding animal exposure experiments, sample preparation and LC-MS/MS methods were described previously by Lai et al.10 It is of note that some m/z regions were zoomed in for demonstrating the background signals.



DISCUSSION It is well known that endogenous formaldehyde is an important metabolic intermediate from multiple pathways such as one-carbon pool metabolism, amino acid metabolism, lipid peroxidation and various demethylation processes.29,

30

Because of the high reactivity of

formaldehyde and its common presence in all tissues and body fluids,9, 10, 26 formaldehyde is known as an important source of endogenous DNA damages and the repair of formaldehydeinduced DPCs is important.9,

15

Our group previously implemented stable isotope labeled

formaldehyde and utilized its corresponding synthetic internal standard for dG-Me-Cys to accurately and structurally measure, for the first time, both endogenous and exogenous DPCs in exposed animal tissues.10 In this study, we further advanced this methodology for DPC quantification using state-of-the-art Orbitrap MS with high mass resolution and accuracy. The quantification method presented in this study is based on the targeted acquisition mode, PRM, executed on an Orbitrap instrument. PRM has been widely used for large-scale targeted peptide quantification.25, 31 Unlike the traditional SRM method performed on triple quadrupole mass spectrometry, PRM mode collects the full product ion spectrum using high mass resolution and accuracy. The PRM quantification is achieved by postacquisition extraction of the target product ions with tight mass tolerance for reconstructing the chromatographic peak. Moreover, owing to the product ion spectrum obtained using high mass resolution and accuracy, the selectivity of quantification was significantly improved using PRM over SRM.28 As expected, PRM presents the background signal-free quantification by extracting the exact mass of target product ion(s) with tight mass tolerance, drastically reducing the background noise signal (Figure 4A). Conversely, the low mass resolution of SRM acquisition results in background signals from


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co-eluting compounds that may interference with target ion(s) even after significantly reducing sample complexity by HPLC purification (Figure 4C). The old version of QqQ MS was previously used to detect dG-Me-Cys by Lai et al.,10 and the limit of quantification was 37.5 amol on the column based on S/N ratio >10. The lowest calibrator tested in the current study was 15.625 amol loaded onto the column, with good signal response and linearity covering three orders of magnitude (Figure 2). In addition, the precision and accuracy of this new methodology showed a significant improvement for dG-Me-Cys quantification (Table 2). Moreover, the scan rate of instrument is an important factor for targeted quantification. The scan rate on the Orbitrap MS is based on the settings between maximum injection time and Orbitrap resolution, because the instrument enables the accumulation and measurement of ion population in parallel.32 Collecting the basic criterion of at least 8 to 10 data points over a chromatographic peak ensures precise quantification.31 Only a limited number of precursors were monitored in this PRM method, therefore maximum injection time for each precursor can be increased to 250 ms in order to pursue high detection sensitivity and still have enough data points for quantification (Figure 3). To our knowledge, the amount of formaldehyde-DPCs (dG-Me-Cys) from any cell lines has not been reported to date. In this study, the dG-Me-Cys level in a human epithelial cell line was analyzed by applying our new Orbitrap-based quantification method (Table 3). The amount of exogenous DPCs in the cells significantly increased from 0.93±0.04 to 12.41±0.69 crosslink/108 dG when treated with a higher concentration of labeled formaldehyde (100 µM and 250 µM, respectively). In contrast to the significant changes observed in exogenous DPCs levels in HEK293T cells after treatment with labeled formaldehyde, no significant difference was detected in the amount of endogenous DPCs. Clearly, the exogenous formaldehyde exposure stress did



not affect the endogenous DPCs formation based on exposure data from cell line and animal models. This observation is in agreement with our previous study using animal models exposed to stable isotope-labeled formaldehyde, showing the steady-state concentrations of endogenous DPCs.10 Furthermore, the endogenous DPCs level in HEK293T cells (0.66±0.06 crosslink/108 dG) was at least 2 and up to 23 folds lower than those in tissues analyzed in our previous study including peripheral blood mononuclear cells (1.34±0.25), bone marrow (2.30±0.30), nasal epithelium (3.59±1.01), and liver (15.46±1.98 crosslink/108 dG).10 The endogenous formaldehyde concentration in the blood is ~100 µM and much higher concentrations were observed in the rat liver and nasal mucosa.33 The significantly lower formaldehyde concentrations in cell lines have been reported by Kato et al.34 The concentration differences of endogenous formaldehyde may account for the lower endogenous DPCs level in the cell line compared to tissues, especially in liver. To detect this low level of DPCs in cell lines requires a more sensitive detection platform demonstrated in this study. Moreover, the methodology demonstrated herein can be applied to detect other linkages of formaldehyde-induced DPCs20 and different forms of DNA damages such as interstrand DNA-DNA crosslinks.7, 8 In summary, we demonstrated a sensitive method for DPCs quantification by an Orbitrapbased MS platform, which provides benefits of high mass resolution and accuracy for unambiguous analysis. The high mass resolution and accuracy of the Orbitrap mass analyzer generated an interference-free XIC spectrum for accurate quantification. This quantification method was further applied to detect endogenous and exogenous DPCs levels in HEK293T cells treated with labeled formaldehyde. In addition, the capability of this method to detect low levels of endogenous DPCs will significantly facilitate the understanding of molecular mechanisms for DPCs repair.


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Funding This work was partially supported by the NIH/NIEHS grant (R03ES024147) and a grant from the American Chemistry Council. Notes The authors declare no competing financial interest.

ABBREVIATIONS Cys, cysteine; dG, 2’-deoxyguanosine; DPCs, DNA-Protein Crosslinks; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HCD, higher-energy collisional dissociation; MS/MS, tandem mass spectrometry; PBS, phosphate-buffered saline; PRM, Parallel reaction monitoring; QqQ, triple quadrupole; SRM, selected reaction monitoring; XIC, extracted ion chromatogram



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Accurate Measurement of FormaldehydeâInduced DNAâProtein

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