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. 2023 Nov 20;36(12):1921–1929. doi: 10.1021/acs.chemrestox.3c00207

Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant O6-Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an O6-Alkylguanine Adductome

Rasha Abdelhady †, Pattama Senthong †, Claire E Eyers ‡, Onrapak Reamtong ‡, Elizabeth Cowley †, Luca Cannizzaro ‡, Joanna Stimpson ‡, Kathleen Cain ‡, Oliver J Wilkinson §, Nicholas H Williams §, Perdita E Barran ‡, Geoffrey P Margison †, David M Williams §, Andrew C Povey †,*
PMCID: PMC10731659  PMID: 37983188

Abstract

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Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, O6-methylguanine O6-methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA O6-alkylguanines (O6-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple O6-alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different O6-alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known O6-MethylG (O6-MeG) levels. It was digested with trypsin, and ASPs were detected and quantified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. ASPs containing S-methyl, S-ethyl, S-propyl, S-hydroxyethyl, S-carboxymethyl, S-benzyl, and S-pyridyloxobutyl cysteine groups were detected by incubating MGMT with ODNs containing the corresponding O6-alkGs. The LOQ of ASPs containing S-methylcysteine detected after MGMT incubation with Me-CT-DNA was <0.05 pmol O6-MeG per mg CT-DNA. Incubation of MGMT with human colorectal DNA produced ASPs containing S-methylcysteine at levels that correlated with those of O6-MeG determined previously by HPLC-radioimmunoassay (r2 = 0.74; p = 0.014). O6-CMG, a putative O6-hydroxyethylG adduct, and other potential unidentified MGMT substrates were also detected in human DNA samples. This novel approach to the identification and quantitation of O6-alkGs in human DNA has revealed the existence of a human DNA alkyl adductome that remains to be fully characterized. The methodology establishes a platform for characterizing the human DNA O6-alkG adductome and, given the mutagenic potential of O6-alkGs, can provide mechanistic information about cancer pathogenesis.

Introduction

Alkylating agents (AAs) are known human mutagens and carcinogens whose effects are largely mediated by the formation of alkyl adducts in DNA.1−3 The mutational landscape observed in patients with malignant melanomas and glioblastoma multiformes following treatment with the chemotherapeutic methylating agent, Temozolomide, consists primarily of G-A transitions attributed to the generation of O6-methylguanine (O6-MeG) in DNA.4 A similar mutational signature has recently been described in colorectal cancer implicating AA exposure as a causal factor in this disease.5O6-MeG and other O6-alkylguanine (O6-alkG) adducts are repaired by the DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT), which provides protection against the toxic, mutagenic, and carcinogenic effects of AA exposure.6,7 Increased methylation of CpG islands in the MGMT promoter region has been described in human tumors8,9 and is associated with G-A transitions in colorectal,10 lung,11 and brain tumors12 and the Temozolomide-induced mutational signature.13 Furthermore, MGMT promoter methylation improves overall survival in glioblastoma patients treated with Temozolomide.14 As promoter methylation downregulates MGMT expression,12,15 these results are entirely consistent with persistence of O6-alkGs in DNA as a cause of toxicity and mutational events and provide compelling evidence that AAs are involved in the etiology of some cancers.

In addition to this indirect evidence for the presence of O6-alkGs in human DNA, there is increasing direct evidence for their presence in human DNA as described in several reviews16−18 of alkyl DNA adductomes. Thus, O6-MeG,19O6-ethyl (O6-EtG),20O6-propyl (O6-PrG),21O6-butyl (O6-BuG),21 and O6-carboxymethyl (O6-CMG)22,23 as well as 7-alkylguanines16 and methyl DNA phosphate adducts24 have all been detected in human DNA. The presence of a wider spectrum of alkyl adducts is not surprising given the wide range of AAs present in the human environment25 and that AAs can also be generated endogenously26 from the many varied and abundant dietary and luminal amines and other substrates.27,28 These processes likely result in AA exposure that significantly increases mutational and cancer risk by the formation of a range of associated O6-alkGs.29 Previous studies have largely focused on quantifying the presence of O6-MeG in human DNA using radioimmunoassays (RIA),1932P-postlabeling,20,30 and mass spectrometry (MS), e.g., high-resolution gas chromatography-MS with selected ion recording21 and ultrahigh-performance liquid chromatography-high resolution MS/MS,23 approaches with differing sensitivities and specificities. Some caution may be needed in some cases, for example, antibodies may recognize a range of O6-alkGs and hence the levels of O6-MeG may be overestimated if the O6-MeG is not completely separated from other possible O6-alkGs before the immunoassay quantitation. MS, in particular, has been routinely used in clinical settings and is increasingly used to detect with high sensitivity and specificity a wide range of different DNA adducts using relatively simple procedures that do not need radioactive materials or antibodies.31−33

In the present paper, we describe a novel method to assess the O6-alkG adductome by the MS analysis of alkylated MGMT active site peptides (ASPs) following in vitro incubation of MGMT with extracted DNA, which results in the irreversible transfer of the alkyl group from the O6 position of the modified guanine bases to the active site cysteine residue in MGMT.

Materials and Methods

Samples

A cross-sectional study of patients presenting with colorectal carcinoma at hospitals within Greater Manchester, U.K., was undertaken. Patients were included if they were undergoing surgery for treatment, and human colorectal tumor (n = 10; obtained from colorectal carcinoma tissue) and macroscopically normal (n = 3; taken ∼5 cm from the tumor edge) tissues were obtained from individuals (six men, two women, and two participants of unknown sex). The age of the eight individuals was 69 ± 14 (mean ± SD). DNA was extracted by a phenol/chloroform procedure and analyzed for O6-MeG by an HPLC-radioimmunoassay (RIA) using a [3H]-O6-methyldeoxyguanosine tracer and mouse monoclonal α-O6-MedG following Aminex chromatography,19 and the remaining DNA was stored at −80 °C until it was analyzed in the present study. Ethical approval was obtained from East Midlands-Derby Research Ethics Committee, Health Research Authority, NHS (REC reference: 15/EM/0505).

Materials

Synthetic methylated (GNPVPILIPMe-CHR) MGMT-ASP and light and heavy isotope (13C15N proline)-labeled methylated MGMT-ASP corresponding to positions 136–147 of MGMT were purchased from Cambridge Research Biochemicals, Cleveland, U.K. Other chemicals used in this work were purchased from Sigma-Aldrich (Poole, Dorset, U.K.) unless otherwise stated. Calf thymus (CT) DNAs containing various levels of O6-MeG (0.050, 0.125, 0.250, and 0.50 pmol/mg-CT-DNA) were prepared by incubating Temozolomide with CT-DNA, and levels were determined by a competitive radioisotope-based assay involving preincubation of incrementally increasing amounts of the Temozolomide-methylated CT DNA with a fixed amount of MGMT and then post incubation with excess N-[3H]methyl-N-nitrosourea (Hartmann Radiochemicals: specific radioactivity 80 Ci/mmole) methylated CT DNA. The decrease in the amount of radioactivity transferred to the MGMT was used to determine the amount of O6-MeG in the Temozolomide-methylated DNA, as described previously.34

Expression and Purification of MGMT

Human MGMT was expressed as a maltose-binding protein (MBP) fusion protein from pMAL-2c expression vector constructs and affinity-purified using amylose resin (New England Biolabs Inc., USA) essentially as described previously.35 For some studies, the MBP-MGMT fusion protein was cleaved with factor Xa, and MGMT was purified using DEAE-sepharose ion exchange chromatography. Human MGMT was also expressed as a hexahistidine (His) fusion protein from pQE30Xa (Qiagen) and purified by nickel affinity chromatography using a complete His-Tag purification resin (Sigma-Aldrich). MGMT activity was subsequently assayed by measuring the transfer of [3H] from N-[3H]-methyl-N-nitrosourea (Hartmann, Germany; specific activity 80 Ci/mmol) methylated CT-DNA to the MGMT fusion protein.36 In addition, for the Vion IMS QToF analysis, MGMT was synthesized by GeneArt (ThermoFisher), cloned into pNic28-Bsa4 linearized with BsaI-HF (NEB) using In-Fusion ligation independent cloning. Competent BL21(DE3) cells (NEB) were transformed with the vector, His-MGMT expressed, and purified by nickel affinity chromatography.

Synthesis of Oligodeoxyribonucleotides Containing a Single O6-alkG Adduct

O6-alkG-containing 12- or 23 mer-oligodeoxyribonucleotides (ODNs) that contained a single O6-alkG adduct in the following sequences, 5′ -SIMA-GCC ATG XCT AGTA or 5′-GAA CTY CAG CTC CGT GCT GGC CC-3′, were synthesized as described.37−40 X was unmodified G, O6-MeG, O6-EtG, O6-PrG, O6-hydroxyethyl (O6-HOEtG), O6-benzylG (O6-BnG), O6-pyridyl-oxobutylG (O6-pobG), 2,6-diaminopurine, O6-aminoethylG, N6-hydroxypropyl-2,6-diaminopurine, or O6-methyladamantylG, and Y was unmodified G or O6-MeG or O6-CMG. Modified 12-mer ODNs were characterized by ESI-MS as previously described for the 12-mers39 and 23-mers in Figure s1.

Control ODNs (with unmodified G bases) as well as complementary ODNs were synthesized by Sigma-Aldrich, U.K. Single-stranded ODNs were annealed to equimolar amounts of the ODN complement by heating to 95 °C in 50 mM NaCl for 20 min and then cooling slowly to room temperature for >1 h. ODNs were stored at −20 °C.40

Preparation of MGMT Tryptic Peptides following Incubation of MGMT with ODNs and Methylated CT-DNA

In a typical assay, double-stranded 23-mer ODNs (20 pmol) containing G, O6-MeG, or O6-CMG or Temozolomide-methylated CT DNA (Me-CT-DNA) were incubated with MBP-MGMT (2 pmol by activity) for 6 h at 37 °C in IBSA buffer (1 mg/mL BSA in 50 mM Tris-HCl pH 8.3 containing 1 mM EDTA and 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)). Trypsin (ratio of MBP-MGMT:trypsin, 20:1) was added, the sample was incubated overnight (18 h) at 37 °C with shaking, and then 1% formic acid was added to give a final concentration of 0.1%. MGMT tryptic peptides were desalted, concentrated using Millipore C18-Ziptips (Merck Millipore, Ireland) according to manufacturer’s instructions, and eluted in 5 μL of 0.1% formic acid in 50% acetonitrile/water.

Preparation of MGMT Tryptic Peptides following Incubation of MGMT with Human DNA

His-MGMT (30 pmol by activity) was incubated with 2 mg of human colorectal DNA for 6 h at 37 °C in 50 mM Tris-HCl pH 8.3 containing 1 mM EDTA and 2 mM TCEP on a shaker incubator. Prewashed Ni-coated magnetic beads (PureProteome Magnetic Beads, Merck Millipore, U.K.) were resuspended in equilibration buffer, vortex-mixed, added to the His-MGMT/DNA solution, and incubated at 4 °C overnight on a rotor mixer (Blood Tube Rotator, SB1, Stuart Scientific, U.K.). The sample was then centrifuged (Fisher Scientific accuSpin Microcentrifuges), and the supernatant was aspirated. Beads were resuspended in 40 μL of buffer containing 50 mM sodium phosphate, 300 mM sodium chloride, and 300 mM imidazole, pH 8. Trypsin was added (as above), and the sample was incubated overnight (18 h) at 37 °C with shaking. Formic acid (1%) was added to give a final concentration of 0.1%, and the tryptic peptides were desalted and concentrated using Millipore C18-Ziptips and then spiked with 250 fmol of 13C15N proline-labeled methylated ASP internal standard.

MALDI-ToF MS Analysis of MGMT Active Site Peptides and Data Acquisition

Tryptic peptides arising from MGMT, MBP-MGMT, and His-MGMT were spotted on a MALDI plate together with a saturated α-cyano-4-hydroxycinnamic acid (Fluka, Buchs, Switzerland) matrix solution (10 mg/mL in 50% ethanol/acetonitrile). The MALDI-ToF was calibrated by using a J67722 MALDI certified mass spec calibration standard (Alfa Aesar, U.K.). Spectra were acquired over the mass to charge ratio (m/z) range 800–2300 using a Bruker (Germany) Ultraflex II operating at 30% laser intensity and 1000 laser shots per spectrum in reflectron positive ion mode. A signal/noise >10 was required for identification of detected alkylated peptide ions. Peak areas (PAs) of chosen peptides (methylated MGMT-ASP and internal standard) were measured using FlexAnalysis software (Bruker, Germany). Label-free quantitation of O6-CMG adducts was carried out by comparing the PA of carboxymethylated ASP to that of methylated ASP generated from known amounts of the appropriate O6-alkG-containing ODNs and applying this ratio to the PAs of carboxymethylated ASPs found after incubating MGMT with colorectal DNA.

Vion IMS QTof Analysis and Data Acquisition of His-MGMT Active Site Peptides

Control (G) and O6-MeG-containing single-stranded 23-mer ODNs (O6-MeG) (37.5 nmol) were incubated with 50 pmol of His-MGMT in 50 mM Tris-HCl pH 8.3 containing 1 mM EDTA and 5 mM DTT for 90 min at 37 °C. In-solution digestion was performed on the samples with 50 mM DTT, 14 mM iodoacetamide, and trypsin (ratio of His-MGMT:trypsin ≤ 12:1) was added and incubated overnight (18 h) at 37 °C. After in-solution trypsin digestion and concentration, one sample of the single-stranded control ODN (G) was spiked with 112 nmol of synthetic methylated ASP. The samples were dried using an SP Genevac miVac Sample Concentrator and then dissolved in water containing 0.05% acetonitrile and 0.1% formic acid.

Tryptic peptides were resolved by ultraperformance liquid chromatography using an ACQUITY UPLC I-Class System and a 100 × 2.1 mm Hypersil GOLD C18 3 μm column (ThermoFisher) in tandem with a quadruple time-of-flight mass spectrometer (Vion IMS QToF, Waters). Water containing 0.1% formic acid was used as mobile phase A, and acetonitrile containing 0.1% formic acid was used as mobile phase B. The flow rate was 0.2 mL/min, and the total elution time was 60 min. The elution gradient program was as follows: 0 to 3 min, 99% A; 3 to 53 min, 99 to 80% A; 53 to 55 min, 80 to 20% A; 55 to 57 min, 20% A; 57 to 58 min, 20 to 99% A; 58 to 60 min, 99% A. Electrospray ionization was carried out in positive ion mode with an ion source temperature of 120 °C. The mass scan range was from 105 to 2000 m/z, with a scan time of 0.250 s. LockSpray solution containing the peptide leucine/enkephalin was analyzed every 2 min to adjust mass calibration of the instrument during analysis. Data were collected in MSE mode41 where the instrument alternated between low (6 eV for precursor ion collection) and high (15–40 eV ramp for fragment ion collection) collision energies throughout the entire chromatographic run. Data were analyzed using UNIFI software version 1.9.4.053 (Waters Corporation); the selected amino acid modifiers were methyl (cysteine), carbamidomethyl (cysteine), oxidation (methionine), and deamidation (asparagine).

Results

MS Analysis of MGMT Active Site Tryptic Peptides following Incubation of MGMT with O6-alkG-Containing ODNs

Qualitative MS analysis of tryptic fragments of MBP-MGMT following incubation with O6-MeG- and O6-CMG-containing DS ODNs confirmed the transfer of the methyl and carboxymethyl groups from O6-MeG and O6-CMG, respectively, to MGMT: modified MGMT-ASPs were detected by the ions formed from methylated MGMT-ASP (m/z 1329.7 [M + H]+) and carboxymethylated MGMT-ASP (m/z 1373.7 [M + H]+) (Figure 1A,B, respectively). In addition, MS analysis of tryptic digests of MBP-MGMT incubated with DS control G ODNs generated multiple MGMT and MBP peptides and, as expected, only the nonalkylated MGMT-ASP (m/z 1315.73 [M + H]+), as shown in Figure 1C. Ethyl, propyl, benzyl, pyridyloxobutyl, and hydroxyethyl groups were also transferred to the active site cysteine of MGMT from ODNs containing O6-EtG, O6-PrG, O6-BnG, O6-pobG, and O6-HOEtG, respectively (Figure s2 panel A). In contrast, incubation of MGMT with ODNs containing damage not known to be repaired by MGMT such as N6-hydroxypropyl-2,6-diaminopurine, O6-aminoethylG, O6-methyladamantylG, or 2,6-diaminopurine confirmed as expected the lack of transfer of the corresponding alkyl group to MGMT (Figure s2 panel B).

Figure 1.

Figure 1

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MALDI-ToF mass spectra of tryptic peptides of MBP-MGMT following its incubation with double-stranded 23-mer ODNs containing (A) O6-MeG, (B)O6-CMG, or (C) guanine. 2 pmol of active MBP-MGMT was incubated for 6 h at 37 °C with 20 pmol of 5′-GAA CTY CAG CTC CGT GCT GGC CC where Y is (A) O6-MeG, (B) O6-CMG, or (C) G and then digested with trypsin. Peak intensities are shown in arbitrary units on the y-axis, and the intensity scale is the same for all panels. The tryptic peptides detected included unmodified MGMT-ASP (GNPVPILIPCHR, 136–147) m/z 1315.73, methylated MGMT-ASP (GNPVPILIP(Me-C)HR, 136–147) m/z 329.74, and carboxymethylated MGMT-ASP (GNPVPILIP(CM-C)HR, 136–147) m/z 1373.73. The peak at m/z 1336.544 is an MBP peptide (SYEEELAKDPR, 332–342).

The presence of methylcysteine in the MGMT ASP was further confirmed after incubation of O6-MeG-containing SS ODNs with His-MGMT, with high confidence detection and identification of the methylated ASP by the fragments obtained with MSE. The ASP was identified as a high confidence peptide with a mass error <2.8 ppm and via tandem mass spectrometry (Table s1, Figures s3–s6).

We then investigated the detection limit of the MALDI-ToF MS assay by using serial dilutions of a synthesized methylated ASP. Linear correlations (amount vs peak area) with R2 values of 0.9985 and 0.9998 were obtained for unlabeled methylated and heavy isotope (13C15N proline)-labeled methylated MGMT-ASP standards, respectively (Figure s7). The lower limit of quantification of unlabeled (m/z = 1329.74) and 13C15N proline-labeled (m/z = 1335.74) methylated MGMT-ASP was found to be <20 fmol with a signal/noise ratio of >16.

MALDI-ToF MS Analysis of MGMT Active Site Peptides following Incubation of MGMT with Methylated CT-DNA

Following His-MGMT incubation with methylated CT-DNA, MALDI-ToF MS analysis of tryptic peptides demonstrated the presence of methylated MGMT-ASP (GNPVPILIPMe-CHR, amino acid residues 136–147) in MGMT tryptic peptides. Figure s7A shows the region of the mass spectra of his-MGMT incubated with methylated CT-DNA (containing 0.125, 0.25, 0.5 pmol of O6-MeG/mg methylated CT-DNA) showing both unmodified and methylated MGMT-ASP ions at m/z 1315.72 and 1329.74, respectively. The observed PAs of methylated MGMT-ASP showed a linear correlation with levels of O6-MeG adducts (Figure s7B), and the LLOQ for MGMT-based detection of O6-MeG in methylated CT-DNA was <0.05 pmol O6-MeG per mg CT-DNA. The optimized approach was verified by considering the recovery of methylated MGMT-ASP following His-MGMT incubation with methylated CT-DNA using the 13C15N internal standard. Based on the ratio of PA-methylated ASP to PA Me-ASP STD recovery values of methylated MGMT-ASP fragments were 39.8 ± 0.9%, 41 ± 3.6%, and 48 ± 7.5% (mean ± SD; n = 3) following His-MGMT incubation with methylated CT-DNA that contained 100, 200, and 400 fmol of O6-MeG, respectively.

Identification of O6-alkylG Adducts in Human Colorectal DNA

Both methylated and carboxymethylated MGMT-ASPs were detected following the incubation of his-MGMT with colorectal DNA. Figure 2 shows the mass spectra for His-MGMT incubated with two different human colorectal tumor DNA samples (96T and 25T) or with unmodified CT-DNA. Both methylated and carboxymethylated MGMT ASPs (m/z 1329.74 [M + H]+ and m/z 1373.73 [M + H]+, respectively) were detected in these two samples, indicating the presence of the respective adducts in both DNA samples.

Figure 2.

Figure 2

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MALDI-ToF mass spectra analysis of alkylated ASPs present after incubating his-MGMT with human DNA. His-MGMT (50 pmol) was incubated with 2 mg of human colorectal DNA samples (A) 96T, (B) 25T, or (C) unmodified CT-DNA. Peak intensities are shown in arbitrary units on the y-axis, and the intensity scale is the same for all panels. S/N > 10 for all detected alkylated MGMT ASPs. The tryptic peptides (residues 136–147) identified were (i) unmodified MGMT-ASP (GNPVPILIPCHR; m/z 1315.73), (ii) methylated MGMT-ASP (GNPVPILIPMe-CHR; m/z 1329.74), and (iii) carboxymethylated MGMT-ASP (GNPVPILIPCM-CHR; m/z 1373.73).

Quantification of O6-alkylG Adducts in Human Colorectal DNA

Following identification of O6-alkG adducts present in human colorectal DNA samples, MGMT tryptic digests were spiked with 13C15N proline-labeled methylated MGMT-ASP and O6-MeG was quantified. O6-MeG was present in all human colorectal DNA samples analyzed at concentrations that ranged from 6.7 to 11.1 and from 5.1 to 78.2 nmol O6-MeG/mol dG for normal and tumor DNA, respectively (Table 1). For the two patients for which we had paired normal and tumor DNA, the levels of O6-MeG in the tumor DNA were higher. Table 1 also shows the concentrations of O6-MeG adducts in the same human colorectal DNA samples quantified previously using an HPLC-RIA:12 there was a significant correlation between the results of the two assays, r = 0.86 (p = 0.014).

Table 1. O6-MeG and O6-CMG Levels in CR DNA.

ID CR tissue type nanomol ofO6-alkyl adduct per mole of dG (mean ± SD (n = 3))
 O6-CMG/O6-MeG
O6-MeG O6-MeG O6-CMG
ASP/MALDI-ToF HPLC/RIAa,b ASP/MALDI-ToFb ASP/MALDI-ToF
19 normal 6.7 ± 0.3 NA ND  
19 tumor 20.7 ± 1.8 NA 68.2 ± 5.7 3.3
25 tumor 19.2 ± 0.9 25 26.4 ± 2.8 1.4
31 tumor 20.2 ± 1.3 20 41.0 ± 2.2 2.0
39 tumor 5.1 ± 0.4 NA 5.2 ± 0.2 1.0
44 normal 11.1 ± 0.8 5 ND  
44 tumor 21.8 ± 1.4 5 ND  
50 tumor 47.5 ± 6.9 48 48.8 ± 3.1 1.0
74 tumor 21.4 ± 0.8 26 21.5 ± 2.3 1.0
79 tumor 19.6 ± 0.9 19 26.8 ± 2.2 1.4
96 tumor 78.2 ± 7.9 NA 34.5 ± 2.7 0.4
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a

Levels previously detected by HPLC-RIA.16

b

NA = not available; ND = not detected.

The levels of O6-CMG in human colorectal tumor DNA ranged from 5.2 to 68.2 nmol of O6-CMG/mol of dG (Table 1). There was no association between O6-MeG and O6-CMG levels in human colorectal DNA (P = 0.93), and the O6-CMG/O6-MeG ratio ranged from 0.44 to 3.30.

Evidence for a Human O6-alkG Adductome

In addition to methylated and carboxymethylated ASPs, a number of other ASPs were detected at varying frequencies following incubation of MGMT with paired colorectal normal and tumor DNA samples (Figure 3). These included ASPs with m/z values of 1459.7, 1461.7, 1477.7, 1530.7, 1546.7, and 1555.7, which correspond to alkyl group modifications of mass between 144 and 240. These modifications have not yet been identified, but, in one sample, a peptide with an m/z of 1359.7 was detected and we hypothesized that this ASP was the result of the transfer of the hydroxyethyl (HOEt) group from O6-hydroxyethyl guanine in the DNA. In support of this, when a synthetic DS ODN containing O6-HOEtG was incubated with MGMT, an ASP ion with the same m/z value was detected (data not shown).

Figure 3.

Figure 3

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MALDI-TOF mass spectra analysis of alkylated ASPs present after incubating his-MGMT with paired normal and tumor colorectal DNA. His-MGMT (50 pmol) was incubated with 2 mg of human colorectal DNA samples isolated from paired tumor (T) or normal (N) tissues numbered 19, 44, and 39 and unmodified CT-DNA. Peak intensities are shown in arbitrary units on the y-axis, and the intensity scale is the same for all panels. S/N > 10 for all detected alkylated MGMT ASPs. These included unidentified (U) ASPs with m/z values of 1459.7 (labeled U2) found in four samples, 1461.7 (labeled U3) in one sample, 1477.7 (labeled U4) in two samples, 1530.7 (labeled U5) in two samples, 1546.7 (labeled U6) in three samples, and 1555.7 (labeled U7) in four samples.

Discussion

In the present work, we have used MGMT to irreversibly transfer alkyl groups from O6-alkGs in DNA to the active site cysteine residue of the protein and, following tryptic digestion, detected the resulting alkylated ASPs by MALDI-ToF MS. This method was developed and validated using DS ODNs containing O6-MeG and O6-CMG and was able to detect ASPs at levels as low as 50 fmol. The potential scope of the method was demonstrated by using the ODNs containing other O6-alkGs having widely different alkyl group structures. Further validation came from the analysis of human colorectal DNA, which found levels of O6-MeG that were directly comparable to those found in the same human DNA samples by using HPLC-RIA.

Analysis of CR DNA by this approach revealed the presence of not only O6-MeG and O6-CMG in normal and tumor tissue but also a putative O6-HOEtG and a number of other adducts that are currently unidentified. Previous studies have shown that alkyl adducts are present in human DNA from both normal and tumor tissue. For example, O6-MeG has been detected in DNA from both normal and tumor tissue from the GI tract,19O6-CMG has been detected in colon tumor samples,23 and methyl DNA phosphate adducts have been detected in human lung tumor tissue and adjacent normal tissue.24 In this study, human DNA samples showed varied patterns in the O6-alkG presence, suggesting that individuals have very different patterns of exposure to the responsible AA and/or different levels of expression of endogenous MGMT perhaps as a result of MGMT methylation in tumors.12,15 The source of these AAs is currently unknown, but humans are likely exposed to a plethora of environmental and dietary alkylating agents as well as endogenously formed N-nitroso compounds.25,26 These are very likely to generate numerous mutagenic O6-alkG DNA adducts that may result in a complex array of genomic modifications, some or all of which may contribute to colorectal carcinogenesis. It is also clear that determining the levels of a single O6-alkG adduct will significantly underestimate human exposure and likely that with increased sensitivity, the method will detect many more unknown alkyl groups. Nevertheless, these data clearly demonstrate the presence of an alkyl adductome that is yet to be completely characterized. The importance of such an alkyl adductome is confirmed by the identification of an AA mutation signature in CR cancers.5

The method that we described has two advantages over other current approaches to measure alkyl DNA adducts. First, O6-alkGs are detected independent of the nature of the alkyl group enabling the detection of unknown O6-alkGs in contrast to, for example, antibody-based methods that can only detect known O6-alkGs.42 Furthermore, the potential cross-reactivity of O6-alkG-derived antibodies with different adducts is avoided. Analysis of human DNA in this study clearly indicates that multiple O6-alkG adducts can be detected in one sample at the same time. Second, by targeting O6-alkGs, this approach focuses on adducts that are biologically relevant because of their pro-mutagenicity and pro-carcinogenicity,2,6,43 in contrast to other adductomic approaches that detect a wide range of DNA adducts, including some that have little biological significance.32,33 The assay, however, depends absolutely on the ability of MGMT to repair O6-alkGs by alkyl transfer to ASP cysteine. The range of MGMT repairable O6-alkGs in DNA remains unknown although a range of different O6-alkGs in DNA are known substrates, though not necessarily repaired at the same rate.44 Optimization of the repair reaction (e.g., by increasing incubation time and the ratio of MGMT to DNA) may increase the ability of the assay to detect poorly repaired substrates, but interestingly, a modified ASP consistent with the presence of O6-HOEt in DNA was detected, and O6-HOEt is a poor MGMT substrate in ODNs.37 Further indications of the range of alkyl groups that are MGMT substrates comes from work with pseudosubstrates (O6-alkGs in the form of free guanine bases) whose alkyl group is removed by MGMT.45 More than 75 pseudosubstrates with alkyl group masses ranging from 40 to 319 Da have been synthesized and have been shown to inhibit MGMT activity potentially as a result of alkyl group transfer though competitive inhibition cannot be ruled out (Margison Pers. commun.) Where this has been assessed, the same pseudosubstrate following its incorporation into an ODN is significantly more potent at MGMT inactivation.37,46O6-alkGs in DNA that are not MGMT substrates, such as O6-methyladamantylG, were not detected by this assay. At present, neither the nature nor the biological significance of MGMT-irreparable O6-alkGs is known, but if they are not repaired by other DNA repair pathways such as nucleotide excision repair47 and are also mutagenic, they may also be deleterious to cells. However, associations between downregulation of MGMT via promoter methylation and increased GC-AT transition mutations in human DNA and an alkylating agent mutation signature10−13 clearly demonstrate that MGMT substrates are important in human mutagenesis and such adducts would be detected by our methodology.

This current study used MALDI-ToF MS analysis for detection of alkylated MGMT-ASPs, and the evidence of the detection of the alkylated peptides following MGMT incubation with human colorectal DNA was based on the following three criteria: (1) m/z of detected ions, (2) recorded change in the molecular weight of the ASP (observed mass shift), and (3) S/N > 10. This strategy allows accurate and reproducible quantification of O6-alkG adducts in terms of quantifying detected alkylated peptides using isotopically labeled (13C15N) internal standards at low cost. Furthermore, the assay offers the necessary sensitivity that is critical to detect the inherently low level of O6-alkG adducts. Though we do not have tandem MS data to confirm the identity of the putative alkylated peptides detected in MGMT digest following incubation with human DNA, levels of O6-MeG detected by this assay were correlated with those detected by an HPLC-RIA. Furthermore, the ready availability of DNA containing O6-alkG adducts using the methods that we have described previously37 allows in principle the generation of any alkylated ASP standard following incubation of the DNA with MGMT. In addition, our novel assay analyzed relatively large amounts of human colorectal DNA, which, although it facilitates the detection of low levels of O6-alkG, may not always be available. To overcome the limitation of low DNA amounts, we are currently investigating the use of targeted MS assays that rely on reaction monitoring, e.g., multiple reaction monitoring (MRM) on a tandem quadrupole instrument and/or parallel reaction monitoring (PRM) on the Thermo Orbitrap series.

In summary, our current work, coupling the action of MGMT with MALDI-ToF MS analysis, provides a novel, sensitive approach for the simultaneous detection of overall DNA O6-guanine alkylation damage in human DNA and, where standards are available, allows the level of known individual adducts to be determined. The sensitivity of the method is limited by the amounts of DNA that can be extracted and analyzed, the ability of MGMT to remove the alkyl groups from the O6-alkGs, and the potential for extremely rare adducts that are potential MGMT substrates that might be present at levels that are less than the lowest limit of quantitation. Furthermore, future development of the methodology should enable the identification of previously unknown O6-alkG adducts in human DNA and hence a comprehensive description of the O6-alkG adductome and its potential contribution to the etiology of human cancer.

Acknowledgments

The authors wish to thank all the participants who provided samples for this study together with clinicians and nurses who helped to facilitate this study.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.chemrestox.3c00207.

  • ESI MS spectra of modified 23-mer ODNs; MALDI-ToF MS analysis of tryptic digests of MGMT incubated with different O6-alkyl-containing ODNs; LC-Vion IMS QToF analysis of His-MGMT tryptic digest following incubation with control SS ODN and SS ODN containing O6-MeG; quantitation of MGMT ASPs; characterization of ASPs formed following incubation with SS ODN containing O6-MeG (PDF)

Author Present Address

ł Faculty of Pharmacy, Fayoum University, Fayoum 63514, Egypt

Author Present Address

◊ Faculty of Science and Industrial Technology, Prince of Songkla University, Surat Thani Campus, Muang, Surat Thani, Thailand 84000

Author Present Address

Δ Centre for Proteome Research, Institute of Systems, Molecular & Integrative Biology, University of Liverpool, Liverpool L69 7ZB, U.K.

Author Present Address

∏ Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand

Author Present Address

⬠ Present address: Elizabeth Blackwell Institute, School of Biochemistry, University of Bristol, Bristol, BS8 1UH, U.K.

Author Contributions

CRediT: Rasha Abdelhady data curation, formal analysis, investigation, methodology, writing-review & editing; Pattama Senthong formal analysis, investigation, methodology, writing-review & editing; Claire E. Eyers investigation, methodology, supervision, writing-review & editing; Onrapak Reamtong investigation, methodology, writing-review & editing; Elizabeth Cowley investigation, methodology; Luca Cannizzaro investigation, methodology, writing-review & editing; Joanna Stimpson investigation, methodology, writing-review & editing; Kathleen Cain investigation, methodology, writing-review & editing; Oliver J. Wilkinson investigation, methodology, writing-review & editing; Nicholas H Williams funding acquisition, investigation, methodology, writing-review & editing; Perdita E. Barran funding acquisition, investigation, methodology, supervision, writing-review & editing; Geoffrey P. Margison funding acquisition, investigation, methodology, writing-review & editing; David M. Williams funding acquisition, investigation, methodology, writing-review & editing; Andrew Clifford Povey conceptualization, formal analysis, funding acquisition, methodology, project administration, writing-original draft, writing-review & editing.

A.C.P., P.B., D.W., N.W., E.C., K.C., and L.C. are supported by Cancer Research UK. R.A. was supported by the Egyptian Ministry of Higher Education and P.S. and O.R. by the Thai government. O.W. was supported by the BBSRC. J.S. is supported by a joint University of Manchester-Weizmann Institute of Science fellowship.

The authors declare no competing financial interest.

Special Issue

Published as part of Chemical Research in Toxicologyvirtual special issue “Mass Spectrometry Advances for Environmental and Human Health”.

Supplementary Material

tx3c00207_si_001.pdf (2MB, pdf)

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Supplementary Materials

tx3c00207_si_001.pdf (2MB, pdf)

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