Identification and Quantification of Human DNA Repair Protein NEIL1

Technical Note pubs.acs.org/jpr

Identification and Quantification of Human DNA Repair Protein NEIL1 by Liquid Chromatography/Isotope-Dilution Tandem Mass Spectrometry Prasad T. Reddy,†,# Pawel Jaruga,†,# Güldal Kirkali,† Gamze Tuna,†,§ Bryant C. Nelson,† and Miral Dizdaroglu*,† †

Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, United States Department of Biochemistry, School of Medicine, Dokuz Eylul University, Izmir, Turkey

§

S Supporting Information *

ABSTRACT: Accumulated evidence points to DNA repair capacity as an important factor in cancer and other diseases. DNA repair proteins are promising drug targets and are emerging as prognostic and therapeutic biomarkers. Thus, the knowledge of the overexpression or underexpression levels of DNA repair proteins in tissues will be of fundamental importance. In this work, mass spectrometric assays were developed for the measurement in tissues of the human DNA repair protein NEIL1 (hNEIL1), which is involved in base excision and nucleotide excision repair pathways of oxidatively induced DNA damage. Liquid chromatography/isotope-dilution tandem mass spectrometry (LC−MS/MS), in combination with a purified and fully characterized recombinant 15Nlabeled analogue of hNEIL1 (15N-hNEIL1) as an internal standard, was utilized to develop an accurate method for the quantification of hNEIL1. Both hNEIL1 and 15NhNEIL1 were hydrolyzed with trypsin, and 18 tryptic peptides from each protein were identified by LC−MS/MS on the basis of their full-scan mass spectra. These peptides matched the theoretical peptides expected from trypsin hydrolysis of hNEIL1 and provided a statistically significant protein score that would unequivocally identify hNEIL1. The product ion spectra of the tryptic peptides from both proteins were recorded, and the characteristic product ions were defined. Selected-reaction monitoring was used to analyze mixtures of hNEIL1 and 15NhNEIL1 on the basis of product ions. Additional confirmation of positive identification was demonstrated via separation of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in-gel tryptic digestion followed by LC−MS/MS analysis. These results suggest that the developed assays would be highly suitable for the in vivo positive identification and accurate quantification of hNEIL1 in tissues. KEYWORDS: human DNA repair protein, NEIL1, LC−MS/MS

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INTRODUCTION DNA repair is an essential life function and protects genomic stability and thus contributes to prevention of disease processes.1,2 DNA repair deficiencies predispose cells to accumulation of DNA damage and consequently mutations, resulting in genomic instability, which is a major factor in carcinogenesis.3−8 Germline mutations and polymorphisms in DNA repair genes also contribute to genomic instability and predisposition to cancer.4,6,8−10 Mounting evidence shows that cancer cells accumulate mutations in their DNA repair proteins, as cancer progresses.6,8 For example, 60% of cancer cell lines possess mutated DNA repair genes (see http://www.sanger.ac. uk.genetics/CGP). Although some cancer cells can lose their capacity to repair various forms of DNA damage, they acquire alternative mechanisms that can lead to therapeutic resistance.6,8,11 On the other hand, recent findings suggest that some types of malignant tumors possess increased DNA repair capacity that may affect the therapy and outcome of cancer.8,12−17 Although additional mutations may provide cancer cells with survival advantage, they may also cause cell © 2012 American Chemical Society

death late in tumor evolution. However, tumors that overexpress DNA repair genes may be favored by natural selection to increase their survival and consequently their DNA repair capacity. Accumulated evidence strongly suggests that DNA repair capacity will be an important factor in predicting patient response to DNA-damaging agents such as chemotherapeutic drugs and ionizing radiation.8 The determination of the overexpression or underexpression levels of DNA repair proteins in tumors will help develop and guide treatment strategies that will likely lead to the best treatment results for patients. In this context, DNA repair proteins are becoming predictive, prognostic and therapeutic factors in cancer, and also promising drug targets for cancer treatment as DNA repair inhibitors are being developed to increase the efficacy of cancer therapy.4,6,8 DNA repair consists of various pathways that involve a plethora of enzymes. Oxidatively induced DNA damage caused Received: November 2, 2012 Published: December 26, 2012 1049

dx.doi.org/10.1021/pr301037t | J. Proteome Res. 2013, 12, 1049−1061

Journal of Proteome Research

Technical Note

Production and Purification of hNEIL1 and

by endogenous and exogenous sources such as free radicals in living cells is mainly repaired by base excision repair (BER) and also by nucleotide excision repair (NER), albeit to a lesser extent.2,18 Among the DNA glycosylases involved in the first step of BER, mammalian NEIL1 protein is a bifunctional enzyme with an additional β,δ-elimination activity.19,20 It possesses a unique substrate specificity and specifically removes 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA; however, it exhibits no significant activity toward 8-hydroxyguanine (8-OH-Gua).19−28 Some pyrimidine-derived lesions such as thymine glycol are also excised albeit to a lesser extent. In addition, there is evidence for the involvement of NEIL1 in NER.29 Since its discovery, accumulated evidence has pointed to a significant role of this unique enzyme in the prevention of disease processes including carcinogenesis.21,25,26,28−33 Polymorphic variants of human NEIL1 (hNEIL1) with reduced or no activity have been discovered, suggesting that individuals carrying neil1 mutations may be at risk for disease development.25,30,34 The knowledge of the overexpression or underexpression levels of DNA repair proteins in tumors will be important, if these proteins are to be used as disease biomarkers, as guides and predictors for development of treatments, and as therapeutic response indicators in patients undergoing antitumor treatments. Our laboratory developed a program for the accurate and reproducible identification and quantification of DNA repair proteins by mass spectrometric techniques such as liquid chromatography-tandem mass spectrometry (LC−MS/MS) with isotope-dilution using fulllength and fully 15N-labeled analogues of these proteins as internal standards.35,36 In the present work, we report on the development of mass spectrometric assays for the measurement by LC−MS/MS of hNEIL1 using full-length and fully 15Nlabeled hNEIL1 (15N-hNEIL1) as an internal standard.

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15

N-hNEIL1

The human neil1 gene in the expression vector pET22b for Histagged production of the protein was kindly provided by Dr. Stephen Lloyd (Oregon Health and Science University, Portland, Oregon). The sequence analysis of the gene resulted in the protein sequence with 390 amino acids that matched the previously published sequence of human NEIL119,20,37,38 (see also, http://www.ncbi.nlm.nih.gov/protein/Q96FI4.3). This is the edited form of NEIL1 with arginine at position 242.38−40 The sequence of the C-terminal His-tag was found to be KLAAALEHHHHHH. E. coli BL21 (DE3) cells harboring the plasmid were grown at 37 °C in LB medium supplemented with ampicillin and chloramphenicol to a final concentration of 100 μg/mL and 40 μg/mL, respectively. At midlogarithmic phase of growth (A600 ∼ 0.6), cells were briefly cooled in ice and hNEIL1 production was induced with 100 μmol IPTG/mL at 25 °C for 16 h. Cells were harvested at 6000g for 20 min and washed with 25 mM Tris buffer (pH 7.5). The wet weight of cells obtained in this procedure was 3.5 g/L culture. Minimal medium was prepared as described.41 The composition of the medium was 6 g of NaH2PO4, 3 g of K2HPO4, 0.5 g of NaCl, and 1 g of 15N-NH4Cl, 5 g of glucose, 246 mg of MgSO4·7H2O per L. Ampicillin and chloramphenicol were added to a final concentration of 50 μg/mL and 20 μg/mL, respectively. E. coli BL21 (DE3) harboring pET22b/ human neil1 recombinant plasmid was grown at 37 °C for 20 h on LB agar plate containing 100 μg of ampicillin and 40 μg of chloramphenicol/mL. A colony was carefully (without touching into the LB medium) transferred to 10 mL minimal medium containing 1 mg of 15N-NH4Cl/mL and 50 μg of ampicillin and 20 μg of chloramphenicol/mL. Cells were grown for 16 h at 37 °C at 250 rpm in a 50 mL tube. This inoculum was transferred to 90 mL minimal medium, in a 250 mL flask, containing 15NNH4Cl, ampicillin, and chloramphenicol as above. This culture was grown at 37 °C for 9 h. Next, this seed culture was transferred to 900 mL of minimal medium containing 15NNH4Cl, ampicillin, chloramphenicol in a 2 L flask. This culture was grown at 37 °C for 4 h. Cells (A600 = 0.45) were briefly cooled in ice, and 15N-hNEIL1 production was induced with 100 μmol IPTG/mL at 25 °C for 16 h. Cells were harvested at 6000g for 20 min and washed with 25 mM Tris buffer (pH 7.5). The wet weight of cells obtained in this procedure was 2.8 g/L culture. Cell pellet was suspended at a rate of 1 g/10 mL of 50 mM Na2HPO4−NaH2PO4 buffer (pH 8.0), 10 mM β-mercaptoethanol, 10 mM imidazole, and 300 mM NaCl (lysis buffer). One tablet of protease inhibitors was added to the cell suspension. Cell suspension was passed through a French Press at 7 × 104 kPa twice. The cell-free extract was centrifuged at 36000g for 30 min. Meanwhile, 1 mL of nickel-agarose slurry (0.5 mL resin) was washed with the lysis buffer in a 30 mL BioRad polypropylene column. The supernatant was added to the resin and mixed on a rocker for 2 h at 4 °C. The flow through was collected, and the column was washed successively with three 10 mL aliquots of the lysis buffer. Next, the resin was washed successively two times with 10 mL aliquots of the lysis buffer containing an additional 30 mM imidazole (total 40 mM). Next, the resin was washed successively three times with 5 mL aliquots of the lysis buffer containing an additional 70 mM imidazole (total 80 mM). The first imidazole80 wash did elute about 30% of 15 N-hNEIL1 with a number of contaminating proteins as judged by SDS-PAGE. The second and third imidazole80 washes eluted about 30% of nearly

MATERIALS AND METHODS

Reagents

Trypsin (Proteomics grade), acetonitrile (HPLC-grade), and water (HPLC-grade) for analysis by LC−MS/MS were purchased from Sigma (St. Louis, MO). Water purified through a Milli-Q system (Millipore, Bedford, MA) was used for all other applications. Acrylamide, bisacrylamide, and protease inhibitor cocktail tablets were obtained from Sigma-Aldrich (St. Louis, MO). 15N-NH4Cl was purchased from Cambridge Isotope Laboratories (Andover, MA). Nickel agarose resin was from Qiagen (Valencia, CA). 4,6-Diamino-5-formamidopyrimidine-13C,15N2 (FapyAde-13C,15N2), 2,6-diamino-4-hydroxy-5-formamidopyrimidine-13C,15N2 (FapyGua-13C,15N2), and 8-hydroxy-2′-deoxyguanosine-15N5 were purchased from Cambridge Isotope Laboratories (Andover, MA). 8-Hydroxyguanine-15N5 (8-OH-Gua-15N5) was obtained by hydrolysis of 8-hydroxy-2′-deoxyguanosine-15N5 with 60% formic acid at 140 °C for 30 min followed by lyophilization. Subsequently, 8-OHGua-15N5 was dissolved in 10 mM NaOH before use. The Laemmli sample buffer, 12.5% Tris-HCl Criterion precast gels, 1×Tris/glycine/SDS running buffer and Coomassie Brilliant Blue R-250 Staining Solutions kit were purchased from Bio-Rad Laboratories (Hercules, CA). The In-Gel Tryptic Digestion kit was from Pierce (Rockford, IL). Prestained protein markers were from New England BioLabs (Ipswich, MA). 1050



Technical Note

homogeneous 15N-hNEIL1. Next, 15N-hNEIL1 was eluted successively with three 5 mL aliquots of the lysis buffer containing 150 mM imidazole. The first 5 mL contained about 30% of 15N-hNEIL1. The subsequent two elutions contained the remaining 15N-hNEIL1. The second and third imidazole80 elutions and all the imidazole150 elutions were pooled and dialyzed overnight against 1 L of 50 mM Na2HPO4−NaH2PO4 buffer (pH 8.0) and 300 mM NaCl. Slight precipitate of hNEIL1 appeared after dialysis even in the presence of 300 mM NaCl. The precipitate was centrifuged off from the dialyzed pool, and the clear supernatant was concentrated on NMLW5 membrane in an Amicon cell. The above purification procedure developed for cells from 1 L culture was proportionately scaled up to 5 L as and when needed. Protein concentration was determined by the Lowry method using BSA as the standard.42 For the production of hNEIL1, NH4Cl was used instead of 15NNH4Cl. Otherwise the procedure was the same as that for 15NhNEIL1. The final yields of 15N-hNEIL1 and hNEIL1 were ∼2 mg/L culture.

The column, mobile phases, and gradient analysis were as given above for LC/MS measurements. The autosampler and column temperature were kept at 15 and 30 °C, respectively. The flow rate was 300 μL/min. Experimental conditions for the automated mass calibration and operating parameters of the MS/MS system in the positive ion mode were as previously described,36 except for sheath gas (nitrogen) pressure and scan width being 60 (arbitrary units) and m/z 2, respectively. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and In-Gel Tryptic Digestion

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described,44 using the gel electrophoresis system Criterion Cell (Bio-Rad Laboratories, Hercules, CA). Separations and in-gel trypsin digestions were performed according to previously published procedures.45,46 Briefly, the protein samples were denatured with Laemmli sample buffer in a 1:1 ratio. The samples (10 μg each) were loaded into a 12.5% Tris-HCl Criterion precast gel (13.3 × 8.7 cm (W × L), 1 mm thick and run at 180 V in 1× Tris/glycine/ SDS running buffer for 90 min). Prestained protein standards were used as protein markers. The gels were stained and destained with Coomassie Brilliant Blue R-250 Staining Solutions kit. The in-gel tryptic digestion was carried out using an In-Gel Tryptic Digestion kit. The proteins bands were on the order of 2 mm to 4 mm in diameter with the gel thickness of 1 mm. The bands were cut from the gel with a sharp scalpel and divided into smaller pieces (1 × 1 mm to 2 × 2 mm). According to the kit protocol, the bands of interest from the gel were destained, and then reduced and alkylated. Subsequently, the buffers were exchanged to wash the reduction and alkylation reagents out of the samples. Prior to trypsin hydrolysis, the gel pieces were dehydrated in 100% acetonitrile, completely shrank and were dried by vacuum centrifugation for 5 min. In the final step, the hydrolysis was performed using activated modified trypsin. The samples were incubated overnight at 30 °C with shaking and then centrifuged. The supernatant fractions were separated and analyzed by LC−MS/MS.

Gas Chromatography/Tandem Mass Spectrometry

The enzymic activities of hNEIL1 and 15N-hNEIL1 were measured using gas chromatography/tandem mass spectrometry (GC-MS/MS) with isotope-dilution. Calf thymus DNA samples were prepared, treated with hNEIL1 or 15N-hNEIL1, and then analyzed by GC-MS/MS as described.35,43 The details of the sample preparation and GC-MS/MS are given in the Supporting Information. Hydrolysis with Trypsin

An aliquot of 100 μg of hNEIL1 or 15N-hNEIL1 was incubated with 2 μg trypsin in 500 μL Tris-HCl buffer (30 mM, pH 8.0) at 37 °C for 2 h. Then, an aliquot of 2 μg trypsin was added again. After another 22 h of incubation, the sample was heated at 95 °C for 5 min to deactivate trypsin prior to analysis by LC−MS/MS. Liquid Chromatography/Mass Spectrometry

Analyses were performed using a liquid chromatograph−mass selective detector (1100 Series, Agilent Technologies, Wilmington, DE) equipped with an automatic injector and atmospheric pressure ionization-electrospray in the positive ionization mode. The flow and temperature of the drying gas (nitrogen) were 10 L/min and 350 °C, respectively. The nebulizing gas pressure was 308 kPa. The capillary potential was 4000 V. The fragmentor and electron multiplier potential were 100 and 2600 V, respectively. A Zorbax Extend-C-18, Rapid Resolution HT column (2.1 mm × 100 mm, 1.8 μm particle size) (Agilent Technologies, Wilmington, DE) with an attached Agilent Eclipse XDB-C8 guard column (2.1 mm × 12.5 mm, 5 μm particle size) was used. The autosampler and column temperature were kept at 5 and 40 °C, respectively. Mobile phase A was water plus 2% acetonitrile and 0.1% formic acid (v/v). Mobile phase B consisted of acetonitrile plus 0.1% formic acid (v/v). A gradient analysis starting from 1% B and linearly increasing to 51% B in 25 min was used. Afterward, B was increased to 90% in 0.1 min and kept at this level for 5 min and then decreased to 1% to equilibrate the column for 20 min. The flow rate was 250 μL/min.

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RESULTS

Production, Purification, and Characterization of hNEIL1 and Fully 15N-Labeled hNEIL1

Both hNEIL1 and 15N-hNEIL1 were overproduced in E. coli as described in the experimental procedures. Both proteins were purified according to the steps shown in Supplemental Figure 1, Supporting Information. First, the enzymic activity of these proteins was tested. Among other properties, the enzymic activity of a stable isotope-labeled protein must be identical to that of its analogous analyte protein. This will ensure that the active site of the labeled protein was not perturbed by 15Nlabeling and other isolation procedures. The glycosylase activity of hNEIL1 is well-known and efficiently removes FapyAde and FapyGua from DNA containing multiple lesions; however, this enzyme exhibits no significant activity toward 8-OHGua.19,22,24−26 The enzymic activities of both hNEIL1 and 15 N-hNEIL1 were tested using a DNA sample containing multiple lesions as substrates. The details and applications of this approach that is used to accurately determine the substrate specificities of DNA glycosylases have previously been described.47 In the present work, GC-MS/MS with isotopedilution was used for the measurement of the levels of FapyAde, FapyGua, and 8-OH-Gua after treatment of DNA

Liquid Chromatography/Tandem Mass Spectrometry

LC−MS/MS analyses were performed using an Agilent 1290 Infinity LC system coupled to a Thermo-Scientific Finnigan TSQ Quantum Ultra AM triple quadrupole MS/MS system with an installed heated electrospray-ionization (HESI) source. 1051



Technical Note

Figure 1. DNA glycosylase activities of hNEIL1 and 15N-hNEIL1. Each data point represents the mean of three independently measured values. The uncertainties are standard deviations.

Figure 2. (A, B) Total-ion current profiles of the tryptic hydrolysates of hNEIL1 and 15N-hNEIL1, respectively. Identities of the peaks are given in Table 1.

FapyAde-13C,15N2, m/z 372 → m/z 283, m/z 372 → m/z 357 and m/z 372 → m/z 371; for FapyGua: m/z 457 (M+•) → m/z 368 (M+• − TMSO•) and m/z 457 → m/z 442 (M+• − •CH3); for FapyGua-13C,15N2; m/z 460 → m/z 371 and m/z 460 → m/z 445; for 8-OH-Gua, m/z 455 (M+•) → m/z 440 (M+• − • CH3), and for 8-OH-Gua-15N5, m/z 460 → m/z 445. FapyAde, FapyGua, and 8-OH-Gua were separately analyzed by varying the collision energies from 4 to 34 V in 2 V increments to determine the optimum collision energy for each

samples with hNEIL1 and 15N-hNEIL1. The multiple-reaction monitoring (MRM) mode of the GC-MS/MS instrument was used to monitor the product ion transitions, which were chosen among characteristic ions from the previously published mass spectra of trimethylsilyl (TMS) derivatives of FapyAde, FapyGua, and 8-OH-Gua, and their stable isotope-labeled analogues.43,48,49 The transitions were for FapyAde, m/z 369 (M+•) → m/z 280 (M+• − TMSO•), m/z 369 → m/z 354 (M+• − •CH3) and m/z 369 → m/z 368 (M+• − H•); for 1052



Technical Note

Table 1. Identification of the Tryptic Peptides in Figure 4 and the m/z Values of the Monoisotopic Masses of their MH+ and (M + 2H)2+ Ions 15

unlabeled peptide +

peak

peptide

MH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

NLADK FGR ISASAR ENVLR EELPR WQPGR ATQLSPEDR EVVQLGGR AWLR AEILYR FYTAPPGPR WDLGGK NPEVPFESSAYR GYGSESGEEDFAAFR TIWFQGDPGPLAPK FGMSGSFQLVPR FFNGIGNYLR LILSPLPGAQPQQEPLALVFR

560.30 379.21 604.34 630.35 643.33 643.33 1016.50 857.48 545.33 764.42 1005.51 675.34 1395.64 1621.67 1526.79 1325.65 1200.60 2287.29

(M + 2H)

N-labeled peptide

2+

MH

280.65 190.10 302.67 315.67 322.16 322.16 508.75 429.24 273.16 382.71 503.25 338.17 698.32 811.33 763.89 663.35 600.80 1144.14

(M + 2H)2+

+

567.30 385.21 613.34 639.35 651.33 653.33 1029.50 869.48 552.32 773.42 1017.51 683.34 1411.64 1639.67 1543.79 1341.65 1215.60 2314.29

Figure 3. (A, B) Full-scan mass spectra of GYGSESGEEDFAAFR (represented by peak 14 in Figure 2A) and (represented by peak 14 in Figure 2B), respectively.

284.15 193.10 307.17 320.17 326.16 327.16 515.25 435.24 277.16 387.21 509.25 342.17 706.32 820.33 772.39 671.35 608.30 1157.64

15

N-GYGSESGEEDFAAFR

8-OH-Gua-15N5 transitions. The data in Figure 1 show that

of the ion transitions to be used for MRM. Supplemental Figure 2, Supporting Information illustrates the plots of the collision energies versus the intensities of the monitored transitions. The optimum collision energies were 14 V for FapyAde and FapyAde-13C,15N2 transitions, 14 and 20 V for two FapyGua and FapyGua-13C,15N2 transitions, and 18 V for 8-OH-Gua and

15

N-hNEIL1 removed FapyAde and FapyGua from DNA as

efficiently as hNEIL1. As expected, both proteins exhibited no activity toward 8-OH-Gua. These results unequivocally demonstrate that the 1053

15

N-labeling did not affect the enzymic



Technical Note

Figure 4. (A, B) MS/MS spectra of GYGSESGEEDFAAFR and 15N-GYGSESGEEDFAAFR, respectively.

profile of the trypsin hydrolysate of hNEIL1. The full-scan mass spectra of the peptides represented by the peaks in Figure 2A were recorded. On the basis of their mass spectra, 18 tryptic peptides were identified that matched the theoretical peptide fragments expected from the trypsin hydrolysis of hNEIL1. As Figure 2B illustrates, the analysis of the trypsin hydrolysate of 15 N-hNEIL1 yielded an essentially identical TIC profile with 18 identified analogous 15N-labeled tryptic peptides. The identities of the peptides are given in Table 1 along with the m/z values of the monoisotopic masses of their protonated molecular ions (MH+ ion) and doubly protonated (charged) molecular ions [(M + 2H)2+ ion]. Supplemental Figure 3, Supporting Information illustrates the extracted (M + 2H)2+ ions from the data shown in Figure 2A, clearly revealing the presence of

activity of hNEIL1, indicating no perturbation of its active site. The C-terminal His-tag had no effect on this function, either. Separation and Identification of the Tryptic Peptides by LC/MS

hNEIL1 and 15N-hNEIL1 were subjected to hydrolysis with trypsin.50 The hydrolysates were analyzed by LC/MS to separate the resulting tryptic peptides and to obtain their fullscan mass spectra for identification. Theoretically, the trypsin hydrolysis of hNEIL1 yields 62 fragments, 12 of which are single amino acids, 5 arginines and 7 lysines. The lengths of the tryptic peptides vary from 2 to 22 amino acids (see, http://au. expasy.org/tools/pi_tool.html). The His-tag does not affect the masses of the tryptic peptides except for the last one at the Cterminus. Figure 2A illustrates the total-ion-current (TIC) 1054



Technical Note

Figure 5. Selected-reaction monitoring of seven unlabeled peptides and their 15N-labeled analogues from the tryptic hydrolysate of a mixture of hNEIL1 and 15N-hNEIL1. Peptides and measured transitions are shown.

the identified peptides. Using the “MASCOT” search engine (http://www.matrixscience.com) and the 18 identified tryptic peptides, a protein score of 181 was obtained. According to this search engine, protein scores greater than 56 are significant (p < 0.05). This means that the 18 tryptic peptides would unequivocally identify hNEIL1.

of low abundance at m/z 1621.7. A mass shift of 18 Da in the mass of the MH+ ion was observed in the full-scan spectrum of the labeled analogue of this peptide (Figure 3B), which is represented by peak 14 in Figure 2B (see also Table 1). This is on a par with the 18 15N atoms in this molecule. Other examples of the full-scan spectra of the tryptic peptides are illustrated in Supplemental Figures 4A-7A, Supporting Information. These spectra and those of the other peptides listed in Table 1 were also dominated by the (M + 2H)2+ ion as the base peak and the MH+ ion of low abundance. The 15Nlabeled peptides had correct mass shifts according to the number of their 15N atoms (Supplemental Figures 4B−7B, Supporting Information). No masses of unlabeled materials

Full-Scan Spectra of the Tryptic Peptides

An intense (M + 2H)2+ ion and an MH+ of low abundance were observed in the full-scan spectra of the tryptic peptides. As an example, the spectrum taken from the peptide represented by peak 14 in Figure 2A is shown in Figure 3A. The full-scan spectrum of this peptide (GYGSESGEEDFAAFR) contains an (M + 2H)2+ ion as the base peak at m/z 811.3 with an MH+ ion 1055



Technical Note

Figure 6. Ion-current profiles of six transitions of GYGSESGEEDFAAFR.

were observed in the spectra of all 15N-labeled peptides, confirming the full 15N-labeling of hNEIL1.

Figure 4A illustrates the MS/MS spectrum of GYGSESGEEDFAAFR (represented by peak 14 in Figure 2A; see also Table 1). The spectrum was dominated by the y-ion series (Supplemental Table 1, Supporting Information) from the y2ion to the y11-ion with high intensities. In the typical b-ion series (Supplemental Table 2, Supporting Information), the b2-, b3-, and b4-ions only were observed with some discernible intensity. The 15N-labeled analogue of this peptide gave an essentially identical MS/MS spectrum (Figure 4B) with mass shifts according to the 15 N-content of the fragments (Supplemental Tables 1 and 2, Supporting Information). As further examples, the MS/MS spectra of EVVQLGGR, FYTAPPGPR, NPEVPFESSAYR, and TIWFQGDPGPLAPK, and their 15N-labeled analogues (represented by peaks 8, 11, 13 and 15, respectively, in Figure 2A,B) are given in Supplemental Figures 8−11, respectively. These MS/MS spectra were dominated by the y-ion series, whereas only a few ions of the b-ion series were observed. The shifts in the masses of the fragments in the spectra of the 15N-labeled analogues were in full agreement with the 15N-contents of the fragments (Supplemental Figures 8B−11B, Supporting Information). In some cases, the ions resulting from loss of NH3 from y-ions (yNH3 ion) were also observed as indicated in the spectra.

Product-Ion Spectra of the Tryptic Peptides

Trypsin hydrolysates of hNEIL1 and 15N-hNEIL1 were analyzed by LC−MS/MS. The full-scan mass spectra of the tryptic peptides and their 15N-labeled analogues were essentially identical to those obtained using LC/MS. Next, the production spectra (MS/MS spectra) were obtained. We calculated the theoretical masses of the typical b- and y-series ions46 as the product ions that are expected to result from the collisioninduced fragmentation of these peptides (Supplemental Tables 1 and 2, Supporting Information). As an example, the calculation of the masses of the b- and y-ions of the peptide GYGSESGEEDFAAFR and its 15N-labeled analogue is shown in Supplemental Table 3, Supporting Information. The values of the b- and y-ions for the unlabeled peptides were in agreement with those calculated using the “ProteinProspector” database of the University of California (http://prospector.ucsf. edu/prospector/cgi-bin/msform.cgi?form=msproduct). In order to obtain the MS/MS spectra of the tryptic peptides, optimum collision energies for the fragmentation of these peptides must be measured experimentally. However, the relationship between the collision energies and the m/z values can also be determined empirically as was previously described for (M + 2H)2+ ions in a tandem quadrupole instrument.46 The collision energy for each identified tryptic peptide of hNEIL1 was initially chosen according to this empirically determined relationship using the m/z value of their (M + 2H)2+ ions. Subsequently, several experiments were performed to determine the optimum collision energy. This was followed by the recording of the MS/MS spectra at the chosen collision energy for each peptide.

Selected-Reaction Monitoring for Identification and Quantification

For the positive identification and accurate quantification of peptides at low concentrations in a complex mixture such as trypsin hydrolysates of proteins, the mode of selected-reaction monitoring (SRM) [also called multiple-reaction monitoring (MRM)] of an LC−MS/MS instrument is used.46,51 For this purpose, it is necessary to choose the (M + 2H)2+ ions or the MH+ ions of the tryptic peptides of a given protein as the precursor ions and some of their product ions of high 1056



Technical Note

abundance. Subsequently, the transitions of each precursor ion to its chosen product ions are monitored. For quantification, this process is performed for the corresponding transitions of the 15N-labeled analogues of the tryptic peptides. The monitored transitions along with the typical retention time of the tryptic peptides enable a positive identification and accurate quantification of these peptides and consequently the corresponding protein.46,51 For this purpose, however, the knowledge of the full-scan mass spectra and MS/MS spectra of the tryptic peptides of an analyte protein is necessary to determine the appropriate transitions. Furthermore, the retention time of a given peptide on the LC column must be known under the experimental conditions used. In order to achieve the optimum sensitivity and selectivity, the transitions of a targeted peptide are recorded within a time window around its known retention time. Having identified the tryptic peptides of hNEIL1 at their respective retention times and determined their product ions as described above, the trypsin hydrolysates of the mixtures of hNEIL1 and 15N-hNEIL1 were analyzed using SRM in order to check the suitability of this technique for the identification and quantification of hNEIL1 using 15N-hNEIL1 as an internal standard. The (M + 2H)2+ ion was chosen as the precursor ion for transitions, because this ion generally represents the highest charge state of the tryptic peptides.46 In addition, the (M + 2H)2+ ion was more prominent than the MH+ ion in the fullscan spectra (Figure 3, and Supplemental Figures 4−7, Supporting Information). The ion-current profiles of the transitions of seven tryptic peptides from the trypsin hydrolysate of a mixture of hNEIL1 and 15N-hNEIL1 are illustrated in Figure 5. A baseline separation of the monitored peptides was achieved with excellent peak shapes of the transition profiles. As expected, each unlabeled peptide and its respective 15 N-labeled analogue exactly coeluted at the same retention time. In addition to characteristic transitions, the retention time of a peptide under defined experimental conditions is an absolutely essential criterion for a positive identification. It should be pointed out that, if there is any doubt about the specificity of a monitored product ion, the identity of the targeted peptide can be confirmed by monitoring multiple transitions that are characteristic for the targeted peptide.51 An example of this approach is shown in Figure 6 in the case of the peptide GYGSESGEEDFAAFR. The ion-current profiles of six characteristic transitions for this peptide are lined up at its appropriate retention time, confirming its identity. In addition, the intensities of the transitions agree with those of the product ions in the MS/MS spectrum of this peptide (Figure 4). Two other examples of this approach are given in Supplemental Figure 12, Supporting Information, which illustrates four transitions of each of the peptides EVVQLGGR and FYTAPPGPR with retention times only 0.4 min apart. If necessary, additional validation would be provided by monitoring the corresponding transitions for the coeluting 15 N-labeled analogue of the targeted peptide.

Figure 7. Determination of optimum collision energies for the most intense transitions of nine tryptic peptides. The (M + 2H)2+ ions were used as the precursor ions (see Table 1 for identification of the peptides).

the collision energy for the transitions of nine representative tryptic peptides. The collision energies at which the transitions had the maximum intensity were plotted as a function of the m/ z values of the precursor ions [(M + 2H) 2+ ions] (Supplemental Figure 13, Supporting Information). The maximum collision energies of three additional ions are shown here, which are not given in Figure 7. A linear relationship between these two parameters was observed. This plot is in excellent agreement with that obtained empirically for a large number of peptides.46 Calibration Plots

The response of the MS/MS instrument to the increasing concentrations of the tryptic peptides was tested using SRM with typical transitions of various tryptic peptides. The amount of hNEIL1 was varied from 0.023 pmol to 23 pmol injected oncolumn. A linear response was obtained as shown in Figure 8 in

Figure 8. Calibration plots obtained using tryptic hydrolysates of varying amounts of hNEIL1. SRM transitions leading to the most intense ions in the MS/MS spectra of 10 tryptic peptides were chosen. The inset shows the range of the x-axis up to 3 pmol of the same plot. Linear regression analyses yielded correlation coefficients (R2) > 0.99 for all transitions.

Determination of the Optimum Collision Energies for Quantification

The optimum collision energies were determined for the most intense ions in the MS/MS spectra in order to develop the most sensitive method capable of identifying and quantifying low levels of hNEIL1. For this purpose, the collision energies were varied from 5 to 55 V in 5 V increments. As examples, Figure 7 illustrates the plots of the measured intensity versus

the case of 10 peptides. The inset shows the peptide responses for low amounts (0−3 pmol) of each peptide injected oncolumn. The MS/MS response was also tested for the increasing ratio of concentrations of hNEIL1 and 15NhNEIL1. As Supplemental Figure 14, Supporting Information illustrates in the case of six tryptic peptides of both proteins, a 1057



Technical Note

this protein must be used as an internal standard. A fully stable isotope-labeled analogue would possess identical chemical and physical properties of the analyte protein and thus compensate for eventual losses and drifts during all stages of analysis. The use of such a labeled internal standard may also overcome eventual problems with fluctuations in signal intensity. Another important reason for this approach is that trypsin hydrolysis can often be inefficient leading to incomplete yields of tryptic peptides and thus to measurement bias. Since the labeled analogue of the analyte protein would undergo the same trypsin hydrolysis in a given sample, its use as an internal standard would minimize the adverse effect of the possible inefficient hydrolysis. Moreover, the labeled analogue of a protein as an internal standard would be absolutely essential for its accurate quantification when one- or two-dimensional gel electrophoresis and “in-gel tryptic digestion” are used prior to LC− MS/MS analysis. Previously, full-length expressed proteins containing 13Cand/or 15N-labeled lysine and arginine have been used as internal standards for quantification of proteins by mass spectrometry.56−60 In our laboratory, full-length and fully 15 N-labeled proteins were used as internal standards for the quantitative measurement of E. coli Fpg and human OGG1 proteins by LC−MS/MS.35,36 In the present work, 15NhNEIL1 was overexpressed, purified, and characterized as an internal standard. Its DNA glycosylase activity was essentially identical to that of the unlabeled protein, indicating no perturbation of its active site during isolation and purification. The identification of 18 tryptic peptides of hNEIL1 and 15NhNEIL1 was achieved. The full-scan mass spectra of these peptides contained intense (M + 2H)2+ and MH+ ions. The shifts in the masses of these ions observed in the full-scan mass spectra of the labeled peptides were in full agreement with the number of the N-atoms in each peptide. No discernible ions corresponding to unlabeled peptides were observed in the fullscan mass spectra of labeled peptides, evidencing that a full 15Nlabeling of hNEIL1 was achieved. This meets an essential requirement for an ideal stable isotope-labeled internal standard. The MS/MS spectra of the tryptic peptides provided the ions of the typical b- and y-ion series. The masses of these ions matched those expected from the sequences of the peptides. The mass shifts observed in the MS/MS spectra of the labeled peptides were in full agreement with the 15Ncontent of each fragment ion. Again, no ion corresponding to an unlabeled fragment was observed, confirming the results obtained with the full-scan spectra of the tryptic peptides of 15 N-hNEIL1. The isolation of hNEIL1 and 15N-hNEIL1 was also achieved from gels after SDS-PAGE. This is a crucial step prior to LC−MS/MS for the identification and quantification of hNEIL1 in complex tissue samples. The neil1 gene has been sequenced by the National Institute of Environmental Health Sciences Environmental Genome Project at the University of Washington in a variety of individuals. Variants have been identified that predicted changes in the amino acid sequence of hNEIL1, i.e., Ser82Cys, Gly83Asp, Cys136Arg, Asp252Asn, and I182M. The variants of hNEIL1 with Cys82, Asp83, Arg136, and Asn252 have been cloned, expressed in E. coli, and purified.25 hNEIL1-Met182 was not isolated. Two of the variants, hNEIL1-Cys82 and hNEIL1Asn252, exhibited wild type activity, whereas the other two were devoid of glycosylase activity. The positions 82 (Ser) and 83 (Gly) are located in the tryptic peptide FGMSGSFQLVPR, whereas the tryptic peptides AEILYR and GYGSESGEED-

linear response was obtained within a concentration ratio ranging from 0.1 to 4.0. Analytical Sensitivity of LC−MS/MS for Tryptic Peptides

The analytical sensitivity of the instrument was examined by analyzing a tryptic hydrolysate of hNEIL1, the amount of which ranged from 5 fmol to 100 fmol injected on-column. The limit of detection (LOD) was approximately 10 fmol with a signalto-noise ratio (S/N) of at least 3. The limit of quantification (LOQ) was approximately 30 fmol with an S/N of 10 (data not shown). However, it should be mentioned that the sensitivity will depend on the monitored transition of a peptide and thus may vary from transition to transition of each peptide and from peptide to peptide. SDS-PAGE Analysis and in-Gel Tryptic Digestion

The analysis of both hNEIL1 and 15N-hNEIL1 was also performed using isolation by SDS-PAGE, and subsequent in-gel tryptic digestion and LC−MS/MS. Supplemental Figure 15, Supporting Information shows the SDS gel where the proteins were separated. E. coli Fpg and human OGG1, and their 15Nlabeled analogues were also separated for comparison. The isolation of 15N-labeled E. coli Fpg and 15N-labeled human OGG1 has been reported in our previous work.35,36 The band (lane 6) containing the mixture of hNEIL1 and 15N-hNEIL1 was excised and subjected to in-gel tryptic digestion. The hydrolysate was analyzed by LC−MS/MS. Supplemental Figure 16, Supporting Information illustrates the ion-current profiles of several transitions of the unlabeled and labeled peptides, revealing a successful isolation from the SDS gel and in-gel tryptic digestion of both proteins.

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DISCUSSION Since its discovery, isolation and characterization,19,20,23 hNEIL1 has become the focus of intense studies. This is due to its unique substrate specificity as a bifunctional DNA glycosylase in BER, the evidence for its critical role in maintaining the genomic stability and in prevention of diseases such as cancer and metabolic syndrome-associated diseases, and its involvement in replication-associated repair (reviewed in ref 52). Moreover, the known mutagenic effects and other properties of its substrates added a new dimension to the importance of this protein. hNEIL1 increases during the S phase indicating its involvement in replication repair and is variably expressed in human tissues with the highest expression observed in the liver, pancreas, and thymus followed by moderate expression in the brain, spleen, prostate, and ovary and by low expression in the testis and leukocytes.19,53 Thus far, all evidence accumulated in the past decade strongly points to a critical role of hNEIL1 in the DNA repair capacity of a living human cell. Knowledge of the expression level of this protein may provide a greater understanding of the DNA repair capacity of a given tissue and may be a critical prerequisite for determining whether DNA repair pathways are altered in cancer (or any other type of disease) tissues. DNA repair alterations may in turn serve as predictive cancer biomarkers. To understand its role in disease processes and its use as a reliable biomarker, hNEIL1 must be positively identified and accurately quantified in relevant human tissues by proper chemical and physical techniques. LC−MS/MS with isotope-dilution is the choice of technique for protein measurements.46,54−60 In order to achieve high precision and accuracy for the quantitative measurement of a protein by this technique, a stable isotope-labeled analogue of 1058



Technical Note

be a great advantage for accurate quantification when complex tissue samples are used. The assays described in this work are likely to be highly suitable for the positive identification and accurate quantification of hNEIL1 in vivo. The accurate and reliable measurement of expression levels of DNA repair proteins in human tissues will be of fundamental importance for understanding of their role in disease processes, for development of DNA repair inhibitors, and for their use as disease biomarkers.

FAAFR contain the positions 182 (Ile) and 252 (Asp), respectively. These three peptides were identified in the present work. On the other hand, the theoretical peptide with the position 136 (Cys) (GPCVLQEYQQFR) was not found. It should be pointed out that, when Cys is replaced by Arg, this peptide is expected to produce two peptides upon trypsin hydrolysis because of the cleavage of the peptide bond between Arg and the next amino acid. Inactivating neil1 mutations reduced hNEIL1 expression, and three rare polymorphisms in the neil1 promoter region have been identified in human gastric cancers.30,34 Such polymorphisms or other mutations may lead to differences in the expression level of hNEIL1,34 as there is evidence for nucleotide changes causing differences in protein expression levels.61 One of the identified mutations in hNEIL1 in gastric cancer is Gly245Arg.30 The tryptic peptide GYGSESGEEDFAAFR identified in the present work contains this position. Because of the replacement of Gly245 by Arg in the mutated hNEIL1, this peptide would undergo the hydrolysis of the peptide bond between Arg and the next amino acid, leading to the peptides GYR and SESGEEDFAAFR. The methodology developed in the present work would readily identify the disappearance of GYGSESGEEDFAAFR and perhaps the subsequent appearance of GYR and SESGEEDFAAFR in the trypsin hydrolysate of the mutated hNEIL1, proving the presence of the said mutation. For the reasons outlined here, it is fair to assume that, in addition to the identification and quantification of wild type hNEIL1, the methodology developed in this work may permit the identification of the known variants of hNEIL1 and help discover unknown variants containing amino acid replacements in one of the 18 peptides identified in this work. In such cases, 15 N-hNEIL1 may serve not only as the ideal internal standard for accurate quantification but also help validate the identity of tryptic peptides with amino acid replacements. In a relevant context, NEIL1 mRNA may be altered via RNAediting, which may modulate the sequence and activity of NEIL1.40 hNEIL1 used in this work was the edited form with arginine at position 242 as originally established.19,20,37,38 The unedited form of hNEIL1 with lysine at position 242 has been observed in untreated U87 cells, and it has been overexpressed and purified.40 The unedited and edited forms of hNEIL1 exhibited different glycosylase activities and lesion specificity toward a number of substrates in different sequence contexts. These two forms may be present in mammalian cells under certain conditions. The methodology described in this work may permit the distinction of these two proteins from each other. The tryptic peptide of EVVQLGGR with arginine at position 242 was identified in this work (see Table 1; peak 8 in Figure 2; Supplemental Figures 4 and 8). Replacing arginine with lysine at position 242 would change the mass of the MH+ ion from 857.48 to 829.47 Da and also the masses of all the yions, but not those of the b-ions. Therefore, it would be possible to identify the unedited form of hNEIL1 by the methodology described here. The use of the 15N-labeled edited form of hNEIL1 will also help identify and quantify the unedited form. The identification and quantification of both these forms of hNEIL1 may provide important insight into the extent that editing occurs in mammalian cells. In conclusion, mass spectrometric assays were developed for the identification and quantification of hNEIL1. The full-scan and MS/MS spectra of a large number of tryptic peptides of this protein and its 15N-labeled analogue were described. The use of the fully 15N-labeled hNEIL1 as an internal standard will

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ASSOCIATED CONTENT

S Supporting Information *

Supplemental Tables 1−3 and Supplemental Figures 1−16. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: 100 Bureau Drive, MS 8311, Gaithersburg, MD 20899. Tel: 301-975-2581. Fax: 301-975-8505. E-mail: miral@ nist.gov. Author Contributions #

P.T.R. and P.J. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Certain commercial equipment or materials are identified in this paper in order to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

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REFERENCES

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