Miral Dizdaroglu Measurement of DNA repair proteins in human tissues by liquid chromatography-tandem mass spectrometry with isotope-dilution National Institute of Standards and Technology Gaithersburg, Maryland, USA

Sites of oxidatively induced damage in DNA  OH, e aq ―, H   OH DNA base damage DNA sugar damage 8,5'-cyclopurine-2'-deoxynucleosides Tandem lesions Clustered sites DNA-protein cross-links Single- and double-strand breaks Abasic sites Reviewed in: Dizdaroglu, M. and Jaruga, P., Free Radic. Res. 46, , 2012

Products of oxidatively induced damage to DNA bases Reviewed in: Dizdaroglu, M. and Jaruga, P., Free Radic. Res. 46, , 2012 guanine-derived products (5'R)-8,5'-cyclo-2'-deoxyguanosine(5'S)-8,5'-cyclo-2'-deoxyguanosine adenine-derived products (5'R)-8,5'-cyclo-2'-deoxyadenosine(5'S)-8,5'-cyclo-2'-deoxyadenosine cytosine-derived products thymine-derived products

Reviewed in: Madhusudan, S. and Middleton, M. R., Cancer Treatment Reviews 31, , Helleday, T., Petermann, E., Lunding, C., Hodgson, B. and Sharma, R.A., Nature Reviews Cancer 8, , 2008 Helleday, T., European J. Cancer 44, , 2008 Raffoul, J.J., Heydari, S.R. and Hillman, G.G., Journal of Oncology, 2012 Kelley, M., DNA Repair in Cancer Therapy, Elsevier, 2012 One important mechanism by which cancer cells can develop resistance to therapy is to increase their DNA repair capacity. DNA repair in cancer therapy The efficacy of anticancer drugs and radiation can be reduced in cancer cells by increased DNA repair that remove DNA lesions before they become toxic. DNA repair pathways are promising targets for novel cancer treatments Inhibition of DNA repair

Poly(ADP-ribose) polymerase 1 (PARP1) PARP1 is required for the efficient repair of AP sites and single-stranded DNA breaks. Cells deficient in BRCA1 or BRCA2 are highly sensitive to PARP1 inhibition. Apurinic/apyrimidic endonuclease 1 (APE1) APE1 hydrolyzes the phosphate bond at 5' to AP site, causing a strand breaks and leaving a 3'-OH group and a 5‘-deoxyribose-phosphate terminus. Targeted DNA repair proteins in base excision repair pathway MTH1 MTH1 dephosporylates modified 2‘-deoxynucleoside triphosphates in the nucleotide pool to prevent incorporation of DNA lesions during DNA replication. Reviewed in: Helleday, T., Petermann, E., Lunding, C., Hodgson, B. and Sharma, R.A., Nature Reviews Cancer 8, , 2008 Wilson III, D.M. and Simeonov, A., Cell. Mol. Life Sci. 67, , 2010 Kelley, M., DNA Repair in Cancer Therapy, Elsevier, 2012 Gad, H., et al. Nature 508, , 2014 DNA glycosylases NEIL1, NEIL2, NEIL3, OGG1, NTH1

DNA repair in cancer therapy To use DNA repair proteins as disease biomarkers or to determine the DNA repair capacity in tissues, the measurement of the levels of DNA repair proteins in vivo will be necessary. Mass spectrometric techniques with isotope-dilution will be the techniques of choice for accurate measurement of DNA repair proteins in tissues. “A knowledge of DNA repair proteins’ overexpression or underexpression in cancers will help predict and guide development of treatments, and yield the greatest therapeutic response.” Kelley, M. R., DNA Repair in Cancer Therapy, Elsevier, 2012

Apurinic/apyrimidinic endonuclease 1 (APE1) Apurinic/apyrimidinic endonuclease 1 (APE1) DNA repair activity of APE1 is critical for cell viability. 1. Complete absence of APE1 is associated with embryonic lethality in mice 2. APE1 depletion hypersensitizes cells to DNA damage 3. Overexpression of APE1 protects cells from DNA damage APE1 expression is increased in human cancers 1. Nuclear and/or cytoplasmic overexpression of APE1 occurs in breast, cervical, colon, head & neck, lung, melanoma, ovarian, prostate, etc., cancers 2. Increased APE1 expression is associated with resistance to chemo- and radiation therapies APE1 as a predictive and prognostic biomarker 1. Alterations in APE expression levels and subcellular localization may have predictive and prognostic significance in many human cancers 2. Nuclear localization is associated with good prognostic features 3. Cytoplasmic localization is associated with poor survival outcomes Positive identification and accurate quantification of APE1 in tissues is essential for its use as an efficient biomarker Positive identification and accurate quantification of APE1 in tissues is essential for its use as an efficient biomarker Reviewed in: Friedberg, E. C., Walker, G. C., Siede, W., Wood, R. D., Schultz, R. A. and Ellenberger, T., DNA Repair and Mutagenesis, 2006 Abbotts, R. and Madhusudan, S., Cancer Treat Rev. 36, , 2010

repaired DNA DNA-damaging agent APE1 DNA repair activity of APE1 DNA repair activity of APE1 Reviewed in: Friedberg, E. C., Walker, G. C., Siede, W., Wood, R. D., Schultz, R. A. and Ellenberger, T., DNA Repair and Mutagenesis, 2006 APE1 hydrolyzes the O-P bond 5' to the AP site, yielding 2'-deoxyribose- 5'-phosphate and 3'-OH end APE1 AP site excision of a modified base Approximately AP sites are formed per day per cell spontaneous hydrolysis base excision repair pathway

Production, isolation and purification of 15 N-hAPE1 protein markers (kDa) 15 N-hAPE Lane 1: Uninduced cell extract Lane 2: Induced cell extract Lane 3: 70,000 x g supernatant fraction Lane 4: Flow through from DEAE cellulose column Lane 5: Flow through from CM cellulose column Lane 6: Purified 15 N-hAPE1 6 Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013 hAPE1 15 N-hAPE1 protein markers (kDa) kDa kDa SDS-PAGE analysis of hAPE1 and 15 N-hAPE1

Measurement of molecular masses of hAPE1 and 15 N-hAPE1 by Orbitrap mass spectrometry mass-to-charge (m/z) 15 N-hAPE1 mass-to-charge (m/z) hAPE1 Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013

Calculation of fragment masses of tryptic peptides using NIST Mass and Fragment Calculator

Measurement of human APE1 by LC-MS/MS with isotope-dilution unlabeled peptide 15 N-labeled peptide peakpeptide MH + (M+2H) 2+ MH + (M+2H) 2+ 1GLASR TSPSGKPATLK GLVR NAGFTPQER NVGWR GAVAEDGDELR VSYGIGDEEHDQEGR AWIK EAAGEGPALYEDPPDQK WDEAFR GLDWVK EGYSGVGLLSR EEAPDILCLQETK QGFGELLQAVPLADSFR intensity 20e7 40e7 60e7 80e7 100e time (min) N-hAPE time (min) 5e7 15e7 25e7 35e7 45e7 intensity hAPE1 Total-ion-current profiles of tryptic peptides Identified peptides Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013 Molecular mass 35.5 kDa Sequence of hAPE1 MPKRGKK GAVAEDGDELR TEPEAK K SK TAAK K NDK EAAGEGPALYEDPPDQK TSPSGKPATLK ICSWNVDGLR AWIK K K GLDWVK EEAPD ILCLQETK CSENK LPAELQELPGLSHQYWSAPSDK EGYSGVGLLSR QCPLK VSYGIGDEEHDQEGR VIVAEFDSFVLVTAYVPNAGR GLVR LEYR QR WDEAFR K FLK GLASR KPLVLCGDLNVAHEEIDLR NPK GNK K NAGFTPQER QGFGELLQAVPLADSFR HLYPNTPYAYTFWTYMMNAR SK NVGWR LDYFLLSHSLLPALCDSK IR SK ALGSDHCPITLYLAL 318

VQEGETIEDGAR (M+2H) 2+ MH + mass-to-charge (m/z) Relative Abundance (%) N-VQEGETIEDGAR (M+2H) 2+ MH + mass-to-charge (m/z) Relative Abundance (%) Full scan-mass spectrum of a tryptic peptide of hAPE1 and its 15 N-labeled analog Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013

relative abundance (%) mass-to-charge (m/z) y5y5 y6y6 y7y7 y8y8 b2b2 b3b3 y9y9 y3y3 y 10 –NH 3 y 10 y 11 y 12 y a9a b 6 –H 2 O b 5 –H 2 O b7b7 y7y7 Q–G–F–G–E–L–L–Q–A–V–P–L–A–D–S–F–R y3y3 y 13 y5y5 y6y6 b3b3 y8y8 b4b4 y9y9 y 10 y 12 y 11 b7b7 Product ion spectra of a tryptic peptide of hAPE1 and its 15 N-labeled analog relative abundance (%) mass-to-charge (m/z) y5y5 y6y6 y7y7 y8y8 b2b2 b3b3 y9y9 y3y3 y 10 –NH 3 y 10 y 11 y 12 y a9a b 6 –H 2 O b 5 –H 2 O b7b7 y7y7 15 N- Q–G–F–G–E–L–L–Q–A–V–P–L–A–D–S–F–R y3y3 y 13 y5y5 y6y6 b3b3 y8y8 b4b4 y9y9 y 10 y 12 y 11 b7b7 MH + m/z (M + 2H) 2+ m/z Mass transitions m/z → m/z m/z → m/z m/z → m/z m/z → m/z m/z → m/z m/z → m/z m/z → m/z MH + m/z (M + 2H) 2+ m/z Mass transitions m/z → m/z m/z → m/z m/z → m/z m/z → m/z m/z → m/z m/z → m/z m/z → m/z Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013

M. Kinter and N.E. Sherman, Protein Sequencing and Identification Using Tandem Mass Spectrometry, Wiley, 2000 Determination of optimal collision energies Reddy, P.T., Jaruga, P., Kirkali, G., Tuna, G., Nelson, B.C. and Dizdaroglu, M., J. Proteome Res. 12, , 2013

Time (min) Absorbance (220 nm) cytoplasmic extract MCF-7 cells Time (min) Absorbance (220 nm) nuclear extract MCF-7 cells Enrichment of APE1 by HPLC from protein extracts of human cells hAPE1 Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013

Intensity m/z → m/z NAGFTPQER m/z → m/z N-NAGFTPQER m/z → m/z GAVAEDGDELR m/z → m/z N-GAVAEDGDELR m/z → m/z EAAGEGPALYEDPPDQK m/z → m/z N-EAAGEGPALYEDPPDQK m/z → m/z NVGWR m/z → m/z N-NVGWR Time (min) Time (min) m/z → m/z GLDWVK m/z → m/z N-GLDWVK m/z → m/z EGYSGVGLLSR m/z → m/z N-EGYSGVGLLSR m/z → m/z QGFGELLQAVPLADSFR m/z → m/z N-QGFGELLQAVPLADSFR m/z → m/z WDEAFR m/z → m/z N-WDEAFR Intensity Identification and quantification of APE1 in human MCF-10A cells Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013

Identification and quantification of APE1 in human cultured cells and mouse liver Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013 MCF-10A MCF-7 HepG-2 MCF-10A:mammary gland epithelial cell line MCF-7: mammary gland epithelial adenocarcinoma cell line HepG-2:hepatocellular carcinoma cell line hAPE1 level (ng/μg protein) p  p  p  Levels of hAPE1 in human normal and cancer cell lines p  hAPE1 level (ng/μg protein) Levels of hAPE1 in mouse liver

Identification and quantification of APE1 in human breast tissues Unpublished results p  Levels of hMTH1 in human disease-free breast tissues and malignant breast tumors disease-free breast tissuemalignant breast tissue

Sanitation of the nucleotide pool MTH1 MTH1 hydrolyzes oxidized 2’-deoxynucleoside triphosphates to monophosphates in the nucleotide pool. As a result, DNA polymerases cannot insert the wrong base across from the normal base, maintaining transcription fidelity, thus inhibiting mutagenesis. 8-OH-dGMP8-OH-dGTP MTH1 hydrolysis 8-OH-dG nucleotidase 8-OH-dGMP cannot be rephosphorylated by guanylate kinase, which phosphorylates dGMP. Reviewed in: Friedberg, E. C., Walker, G. C., Siede, W., Wood, R. D., Schultz, R. A. and Ellenberger, T., DNA Repair and Mutagenesis, 2006

Inhibition of MTH1 in cancer therapy Nature, 508, , 2014

Production, isolation and purification of 15 N-hNTH1 Coskun, E., Jaruga, P., Jemth, A.-S., Loseva, O., Scanlan, L.D., Tona, A., Lowenthal, M.S., Helleday, T. and Dizdaroglu, M., DNA Repair ( in press). SDS-PAGE analysis of hMTH1 and 15 N-hMTH1 Separation of hMTH1 and 15 N-hMTH1 by HPLC. The elution profiles were superimposed.

Measurement of the masses of hMTH1 and 15 N-hMTH1 by QToF LC/MS Coskun, E., Jaruga, P., Jemth, A.-S., Loseva, O., Scanlan, L.D., Tona, A., Lowenthal, M.S., Helleday, T. and Dizdaroglu, M., DNA Repair ( in press).

unlabeled 15 N-labeled peaktryptic peptideMH + (M+2H) 2+ MH + (M+2H) 2+ 1GFGAGR VQEGETIEDGAR FHGYFK WNGFGGK VLLGMK ELQEESGLTVDALHK FQGQDTILDYTLR Measurement of human MTH1 by LC-MS/MS with isotope-dilution Sequences of the identified peptides Coskun, E., Jaruga, P., Jemth, A.-S., Loseva, O., Scanlan, L.D., Tona, A., Lowenthal, M.S., Helleday, T. and Dizdaroglu, M., DNA Repair ( in press). Total-ion-current profiles of tryptic peptides hMTH1 15 N-hMTH1 Sequence of hMTH1 p18 isoform molecular mass kDa 156

Determination of optimal collision energies Coskun, E., Jaruga, P., Jemth, A.-S., Loseva, O., Scanlan, L.D., Tona, A., Lowenthal, M.S., Helleday, T. and Dizdaroglu, M., DNA Repair ( in press).

Identification and quantification of MTH1 in human cultured cells Coskun, E., Jaruga, P., Jemth, A.-S., Loseva, O., Scanlan, L.D., Tona, A., Lowenthal, M.S., Helleday, T. and Dizdaroglu, M., DNA Repair ( in press). MCF-10A cellsMCF-7 cells Levels of hMTH1 in human normal and cancer cell lines p  MCF-10A:mammary gland epithelial cell line MCF-7: mammary gland epithelial adenocarcinoma cell line HeLa:cervix epithelial adenocarcinoma cell line HepG-2:hepatocellular carcinoma cell line

Coskun, E., Jaruga, P., Jemth, A.-S., Loseva, O., Scanlan, L.D., Tona, A., Lowenthal, M.S., Helleday, T. and Dizdaroglu, M., DNA Repair ( in press). Identification and quantification of MTH1 in human breast tissues A: disease-free breast tissues B: malignant breast tumors Levels of hMTH1 in human disease-free breast tissues and malignant breast tumors p 

Conclusions Oxidative stress caused in vivo by endogenous and exogenous DNA-damaging agents leads to the formation of a plethora of lesions in DNA. DNA lesions are repaired in vivo by a variety of DNA repair mechanisms. Cancer cells resist to therapy by greater DNA repair capacity than in normal cells. DNA repair proteins are promising targets for novel cancer treatments. DNA repair inhibitors are being developed worldwide as potential drugs. Accurate measurement of DNA repair proteins’ overexpression or underexpression in cancers may help predict and guide development of treatments, and yield the greatest therapeutic response. LC-MS/MS with isotope-dilution using stable isotope-labeled analogs is well suited for the positive identification and accurate quantification of DNA repair proteins in human tissues.

Pawel Jaruga, NIST Erdem Coskun, NIST Güldal Kirkali, NIST and NIH Prasad T. Reddy, NIST Bryant C. Nelson, NIST Mark S. Lowenthal, NIST Leona D. Scanlan, NIST Alex Tona, NIST Gamze Tuna, NIST Thomas Helleday, Sweden Ann-Sofie Jemth, Sweden Olga Loseva, Sweden Collaborators

Thank you National Institute of Standards and Technology Gaithersburg, Maryland, USA