Miral Dizdaroglu Measurement of DNA repair proteins in human tissues by liquid chromatography-tandem...
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Miral DizdarogluMiral Dizdaroglu
Measurement of DNA repair proteins in human tissues by Measurement of DNA repair proteins in human tissues by liquid chromatography-tandem mass spectrometry with liquid chromatography-tandem mass spectrometry with
isotope-dilutionisotope-dilution
National Institute of Standards and National Institute of Standards and TechnologyTechnology
Gaithersburg, Maryland, USAGaithersburg, Maryland, USA
N
NN
N
NH2
O
HO
HH
HH
O
NH
N
N
O
NH2N
O
H
HH
HHO
OP
O-
O
O
H
HH
HH
N
N
NH2
O
OP
O-
O
O
P
O-
OO
H
HH
HH
HN
N
O
O
O
O
O
P
O-
O
P
O-
O O
CH3
sugar- phosphate backbone
adenine
guanine
cytosine
thymine
Sites of oxidatively induced damage in DNASites of oxidatively induced damage in DNA
OH, eOH, eaqaq――, H, HOH, eOH, eaqaq――, H, H
OHOHOHOHDNA base damageDNA sugar damage 8,5'-cyclopurine-2'-
deoxynucleosidesTandem lesionsClustered sitesDNA-protein cross-linksSingle- and double-strand breaksAbasic sites
Reviewed in:Dizdaroglu, M. and Jaruga, P., Free Radic. Res. 46, 382-419, 2012
Products of oxidatively induced damage to DNA Products of oxidatively induced damage to DNA bases bases
Reviewed in:Dizdaroglu, M. and Jaruga, P., Free Radic. Res. 46, 382-419, 2012
N
N NH2
H2N
O
2,5-diamino-4H-imidazol-4-one
O
N NH2
H2NO
H2N
2,2,4-triamino-5(2H)-oxazolone
HN
N NH
NHN
N NH2
NHCHO
8-hydroxyguanine 2,6-diamino-4-hydroxy-5-formamidopyrimidine
H2N
OH
O O
H2N
guanine-derived products
NH
NN
N
O
O
HO
HH
HH
O
H
DNA
DNANH2
(5'R)-8,5'-cyclo-2'-deoxyguanosine
NH
NN
N
O
O
HO
HH
HH
O
H
DNA
DNA
NH2
(5'S)-8,5'-cyclo-2'-deoxyguanosine
N
N NH
NN
N NH2
NHCHO
H
N
N NH
N
HO
8-hydroxyadenine 4,6-diamino-5-form-amidopyrimidine
2-hydroxyadenine
NH2
OH
NH2
H
NH2
H
adenine-derived productsN
NN
N
NH2
O
HO
HH
HH
O
H
DNA
DNA
(5'R)-8,5'-cyclo-2'-deoxyadenosine
N
NN
N
NH2
O
HO
HH
HH
H
O
DNA
DNA
(5'S)-8,5'-cyclo-2'-deoxyadenosine
NH
N
NH
N
OHNH
N
NH
N
5-hydroxy-6-hydrocytosine
cytosine glycol 5-hydroxycytosine 5,6-dihydroxy-cytosine
NH2
H
OH
OH
H
NH2
O H
NH2
OHH
O H
OH
NH2
O
OH
OHNH
N
5,6-dihydrocytosine
NH2H
OH
H
H
NH
HNO
5-hydroxy-hydantoin
O OHH
NH
HN
OH
uracil glycol
O
H
H
OH
O
NH
HN
O
H
5,6-dihydrouracil
H
HO
H
NH
HN
5-hydroxyuracil
O
O H
OH
NH
HN
5-hydroxy-6-hydrouracil
O
O H
H
H
OH
NH
HN
alloxan
O
O
O
O NH
HN
isodialuric acid
O
O
OH
O
H
NH
HN
dialuric acid
O
OH
O
OH
cytosine-derived products
NH
HN
O
HNH
HN
O
CH2OH
NH
HNO
NH
HN
O
HNH
HN
OH
thymine glycol
3
5-(hydroxymethyl)- uracil
5,6-dihydrothymine
3
5-hydroxy-5-methylhydantoin
3
5-hydroxy-6-hydro- thymine
NH
HN
O
CHO
5-formyluracil
H
OH
O
CH
H
H
H OOCH
O
CH
OHH
OH
O
O
CH3
HO
thymine-derived products
Reviewed in:Madhusudan, S. and Middleton, M. R., Cancer Treatment Reviews 31, 603-617, 2005.Helleday, T., Petermann, E., Lunding, C., Hodgson, B. and Sharma, R.A., Nature Reviews Cancer 8, 193-204, 2008Helleday, T., European J. Cancer 44, 921-927, 2008Raffoul, J.J., Heydari, S.R. and Hillman, G.G., Journal of Oncology , 2012Kelley, 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 therapyDNA 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 Inhibition of DNA repairrepair
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 pathwayTargeted 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, 193-204, 2008Wilson III, D.M. and Simeonov, A., Cell. Mol. Life Sci. 67, 3621-3631, 2010Kelley, M., DNA Repair in Cancer Therapy, Elsevier, 2012Gad, H., et al. Nature 508, 215-221, 2014
DNA glycosylases
NEIL1, NEIL2, NEIL3, OGG1, NTH1
DNA repair in cancer therapyDNA 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 Apurinic/apyrimidinic endonuclease 1 (APE1)(APE1)
DNA repair activity of APE1 is critical for cell viability.1. Complete absence of APE1 is associated with embryonic lethality in mice2. APE1 depletion hypersensitizes cells to DNA damage3. Overexpression of APE1 protects cells from DNA damage
APE1 expression is increased in human cancers1. Nuclear and/or cytoplasmic overexpression of APE1 occurs in breast, cervical, colon, head & neck, lung, melanoma, ovarian, prostate, etc., cancers2. Increased APE1 expression is associated with resistance to chemo- and radiation
therapies
APE1 as a predictive and prognostic biomarker1. Alterations in APE expression levels and subcellular localization may have predictive
and prognostic significance in many human cancers2. Nuclear localization is associated with good prognostic features3. Cytoplasmic localization is associated with poor survival outcomes
Positive identification and accurate quantification of APE1 in tissues is Positive identification and accurate quantification of APE1 in tissues is essential for its use as an efficient biomarker 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, 2006Abbotts, R. and Madhusudan, S., Cancer Treat Rev. 36, 425-435, 2010
repaired DNA
DNA-damaging agent
APE1
DNA repair activity of APE1DNA 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
APE1AP site
excision of a modified base
Approximately 10000 AP sites are formed per day per cell
spontaneous hydrolysis
base excision repair pathway
Production, isolation and purification of Production, isolation and purification of 1515N-N-hAPE1hAPE1
proteinmarkers
(kDa)
15N-hAPE1
175
80
58
46
30
25
54321
Lane 1: Uninduced cell extractLane 2: Induced cell extractLane 3: 70,000 x g supernatant fractionLane 4: Flow through from DEAE cellulose columnLane 5: Flow through from CM cellulose columnLane 6: Purified 15N-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 15N-hAPE1
proteinmarkers
(kDa)
250
150
100
75
50
37
25
20
35.532 kDa35.970 kDa
SDS-PAGE analysis of hAPE1 and 15N-hAPE1
Measurement of molecular masses of hAPE1 and Measurement of molecular masses of hAPE1 and 1515N-N-hAPE1 by Orbitrap mass spectrometryhAPE1 by Orbitrap mass spectrometry
hAPE1 15N-hAPE1average mass 35554 35992
loss of Met 131 13235423 35860
found 35421 35854difference -2 -6
% labeling99.98
200002100022000230002400025000260002700028000290003000031000320003300034000350003600037000380003900040000Mass 35854
mass-to-charge (m/z)
15N-hAPE1
200002100022000230002400025000260002700028000290003000031000320003300034000350003600037000380003900040000Mass 35421
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 Calculation of fragment masses of tryptic peptides using using NIST Mass and Fragment CalculatorNIST Mass and Fragment Calculator
Measurement of human APE1 by LC-MS/MS with isotope-Measurement of human APE1 by LC-MS/MS with isotope-dilutiondilution
unlabeled peptide 15N-labeled peptide
peak peptide MH+ (M+2H)2+ MH+ (M+2H)2+
1 GLASR 503.2 252.1 511.2 256.1
2 TSPSGKPATLK 1086.6 543.8 1099.6 550.3
3 GLVR 444.2 222.6 452.2 226.6
4 NAGFTPQER 1019.4 510.2 1033.4 517.2
5 NVGWR 631.3 316.1 641.3 321.1
6 GAVAEDGDELR 1131.5 566.2 1145.5 573.2
7 VSYGIGDEEHDQEGR 1690.7 845.8 1711.7 856.3
8 AWIK 517.3 259.1 523.3 262.1
9 EAAGEGPALYEDPPDQK 1786.8 893.9 1805.8 903.4
10 WDEAFR 823.3 412.1 833.3 417.1
11 GLDWVK 717.3 359.1 725.3 363.1
12 EGYSGVGLLSR 1137.5 569.2 1151.5 576.2
13 EEAPDILCLQETK 1488.7 744.8 1503.7 752.3
14 QGFGELLQAVPLADSFR 1847.9 924.4 1869.9 935.4
inte
ns
ity
20e7
40e7
60e7
80e7
100e7
2 4 6 8 10 12 14 16time (min)
1
2
3
4
57
8
9
11
12
13
6 14
10
15N-hAPE1
2 4 6 8 10 12 14 16time (min)
5e7
15e7
25e7
35e7
45e7
inte
ns
ity
12
3
4
5
7
8
9
11
12
13
6
14
10
hAPE1
Total-ion-current profiles of tryptic peptidesTotal-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 ALGSDHCPITLYLAL318
100
600 700 800 900 1000 1100 1200 1300 14000
10
20
30
40
50
60
70
80
90
652.1
1303.1
VQEGETIEDGARVQEGETIEDGAR
(M+2H)2+
MH+
mass-to-charge (m/z)
Rel
ati
ve
Ab
un
da
nc
e (
%)
600 700 800 900 1000 1100 1200 1300 14000
10
20
30
40
50
60
70
80
90
100 659.9
1319.1
15N-VQEGETIEDGAR15N-VQEGETIEDGAR
(M+2H)2+
MH+
mass-to-charge (m/z)
Rel
ati
ve
Ab
un
da
nc
e (
%)
Full scan-mass spectrum of a tryptic peptide of hAPE1 and its Full scan-mass spectrum of a tryptic peptide of hAPE1 and its 1515N-N-labeled analoglabeled analog
659.9
1319.1
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
200 400 600 800 1000 1200 14000
10
20
30
40
50
60
70
80
90
100
rela
tive
ab
un
dan
ce (
%)
805.1
975.4
915.7
595.3
409.2
1087.11216.6
186.0
708.31329.8
1458.8
mass-to-charge (m/z)
333.1
904.4
y5 y6
y7
y8
b2
b3
y9
y3
y10 –NH3
y10
y11
y12
y13
1103.5
a9
614.4
b6 –H2O
501.14b5 –H2O
745.3b7
y7
Q–G–F–G–E–L–L–Q–A–V–P–L–A–D–S–F–R
y3y13 y5y6
b3
y8
b4
y9y10y12 y11
b7
Product ion spectra of a tryptic peptide of hAPE1 and its Product ion spectra of a tryptic peptide of hAPE1 and its 1515N-labeled N-labeled analoganalog
200 400 600 800 1000 1200 14000
10
20
30
40
50
60
70
80
90
100
rela
tive
ab
un
dan
ce (
%)
815.2
925.93415.14 987.5336.6
1231.3
1117.4
717.41345.9
603.04
1476.0
mass-to-charge (m/z)
189.1
915.4
y5
y6
y7
y8
b2
b3 y9y3
y10 –NH3
y10
y11
y12
y13
1099.4
a9
620.0b6 –H2O
506.6b5 –H2O
753.4
b7
y7
15N-Q–G–F–G–E–L–L–Q–A–V–P–L–A–D–S–F–R
y3y13 y5y6
b3
y8
b4
y9y10y12 y11
b7
MH+ m/z 1848.0 (M + 2H)2+ m/z 924.5
Mass transitionsm/z 924.5 → m/z 805.4m/z 924.5 → m/z 904.5m/z 924.5 → m/z 975.5
m/z 924.5 → m/z 1103.6 m/z 924.5 → m/z 1216.7 m/z 924.5 → m/z 1329.8 m/z 924.5 → m/z 1458.8
MH+ m/z 1870.0(M + 2H)2+ m/z 935.5
Mass transitions m/z 935.5 → m/z 815.4 m/z 935.5 → m/z 915.5m/z 935.5 → m/z 987.5
m/z 935.5 → m/z 1117.6 m/z 935.5 → m/z 1231.7 m/z 935.5 → m/z 1345.8 m/z 935.5 → m/z 1475.8
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
Collision energy (V)
Inte
nsi
ty
0 5 10 15 20 25 30 35 40 45 50 55 600
2.0107
4.0107
6.0107
m/z 429 m/z 402
m/z 698 m/z 956
m/z 811 m/z 1041
m/z 763 m/z 679
m/z 503 m/z 695
m/z 315 m/z 288
m/z 322 m/z 272
m/z 508 m/z 603
m/z 382 m/z 564
Determination of optimal collision energiesDetermination of optimal collision energies
Reddy, P.T., Jaruga, P., Kirkali, G., Tuna, G., Nelson, B.C. and Dizdaroglu, M., J. Proteome Res. 12, 1049-1061, 2013
m/z
Co
llis
ion
en
erg
y (
V)
300 400 500 600 700 8000
10
20
30
40
0
100
200
300
400
500
600
Time (min)
10 12 14 16 18 20 22 24 26
cytoplasmic extractMCF-7 cells
Time (min)
10 12 14 16 18 20 22 24 26
100
150
200
250
300
350
400
450
500 nuclear extractMCF-7 cells
Enrichment of APE1 by HPLC from protein extracts of human cellsEnrichment of APE1 by HPLC from protein extracts of human cells
hAPE1
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
Inte
nsi
ty
5000
100000
1000
3000
0
10000
20000
0
5000
15000
0
50000
0
50000
0
10000
20000
0
10000
20000
4.84
4.84
5.14
5.13
5.30
5.30
7.06
7.04
m/z 510.2 → m/z 834.4
NAGFTPQER
m/z 517.2 → m/z 845.4
15N-NAGFTPQER
m/z 566.3 → m/z 904.4
GAVAEDGDELR
m/z 573.3 → m/z 915.4
15N-GAVAEDGDELR
m/z 893.9 → m/z 584.3
EAAGEGPALYEDPPDQK
m/z 903.4 → m/z 591.3
15N-EAAGEGPALYEDPPDQK
m/z 316.2 → m/z 418.2
NVGWR
m/z 321.2 → m/z 425.2
15N-NVGWR
0 2 4 6 8 10 12 14 16Time (min)
0
7.36
0 2 4 6 8 10 12 14 16Time (min)
0
2000
4000
0
1000
3000
0
10000
30000
0
20000
40000
0
50000
1000000
20000
40000
0
10000
20000
0
5000
15000
7.37
8.27
8.27
8.80
8.80
14.70
14.69
m/z 359.2 → m/z 547.3
GLDWVK
m/z 363.2 → m/z 553.3
15N-GLDWVK
m/z 569.3 → m/z 545.3
EGYSGVGLLSR
m/z 576.3 → m/z 553.3
15N-EGYSGVGLLSR
m/z 924.5 → m/z 805.4
QGFGELLQAVPLADSFR
m/z 935.5 → m/z 815.4
15N-QGFGELLQAVPLADSFR
m/z 412.2 → m/z 637.3
WDEAFR
m/z 417.2 → m/z 645.3
15N-WDEAFR
Inte
nsi
ty
Identification and quantification of APE1 in human MCF-10A cellsIdentification 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 Identification and quantification of APE1 in human cultured cells and mouse livermouse 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
0
1
2
3
4
hA
PE
1 l
ev
el (
pg
/ g
pro
tein
)
MCF-10A
MCF-7
HepG-2
MCF-10A: mammary gland epithelial cell lineMCF-7: mammary gland epithelial adenocarcinoma
cell lineHepG-2: hepatocellular carcinoma cell line
hA
PE
1 le
vel
(ng
/μg
pro
tein
)
p 0.0001
p 0.0001
p 0.0015
p 0.0001
p 0.0001
Levels of hAPE1 in human normal and cancer cell lines
p 0.0001
0.00
0.04
0.08
0.12
mA
PE
1 l
ev
el
(pg
/ g
pro
tein
)h
AP
E1
leve
l (n
g/μ
g p
rote
in)
Levels of hAPE1 in mouse liver
Identification and quantification of APE1 in human breast Identification and quantification of APE1 in human breast tissuestissues
Unpublished results
p 0.0001
normal cancer0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
AP
E1
leve
l (n
g/
g p
rote
in)
Levels of hMTH1 in human disease-free breast tissues and malignant breast tumors
disease-free breast tissue malignant breast tissue
Sanitation of the nucleotide Sanitation of the nucleotide poolpool
MTH1MTH1 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-dGTPMTH1
hydrolysis8-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 therapyInhibition of MTH1 in cancer therapy
Nature, 508, 215-221, 2014
Production, isolation and purification of Production, isolation and purification of 1515N-N-hNTH1hNTH1
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 15N-hMTH1
Separation of hMTH1 and 15N-hMTH1 by HPLC. The elution profiles were superimposed.
Measurement of the masses of hMTH1 and Measurement of the masses of hMTH1 and 1515N-hMTH1 by QToF N-hMTH1 by QToF LC/MSLC/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 15N-labeled
peak tryptic peptide MH+ (M+2H)2+ MH+ (M+2H)2+
1 GFGAGR 564.2 282.6 573.2 287.1
2 VQEGETIEDGAR 1303.6 652.3 1319.5 660.2
3 FHGYFK 798.3 399.7 807.3 404.2
4 WNGFGGK 765.3 383.1 775.3 388.2
5 VLLGMK 660.4 330.7 667.3 334.2
6 ELQEESGLTVDALHK 1668.8 834.9 1687.7 844.4
7 FQGQDTILDYTLR 1569.7 785.4 1587.7 794.4
Measurement of human MTH1 by LC-MS/MS with isotope-Measurement of human MTH1 by LC-MS/MS with isotope-dilutiondilution
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 peptidesTotal-ion-current profiles of tryptic peptides
hMTH1
15N-hMTH1
Sequence of hMTH1 p18 isoform
molecular mass 17.95 kDa
156
Determination of optimal collision energiesDetermination 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 Identification and quantification of MTH1 in human cultured cellscultured 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 cells MCF-7 cells
MCF-1
0A
MCF-7
HeLa
HepG2
0.00
0.05
0.10
0.15
0.20
leve
l of
MT
H1
(ng
/ g
pro
tein
)
Levels of hMTH1 in human normal and cancer cell lines
p 0.0001
p 0.0001
MCF-10A: mammary gland epithelial cell lineMCF-7: mammary gland epithelial
adenocarcinoma cell lineHeLa: cervix epithelial adenocarcinoma
cell lineHepG-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 Identification and quantification of MTH1 in human breast tissuestissues
A: disease-free breast tissuesB: malignant breast tumors
Levels of hMTH1 in human disease-free breast tissues and malignant breast tumors
normal cancer0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
leve
l o
f M
TH
1 (n
g/
g p
rote
in)
p 0.0001
ConclusionsConclusions
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, NISTErdem Coskun, NISTGüldal Kirkali, NIST and
NIHPrasad T. Reddy, NISTBryant C. Nelson, NISTMark S. Lowenthal, NISTLeona D. Scanlan, NISTAlex Tona, NISTGamze Tuna, NISTThomas Helleday, SwedenAnn-Sofie Jemth, SwedenOlga Loseva, Sweden
Collaborators Collaborators
Thank youThank you
National Institute of Standards and National Institute of Standards and TechnologyTechnology
Gaithersburg, Maryland, USAGaithersburg, Maryland, USA