1. The document describes methods for measuring DNA damage and protein expressions using mass spectrometry. DNA damage from radiation exposure was measured in samples with varying radiation dosages, showing a linear relationship between damage and dosage up to a saturation point. 2. Both gas chromatography/tandem mass spectrometry and liquid chromatography/tandem mass spectrometry were used to measure specific DNA lesions and repair proteins, respectively. Stable isotope labeled standards were added for accurate quantification. 3. Preliminary results found increasing levels of certain DNA lesions corresponding to increased radiation dosage, with some lesions leveling off at higher doses. Future work will apply these methods to study effects of other exposures like nanoparticles.

1. • At the end of each trial, the subjects recorded the accuracy of their
category guess.
Measuring DNA damage and Protein Expressions with
Mass Spectrometry
Zach Kasica1,2, Erdem Coskun1, Pawel Jaruga1 & Miral Dizdaroglu1
1 Biomolecular Measurement Division, MML, NIST, Gaithersburg, MD
2Saint Johns Catholic Prep, Buckeystown, MD
o High-energy ionizing radiation has been linked to the formation
of free radicals within a cell.
o Free radicals have the capacity to cause DNA damage. These
highly reactive molecules bind to DNA bases and cause
structural changes in the DNA, called lesions.
o Cells are equipped with DNA repair proteins that fix the
damaged DNA. There is strong evidence that DNA that remains
damaged plays a fundamental role in carcinogenesis.
Introduction
Figure 1: Free radical formation and DNA damage
Aims
1. To positively identify and accurately
quantify DNA lesions based upon
different levels of ionizing radiation by
GC/MS-MS with isotope dilution using
stable isotope (13C, 15N)-labeled DNA
lesion standards.
2. To positively identify and accurately
quantify DNA repair proteins such as
APE1 and MTH1 in human cell lines and
tissues by LC/MS-MS with isotope dilution
using stable isotope (15N)-labeled protein
standards.
Our Methods
Measuring DNA Repair ProteinsMeasuring DNA Damage
o In order to measure DNA damage, we analyzed the amounts of four
different types of lesions. Our lesions were analyzed from five sets of
DNA:
o Control group (no radiation)
o 5 Gy dosage of radiation
o 10 Gy dosage of radiation
o 20 Gy dosage of radiation
o 40 Gy dosage of radiation
Figure 3: Our four DNA lesions for analysis. Abbreviations (L-R): 5-OH-Cyt, 8-OH-Gua, FapyGua, FapyAde
Sample
Standard
Figure 4: Total Ion Chromatogram (TIC). This displays all the peaks the GC/MS-MS collected for one set of DNA.
Figure 5b: Maintaining a consistent baseline is essential
to producing accurate results.
Figure 5a: We calculate the amount of lesions in
each sample by using the ratio between the areas
of the sample and the internal standard.
DNA Damage Results
0.0000
1000.0000
2000.0000
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6000.0000
7000.0000
0 5 10 20 40
Lesionsper106DNAbases
Radiation (Gy)
8-OH-Gua
0.0000
20.0000
40.0000
60.0000
80.0000
100.0000
120.0000
140.0000
160.0000
180.0000
0 5 10 20 40
Lesionsper106DNAbases
Radiation (Gy)
FapyGua
0.0000
20.0000
40.0000
60.0000
80.0000
100.0000
120.0000
140.0000
160.0000
180.0000
0 5 10 20 40
Lesionsper106DNAbases
Radiation (Gy)
FapyAde
0.0000
20.0000
40.0000
60.0000
80.0000
100.0000
120.0000
140.0000
0 5 10 20 40
Lesionsper106DNAbases
Radiation (Gy)
5-OH-Cyt
Figure 6: (Left) MTH1 and 15N-labeled MTH1 internal standard elute at the same time.
(Right) A closer look at the molecular structure of MTH1.
Figure 7a: TIC profiles of tryptic peptides from
MTH1 (A) and 15N-labeled MTH1 internal
standards (B).
Photo Credit: RCSB Protein Data Bank
Figure 7b: Mass spectra of the peptide
VQEGETIEDGAR (A) and its 15N-labeled internal
standard (B), both represented by peak 2 on A
and B respectively on the left.
Figure 8: Superimposed elution profiles of hundreds of proteins from four cell lines. Collecting only during
the retention time period of MTH1 allows us to enrich our sample with MTH1 and filter out other
proteins.
Conclusions
o We use isotope-dilution methods with tandem mass
spectrometry in our lab
o We add a known amount of a stable isotope labeled
internal standard to each sample before analysis
o To measure DNA lesions, we use gas
chromatography/tandem mass spectrometry
(GC/MS-MS).
o To measure DNA repair proteins, we use liquid
chromatography/ tandem mass spectrometry
(LC/MS-MS).
Figure 2: Diagram of GC/MS. After molecules are separated in the gas column, they are
fragmented and ionized in the MS. Our lab uses tandem MS for further precision.
Separates
molecules
based on their
chemical and
physical
properties
Measures
DNA lesions
Measures DNA
repair proteins
GC/MS-MS LC/MS-MSBoth
Liquid phaseGas phase
o From the DNA damage study we conclude:
o There is a linear dependence between
DNA lesion amount and radiation dosage
o 8-OH-G and FapyGua lesion amounts
level off because the radiation had
destroyed the cell.
o GC/MS-MS delivers accurate and precise
DNA lesion data
o Science is cool.
o Study the effects of ionizing radiation on DNA
repair protein expression
o Study how TiO2 nanoparticle exposure affects
expression levels of APE1 and MTH1
o Our ultimate goal is to evolve our method for
use as a quick screening test for cancer patients
to predict the most suitable treatment schemes
and as a biomarker to evaluate specific cancer
risks in healthy individuals
Figure 9: Mass transitions of four tryptic peptides from hMTH1 and 15N-labeled internal standard
hMTH1 that were extracted from cancer cells (A) and non-cancer cells (B). The internal standards were
added to the protein extracts prior to trypsin digestion.
Future Directions
Photo Credit: theatlantic.com
Photo Credit: chromacademy.com