PhD thesis presentation 2012

CHAPTER 3

Mapping of sites of base modification of isomeric Oligonucleotide adducts and investigating the

sequence specificity of the carcinogens using LC-MS/MS

CHAPTER 2

Ion-pair Reversed-phase liquid chromatography electrospray ionization tandem mass spectrometry

method development for separation and sequencing of isomeric Oligonucleotide adducts

CHAPTER 1

DNA adducts and cancer: a perspective

CHAPTER 4

GenoMass software: a tool based on electrospray ionization tandem mass spectrometry for

characterization and sequencing of Oligonucleotide adducts

CHAPTER 5

Future Research Perspectives

Liquid Chromatography - Tandem Mass Spectrometry Methods For

The Analysis Of Isomeric Oligonucleotide Adducts

Vaneet Kumar Sharma

November 29, 2012

CHAPTER 5


‘transrenal’ DNA

LC-MS/MS data

Oligonucleotide

Sequencing software

Gene identification

Oligonucleotide fragments (12-20)

Nucleotide

Basic Local Alignment Search

Tool (BLAST)

To develop LC- MS/MS based analytical platform for the risk assessment of DNA adducts

Methodology that incorporates mass spectrometry coupled to online separation techniques and

sequencing software will be a significant step forward in probing the risk assessment of DNA

adducts

CHAPTER 1

DNA adducts and cancer: a perspective

What is the relationship between DNA adducts and cancer?

Exogenous chemical are the chemical agents penetrated by respiratory, digestive,

cutaneous or other possible contamination routes in human body to form DNA adducts

Exogenous chemical can either directly react or require metabolic activation to form

electrophilic reactive species that covalently binds to nucleophilic sites in DNA

Not all DNA adducts result in mutation and not all mutations are in critical genes

Exogenous chemical carcinogens show a degree of mutational site specificity, either in

adduct formation or in repair of these adducts

Detection and quantification of DNA adducts, alone, is not sufficient, to be most useful in

risk assessment, the DNA adducts should be structurally identified and their mutagenic

capabilities defined

Exogenous chemical carcinogenesis is an extremely complex multifactorial process

that requires multiple steps or key events over a number of years

Methylating agents

Ethylating agents

Ethylene

Butadiene

Acrylamide

7,12-dimethylbenz[a]anthracene

Styrene oxide

Phenyl glycidyl ether

Aflatoxin

Estrone

Hydroxyl radicals

IQ

PHIP

MeIQx

Ethanol

4-Aminobiphenyl

Peroxynitrite

Ethylating agents


Lipid peroxidation products

-malondialdehyde

-4-hydroxy-2-nonenal

-crotonaldehyde

-2-hexenal

Vinyl chloride

Ethanol

Estrogen

N-Nitrosodiethanolamine

IQ

PHP

MeIQx

Tamoxifen

Ethanol


Benzo{a}pyrene

N-Nitrosodiethanolamine

N-methyl-N-nitrosourea

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Lipid peroxidation

products

-4-hydroxy-2-nonenal

N-nitroso compounds (NOCs) PAHs

Aflatoxins Heterocyclic aromatic amines (HAA)

1. 32P-Postlabeling

2. Immunoassays

3. Fluorescence Spectroscopy

4. Mass Spectrometry Based Methods

Gas Chromatography – Mass Spectrometry (GC-MS)

Capillary Electrophoresis - Mass Spectrometry (CE-MS)

Liquid Chromatography – Mass Spectrometry (LC-MS)

The analysis of DNA adducts: The transition from 32P-postlabeling to mass spectrometry

Joshua J. Klaene, Vaneet K. Sharma, James Glick, Paul Vouros,

Cancer letters, 2012 (DOI: 10.1016/j.canlet.2012.08.007)

The analytical challenge to probe the role of DNA adducts in initiation and

progression of cancers

In the last few years, liquid chromatography interfaced with mass spectrometry [LC-MS]

has emerged as a central analytical technique for the characterization of DNA adducts

In addition to the chemical nature and exposure quantification, the exact position of the

adducts within the DNA may play a role in the risk assessment of the carcinogens

CHAPTER 2

Separation and sequencing of isomeric Oligonucleotide adducts using monolithic

PS-DVB capillary column and Ion-pair Reversed-phase liquid chromatography

electrospray ionization tandem mass spectrometry

Reversed-phase ion-pair liquid chromatography electrospray ionization tandem mass spectrometry for

separation, sequencing and mapping of sites of base modification of isomeric Oligonucleotide adducts using

monolithic column.

Vaneet K. Sharma, James Glick, Paul Vouros, Journal of Chromatography A, (2012), 1245, 65-74

1) Oligonucleotide adduct ?

2) Separation mode ?

3) Column/stationary phase ?

4) LC-MS mobile phase conditions?

5) Oligonucleotide sequencing ?

6) Structural identification ?

7) Chromatographic efficiency ?

To develop high-resolution ion pairing reversed phase liquid chromatography

electrospray ionization tandem mass spectrometry (IP-RP-LC–ESI-MS/MS) method for

separation and sequencing of isomeric Oligonucleotide adducts

N-acetoxy-2-acetylaminoflourene

(AAAF)

Oligonucleotide adduct ?

CCC CGA GCA ATC TCA AT


AAF

AAF

Positional isomers have

same m/z

identical composition

identical oligonucleotide backbone

A single stranded (ss) synthetic Oligonucleotide (CCC CGA GCA ATC TCA AT) adducted with N-

acetoxy-2-acetylaminoflourene [AAAF] was used as a model Oligonucleotide adduct

2-Aminofluorene, has been investigated extensively to understand the role of arylamines in cancer biology.

N-acetoxy-2-acetylaminofluorene (AAAF) is active metabolite of 2-aminofluorene


Anion exchange chromatography is incompatible with ESI-

mass spectrometry i.e. mobile phase contains high

concentration of nonvolatile salts or high strength buffers

Oligonucleotides have negatively charged backbone, thus

they are poorly retained on the non-polar stationary phases

of the reversed phase column

Separation mode ?

N

N

HN

NH

N

N

N

N

Triethylamine (TEA) Tripropylamine (TPA) trans-N,N dimethylcyclohexane-1,2 -

diamine

N,N- dimethylbutylamine

(DMBA)

N,N- dimethylcyclohexylamine

(DMcHA)

1,8-diazabicyclo[5.4.0]undec-7ene

(DBU)

N

N

HN

NH

N

N

N

N


diamine


(DMBA)


(DMcHA)


(DBU)

NH2

Hexylamine (HxA)

N

N

HN

NH

N

N

N

N


diamine


(DMBA)


(DMcHA)


(DBU)

N

N

HN

NH

N

N

N

N


diamine


(DMBA)


(DMcHA)


(DBU)

N

N

HN

NH

N

N

N

N


diamine


(DMBA)


(DMcHA)


(DBU)

N

N

HN

NH

N

N

N

N


diamine


(DMBA)


(DMcHA)


(DBU)

Ion pairing reagent possess dual functionality i.e. positively charged amine and a

hydrophobic component in there structure

Amine used as cationic ion pairing reagent

Anion exchange chromatography has been the most widely used mode

for oligonucleotide analysis because of negatively charged backbone

Reversed-phase liquid chromatography is nowadays the most commonly

used interface with ESI-MS

Column/stationary phase ?

Filled capillary, Thermal initialization

Styrene, divinyl benzene, Decanol, THF, AIBN,

70 °C/ 24 hour Polymerization

Silanization process 3-(trimethoxysilyl) propyl methacrylate/ NaOH

2,2- diphenyl- 1- picrylhydrazyl hydrate/ DMF,

120 °C/ 6 hour

Inner wall silanization

Contact angle (θ) > 80

Polyimide-coated fused silica capillary

Monolithic PS-DVB capillary column

0.25X95 mm

Monolithic column mm

Mobile phase , FM

Viscosity

[mobile

phase]

η, (Pa.s)

Flow rate μL/min

Back pressure Δp,(Pa)

Vc Empty column μL

εo interstitial (flow-through pore) porosity

εi inner (mesopore) porosity

KF column permeability m

dp pore size [mesopore] μm

PS-DVB 0.25X95

50/50 CH3OH/ water

1.5X10-3 6.0 98X105 4.66 0.82 0.08 2.96X10-14 0.709

Characterization

Acetonitrile Washing

LC-MS mobile phase conditions?

Various ion pairing reagents under similar conditions were evaluated not only

for their effect on retention time and relative MS sensitivity but more

importantly in terms of their ability to separate positional isomers

Evaluate the effect of

concentration of ion pairing reagent

flow rate

gradient conditions

organic modifier

mobile phase additives

on the LC-MS separation of AAF adducted oligonucleotides

Structural identification of AAF adducted Oligonucleotide by tandem mass

spectrometry (MS/MS) using the optimized mobile phase conditrions

In all the cases, a peak attributed to the mono AAF adducted 17-mer

Oligonucleotide was observed at m/z 1773.47 corresponding to −3 charge state

of the adducted Oligonucleotide in the mass chromatogram

16.5 mM N,N-dimethylcyclohexylamine / 400 mM HFIP

2.5 mM 1,8-Diazabicyclo[5.4.0]undec-7-ene

(DBU)/100 mM HFIP

25 mM trans-N,N-Dimethylcyclohexane-

1,2-diamine/100 mM HFIP


Model Oligonucleotide adduct : AAF adducted ss- CCC CGA GCA ATC TCA AT Oligonucleotide

Chromatography: Reversed Phase Liquid Chromatography (RPLC)

Stationary phase/ Column: Monolithic PS-DVB capillary column (0.25X95 mm)

Flow rate (6µL/min), Mobile Phase B(Methanol), Linear gradient (1% B per min)

Effect of different amine/ HFIP ion pair system on IP-RP-LC-ESI-MS/MS separation of

AAF adducted isomeric Oligonucleotide

16.5 mM Triethylamine/ 400 mM HFIP

16.5 mM Tripropylylamine/ 400 mM HFIP

16.5 mM Hexylamine/ 400 mM HFIP

16.5 mM N,N-dimethylbutylamine/

400 mM HFIP

5 10 15 20 25 30 35 40 45 50

Time (min)

0

100

0

100

0

100

0

100

0

100

0

100

0

100 38.24

30.89

42.01

31.64

47.78

43.26

30.66

NL: 1.40E6

m/z= 1772.90-1773.90 MS data04_111115155813

NL: 2.50E6

m/z= 1772.90-1773.90 MS data02_111115113942

NL: 2.15E7

m/z= 1772.90-1773.90 MS data05_111102180133

NL: 3.93E7

m/z= 1772.90-1773.90 MS data01_111208120236

NL: 2.93E5

m/z= 1772.90-1773.90 MS data01_111020112154

NL: 3.99E5

m/z= 1772.90-1773.90 MS data04_111019151934

NL: 5.23E5

m/z= 1772.90-1773.90 MS data04_111018150407


RT: 5.00 - 60.00 SM: 13G

5 10 15 20 25 30 35 40 45 50 55 60

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

0

50

10026.07

32.13

54.26

41.53

40.78

25.46

17.89

NL: 4.86E5

m/z= 1772.90-1773.90 MS data05_111103193140

NL: 8.91E5

m/z= 1772.90-1773.90 MS data05_111108172835

NL: 3.00E5

m/z= 1772.90-1773.90 MS data01_111121105642

NL: 1.29E6

m/z= 1772.90-1773.90 MS data01_111109154839

NL: 1.26E6

m/z= 1772.90-1773.90 MS data01_110926121303

NL: 2.16E5

m/z= 1772.90-1773.90 MS data03

NL: 2.88E4

m/z= 1772.90-1773.90 MS data02_111209113537

25 mM triethylammonium bicarbonate

25 mM N,N dimethylbutylammonium

bicarbonate

25 mM N,N hexylammonium bicarbonate

25 mM N,N dimethylcyclohexylammonium bicarbonate

25 mM N,N dimethylcyclohexylammonium acetate

25 mM triethylammonium acetate

25 mM ammonium acetate





Effect of trialkylammonium acetate and trialkylammonium bicarbonate on IP-RP-LC-

ESI-MS/MS separation of AAF adducted isomeric Oligonucleotide

RT: 5.00 - 50.00 SM: 13G

5 10 15 20 25 30 35 40 45 50

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

Re

lative

Ab

un

da

nce

0

50

100

0

50

10013.12

15.5515.20

20.4419.68

31.8330.68

39.7337.88

25.9925.10

NL: 5.52E5

Base Peak m/z= 1772.90-1773.90 F: - p ESI Full ms [700.00-2000.00] MS data04_110906181051

NL: 2.83E5


NL: 3.01E5


NL: 1.68E5


NL: 1.52E5


NL: 4.31E4

Base Peak m/z= 1772.90-1773.90 MS data05_111103193140


Flow rate 6 µL/

minute

Flow

rate

10 µL/

minute

Linear gradient 2.5% B/minute

Linear gradient 1.67 % B/minute



Linear gradient 1.0 % B/minute





Ion pairing reagent: 25 mM triethylammonium bicarbonate (TEAB)


Effect of flow rate and linear gradient on IP-RP-LC-ESI-MS/MS separation of AAF

adducted isomeric Oligonucleotide

Oligonucleotide sequencing ?

LCQ Deqa quadrupole ion trap mass spectrometer, Negative ionization mode, scan range

[MS (m/z 700–2000), MS/MS (m/z 450-2000)], ESI voltage (5.5 kV), Nitrogen sheath gas (15–

20 arbitrary units), Heated capillary temp. (210 °C), Data dependent MS/MS, 30%

Relative collision energy




Ion pairing reagent: 25 mM triethylammonium bicarbonate (TEAB)


D:\01_2012\...\data05_111103193140 11/3/2011 7:31:40 PM

RT: 5.00 - 50.00 SM: 7G

6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

26.07

25.10

NL: 9.57E5

m/z=

1772.98-1773.98 F:

- p ESI Full ms

[700.00-2000.00]

MS

data05_1111031931

40

data05_111103193140 #1 RT: 0.01 AV: 1 NL: 1.55E3

T: - p ESI Full ms [700.00-2000.00]

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

908.47

988.001985.931220.33

1679.271322.13

717.33 1503.601230.93929.87 1436.201389.93 1528.67 1915.871197.601160.40787.53 1278.00741.27 871.20 1729.731076.93 1838.871539.73

1928.93933.40 1035.13 1610.00856.60 1867.001796.001350.80

Peak 1 Peak 2

Structural identification of positional isomers of the AAF adducted

Oligonucleotide by tandem mass spectrometry (MS/MS)

Structural identification of Oligonucleotide adducts by tandem mass

spectrometry (MS/MS)

McLuckey fragmentation Scheme

The principal collision induced dissociation (CID) pathways of polyanionic

Oligonucleotide

D:\01_2012\...\data05_111103193140 11/3/2011 7:31:40 PM

RT: 5.00 - 50.00 SM: 7G

6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

26.07

25.10

NL: 9.57E5

m/z=

1772.98-1773.98 F:

- p ESI Full ms

[700.00-2000.00]

MS

data05_1111031931

40

data05_111103193140 #1 RT: 0.01 AV: 1 NL: 1.55E3

T: - p ESI Full ms [700.00-2000.00]

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

908.47

988.001985.931220.33

1679.271322.13

717.33 1503.601230.93929.87 1436.201389.93 1528.67 1915.871197.601160.40787.53 1278.00741.27 871.20 1729.731076.93 1838.871539.73

1928.93933.40 1035.13 1610.00856.60 1867.001796.001350.80


(Peak 1)


(Peak 2)

Oligonucleotide sequencing ?

AAF AAF

D:\01_2012\...\data05_111103193140 11/3/2011 7:31:40 PM

RT: 0.00 - 39.40 SM: 7G

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

26.07

25.10

NL: 9.57E5

m/z=

1772.98-1773.98 F:

- p ESI Full ms

[700.00-2000.00]

MS

data05_1111031931

40

data05_111103193140 #522-536 RT: 24.81-25.36 AV: 5 NL: 2.32E3

F: - p d Full ms2 [email protected] [475.00-2000.00]

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

1525.00947.67

1540.93

1722.80

1846.60

1253.93634.40 1397.00

1296.27

1703.471620.871379.93964.67 1909.131561.93 1677.73779.60 1990.131736.001505.731044.80

1367.531223.73 1745.27 1970.071083.67675.53 794.471142.87508.27 1028.73901.60819.40770.53617.47

D:\01_2012\...\data05_111103193140 11/3/2011 7:31:40 PM

RT: 0.00 - 39.40 SM: 7G

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

26.07

25.10

NL: 9.57E5

m/z=

1772.98-1773.98 F:

- p ESI Full ms

[700.00-2000.00]

MS

data05_1111031931

40

data05_111103193140 #544-554 RT: 25.71-26.10 AV: 5 NL: 2.86E3

F: - p d Full ms2 [email protected] [475.00-2000.00]

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

1254.00

1525.00

1541.07 1957.071728.47

947.67

964.671648.73

1889.53

1800.001380.47634.53

1142.60 1897.001745.001044.67 1658.071620.73 1851.071969.87

1223.13 1329.67795.60 1405.73 1469.13755.40 812.27 850.80 1010.93 1197.60675.40565.53

b Peak 1 (a6-B6)- W12

2- (a5-B5)

- W5-

W3-

(a4-B4)-

(a8-B8)2-

(a3-B3)-

W2-

W92-

(a9-B9)2-

(a10-B10)2-

c Peak 2 W12

2- W112- (a5-B5)

- W5-

W3-

(a4-B4)-

(a8-B8)2-

(a3-B3)-

W2- W9

2-

(a7-B7)2-

(a10-B10)2-

(a6-B6)-

Column: Monolithic PS-DVB capillary (250 μm ID X 95 mm)

Column temperature: Room temperature

Flow rate: 6 µL/minute

Ion pairing reagent: 25 mM Triethylammonium bicarbonate (TEAB)

Mobile phase A: Aqueous solution containing 25 mM TEAB

Mobile Phase B: Methanol or Acetonitrile

Mass Spectrometer: LCQ Deqa quadrupole ion trap mass spectrometer

Ionization mode: ESI, negative mode

Scan range: MS (m/z 700–2000), MS/MS (m/z 450-2000)

Electrospray voltage: 5.5 kV

Gas flow: Nitrogen sheath gas (15–20 arbitrary units)

Temperature (heated capillary): 210 °C.

LC-MS software: Xcalibur software version 1.4

MS/MS: Data dependent

Relative collision energy: 30%

IP-RP-LC-ESI-MS/MS optimized mobile phase conditions

RT: 5.00 - 30.00 SM: 13G

6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

0

20

40

60

80

100

Re

lativ

e A

bu

nd

an

ce

24.80

24.34

23.89

23.38

22.85

22.27

21.52

NL: 5.35E5

m/z= 1803.97-1804.97 F: - p ESI Full ms [700.00-2000.00] MS data11

NL: 6.89E5


NL: 8.66E5


NL: 6.31E5


NL: 4.58E5


NL: 3.20E5


NL: 9.06E4


Chromatographic efficiency of the monolithic PS-DVB column

Flow Rate (µL/min) 6.5 6.0 5.5 3.50 2.75

Flow velocity (mm/s) 2.20 2.04 1.75 1.39 0.93

HETP [µm] 12.38 10.41 9.84 9.72 29

HETP vs linear velocity

Van Deemeter plot uu/ CBAHETP

5’OH- TTTTTTTTTTTTTTTTTT -3’OH

5’OH- TTTTTTTTTTTTTTTTT -3’OH

5’OH- TTTTTTTTTTTTTTTT -3’OH

5’OH- TTTTTTTTTTTTTTT -3’OH

5’OH- TTTTTTTTTTTTTT -3’OH

5’OH- TTTTTTTTTTTTT -3’OH

5’OH- TTTTTTTTTTTT -3’OH

Isocratic elution of dT16 mobile phase containing 25 %B

Chromatographic separation of the poly(dT)12-18 oligo using monolithic PS-DVB column (0.25X95

mm). Mobile phase A(25 mM TEAB), mobile phase B(methanol), linear gradient 1% B per minute,

flow rate 6 µL/min.

Height Equivalent to a Theoretical Plate (HETP)

CHAPTER 3

Direct detection and mapping of sites of base modification of isomeric

Oligonucleotide adducts by ion-pair reversed-phase Liquid Chromatography

tandem mass spectrometry

Determination of the Site selectivity of arylamine carcinogens for mutational hotspots of

Tp53 gene using IP-RP-LC-ESI-MS/MS method N-OH-4-ABP adducted ds- Oligonucleotide 5’P- ACC CG(1)C G(2)TC157 CG(3)C158 G(4)C

AAF adducted ds- Oligonucleotide 5’P- ACC CG(1)C G(2)TC157 CG(3)C158 G(4)C

Separation and sequencing of positional isomers of AAF adducted Oligonucleotide using

IP-RP-LC-ESI-MS/MS method

Separation and sequencing of mixture of (+)-anti-BPDE adducted ss-Oligonucleotide

fragments containing codon 135 and codon 248 of Tp53 gene (+)-anti-BPDE adducted single stranded oligo mixture containing codon 135 (TG(1) TTT

TG(2)C135 CAA CTG(3) G(4)) & codon 248 (ATG(1) AAC CG(2)G(3)248 AG(4)G(5) CCC)

LC-MS/MS profiling of a mixture containing Tp53 gene fragments

Site selectivity of the carcinogens for mutational hotspots on Oligonucleotide containing

Tp53 gene sequence using nanoLC-nanoESI-MS/MS N-OH-4-ABP adducted ds- Oligonucleotide 5’P- ACC CG(1)C G(2)TC157 CG(3)C158 G(4)C

AAF adducted ds- Oligonucleotide 5’P- ACC CG(1)C G(2)TC157 CG(3)C158 G(4)C

(+)-anti-BPDE adducted ds- Oligonucleotide 5’P- ACC CG(1)C G(2)TC157 CG(3)C158 G(4)C

Determination of the Site selectivity of arylamine carcinogens for mutational

hotspots of Tp53 gene using IP-RP-LC-ESI-MS/MS method

RT: 5.00 - 30.00 SM: 13G

6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

12.66 NL: 1.03E5

m/z= 1494.92-1495.92 F: - c ESI Full ms [500.00-2000.00] MS data04_110215150240

5’P- ACC CG(1)C G(2)TC157 CG(3)C158 G(4)*C -3’OH

m/z 1495.45,

[M-3H]3-

IP-RP-LC-ESI-MS/MS separation & sequencing of AAF adducted Oligonucleotide

14-mer ds- Oligonucleotide (5’P- ACC CG(1)C G(2)TC157 CG(3)C158 G(4)C / 3'- TGG GCG CAG GCG CG- 5'p)

represents a region of exon 5 of the p53 gene and contains the mutational hotspot codon 157 & 158

G(4) position at the 3’end is the preferred site of adduction, suggesting that AAAF

has neither site nor sequence selectivity but a preference for the 3’end probably due

to the steric reasons in the selected adducted oligonucleotide

Determination of the Site selectivity of arylamine carcinogens for mutational

hotspots of Tp53 gene using IP-RP-LC-ESI-MS/MS method

RT: 5.00 - 30.00 SM: 13G

6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

11.2310.73

NL:1.07E5

m/z= 1476.90-1477.90 MS data01_110215111012

5’P- ACC CG(1)C G(2)TC157 CG(3)*C158 G(4)C -3’OH

5’P- ACC CG(1)C G(2)TC157 CG(3)C158 G(4)*C -3’OH

5’P- ACC CG(1)*C G(2)TC157 CG(3)C158 G(4)C -3’OH

m/z 1477.25,

[M-3H]3-

IP-RP-LC-ESI-MS/MS separation & sequencing of N-OH-4-ABP adducted Oligonucleotide,

4-ABP in tobacco smoke is the main cause of human bladder cancer, 4-ABP itself is not carcinogenic but

undergoes Phase 1 metabolic activation in the liver to form N-hydroxy-4-ABP (N-OH-4-ABP) to forms

DNA adducts

Under similar conditions, the two arylamines (AAAF & N-OH-4-ABP) behaved

differently for the same Oligonucleotide. In both cases, G(4) was adducted

preferentially at the 3’end but more importantly a significant amount of 4-ABP was

also adducted at other codons, thus implying that there is a site selectivity in case of

N-OH-4-ABP as compared to AAAF carcinogen.

Separation and sequencing of mixture of (+)-anti-BPDE adducted ss-

Oligonucleotide fragments containing codon 135 and 248 of Tp53 gene

RT: 5.00 - 50.00 SM: 7G

6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

Time (min)

0

20

40

60

80

100

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

20.98

22.54 22.8524.52

20.29 24.96

27.16

26.41

27.73

29.3125.2824.80

NL:7.42E5

m/z= 1629.18-1630.18 MS data03_110502155737

NL:8.38E5

m/z= 1624.81-1625.81 MS data03_110502155737

5’OH ATG(1) AAC CG(2)*G(3) 248AG(4)G(5) CCC

5’OH- TG(1)TTT TG(2)C135 CAA CTG(3) G(4)*

IP-RP-LC-ESI-MS/MS separation & sequencing of (+)-anti-BPDE adducted Oligo mixture

Oligo mixture consists of ss- Oligonucleotide containing hotspot codon 135 (TG(1) TTT TG(2)C135 CAA

CTG(3) (4)G) and codon 248 (ATG(1) AAC CG(2)G(3)248 AG(4)G(5)249 CCC) of the Tp53 gene.

A racemic mixture of (+)-anti-BPDE adducts predominantly at the N2 -position of guanine and forms (+)-

trans, (-)-trans, (+)-cis, and (-)-cis sterioisomers. IP-RP-LC-ESI-MS/MS separation was affected due the

ion suppression caused by the co-elution of the BPDE adducted stereoisomers formed along with the

positional isomers of adducted adenine.

Thus, there is a need to increase the chromatographic resolution, One of the ways to achieve it is by using

a miniaturized LC system

Benzo[a]pyrene is metabolically activated in vivo to form syn and anti isomers of benzo[a]pyrene-7,8-diol

9,10-epoxide (BPDE)

RT: 10.00 - 75.00 SM: 13G

10 15 20 25 30 35 40 45 50 55 60 65 70 75

Time (min)

0

100

0

100

0

100

0

100

0

100

Rela

tive A

bundance

0

100

0

100

0

10021.06

14.34

15.36

16.33

18.85

20.09

21.16

22.32

23.56

RT: 10.00 - 75.00 SM: 13G

10 15 20 25 30 35 40 45 50 55 60 65 70 75

Time (min)

0

100

0

100

0

100

0

100

Rela

tive A

bundance

0

100

0

100

0

10044.2325.37

33.3831.83

39.8837.35

37.52

42.1045.02

46.94 49.42

52.56

NL: 1.84E5

m/z= 1341.43-1342.43 F: - p ESI Full ms [700.00-2000.00] MS data02_111212150833

NL: 2.18E5


NL: 1.92E5


NL: 4.22E5


NL: 6.68E5


NL: 8.12E5


NL: 8.53E5


RT: 30.00 - 100.00 SM: 13G

30 40 50 60 70 80 90 100

Time (min)

0

100

0

100

0

100

0

100

Rela

tive A

bundance

0

100

0

10060.22

70.7754.65

64.98

56.19

68.3966.37

74.45

77.79

88.43

NL: 7.25E5


NL: 7.54E5


NL: 1.08E6


NL: 3.67E5


NL: 2.82E6


NL: 9.89E4


CGCGCCTGCGCC

ACA AAA CGG TTG ACC

GAG GTG CGT GTT TGT

TAC TTG GCC TCC GGG


TG TTT TGC CAA CTG G

ACC CGC GTC CGC GCC

CTC CAC GCA CAA ACA

CGCGCCTGCGCC

ATG AAC CGG AGG CCC


ATG CTT ACC GAA CGA TGG



CGCGCCTGCGCC

ATG AAC CGG AGG CCC


ATG AAC CGG AGG CCC



ATG AAC CGG AGG CCC

ATG AAC CGG AGG CCC

ATG AAC CGG AGG CCC


ATG AAC CGG AGG CCC

CGCGCCTGCGCC CGCGCCTGCGCC



ATG AAC CGG AGG CCC


TG TTT TGC CAA CTG /G

ATG AAC CGG AGG CCC ATG AAC CGG AGG CCC

CGCGCCTGCGCC



ATG AAC CGG AGG CCC

LC-MS/MS profiling of a mixture containing Tp53 gene fragments

Oligo Sequence AAF adduction Molecular weight

CG CGC CTG CGC C Mono 3804.356

CG CGC CTG CGC C bis- 4025.446

CG CGC CTG CGC C tris- 4246.536

CG CGC CTG CGC C tetra- 4467.626

ACC CGC GTC CGC GCC No 4474.947

CTC CAC GCA CAA ACA No 4474.997

TG TTT TGC CAA CTG G No 4574.032

TAC TTG GCC TCC GGG No 4560.008

ACA AAA CGG TTG ACC No 4570.059

TGT TTG TGC GTG GAG No 4670.082

TG TTT TGC CAA CTG G Mono 4796.037

ATG AAC CGG AGG CCC Mono 4808.137

TG TTT TGC CAA CTG G bis- 5017.127

ATG AAC CGG AGG CCC bis- 5029.277

CCC CGA GCA ATC TCA AT No 5099.391

TG TTT TGC CAA CTG G tris- 5238.217

ATG AAC CGG AGG CCC tris- 5250.367

TG TTT TGC CAA CTG G tetra- 5459.307

ATG AAC CGG AGG CCC tetra- 5471.457

ATG AAC CGG AGG CCC penta- 5692.547

ATG CTT ACC GAA CGA TGG Mono 5726.753

ATG CTT ACC GAA CGA TGG bis- 5947.843

The mixture components were separated

and sequenced over a monolithic PS-DVB

capillary column (0.25X95 mm) using a

linear gradient run 0.5% B per min over

100 mins, 25 mM TEAB ion pairing

reagent (Mobile Phase A), methanol as

mobile phase B, flow rate 6 µL/minute

10 µm i.d. distal coated silica tip

Teflon tubing sleeve

HV (1.8 KV)

Injector HPLC

flow splitter

(1000:1 split ratio)

ESI tip XYZ

positioner

Sliding rail mount

NanoLC flow

(200nl/min)

waste

Monolithic

column

0.2ml/min

Waste

Syringe

HPLC

Column

i.d.

Flow rate LC Gain in sensitivity

= (d250 µ i.d./ d75 µ i.d.)2

250 µm 5 µL/min µ Capillary = X

75 µm 250 nL/min nano capillary = 11.11 X

nano Liquid Chromatography-nano Electrospray ionization

(nanoLC-nano ESI)

The previously optimized mobile phase conditions were applied to the nanoLC-nanoESI

format in hopes to lower the detection limits in addition to perhaps increasing

chromatographic resolution and higher sensitivity

Column: Monolithic PS-DVB capillary (75 μm ID X 20 cm)

Column temperature: Room temperature

Flow rate: 200 nL/minute

Ion pairing reagent: 25 mM Triethylammonium bicarbonate (TEAB)

Mobile phase A: Aqueous solution containing 25 mM TEAB

Mobile Phase B: Methanol

Linear gradient: 0.5% B/minute

Mass Spectrometer: LCQ Deqa quadrupole ion trap mass spectrometer

Ionization mode: ESI, negative mode

Scan range: MS (m/z 1000–2000), MS/MS (m/z 450-2000)

Electrospray voltage: 1.8 kV

Temperature: 210 °C.

LC-MS software: Xcalibur software version 1.4

MS/MS: Data dependent

Relative collision energy: 30%

Site selectivity of carcinogens towards ds 14-mer long Oligonucleotide

To determine the relationship between mutational hot spots and carcinogens, the site selectivity of

three different carcinogens was investigated for a ds- 14-mer Oligonucleotide

(5’P- ACC CG(1)C G(2)TC157 CG(3)C158 G(4)C/ 3'- TGG GCG CAG GCG CG- 5'p)

IP-RP-nanoLC-nanoESI-MS/MS mobile phase conditions

0

20

40

60

80

100

G(1) codon 156 G(2) codon 157 G(3) codon 158 G(4)

0

10

20

30

40


0

10

20

30

40

50


Site selectivity of carcinogens towards ds 14-mer long Oligonucleotide

N-acetoxy-2-acetylaminofluorene

N-hydroxy-4-aminobiphenyl

(+)-anti-BPDE

5’P- ACC CG(1)C156 G(2)TC157 CG(3)C158 G(4)C159

3'- TGG GCG CAG GCG CG- 5'p

The site selectivity of the carcinogens based on the relative ratio of carcinogen adducted peak area

obtained from IP-RP-nanoLC-nanoESI-MS/MS

The IP-RP-LC-ESI-MS/MS method developed was evaluated for chromatographic

separation and mass spectrometry based structural characterization of different

carcinogenic adducted Oligonucleotide individually or in a mixture

The site selectivity investigation was carried out using the optimized mobile phase

conditions in a IP-RP-nanoLC-nanoESI-MS/MS format for different carcinogens

and different aspect of adduction was understood for the femtomole amounts of

oligonucleotide adducts

Chapter 3

Conclusion

Detection, separation and mapping of sites of base modification in oligonucleotide adducts using ion-pair reversed-phase

nano-HPLC coupled to ion trap mass spectrometry, Vaneet K Sharma, James Glick and Paul Vouros (To be submitted)

Detection, separation and mapping of sites of base modification in oligonucleotide adducts using ion-pair reversed-phase

nano-HPLC coupled to ion trap mass spectrometry. Vaneet Sharma, James Glick, Paul Vouros, 244th ACS National Meeting

& Exposition - August 19-23, 2012, Philadelphia

Separation and sequencing of isomeric oligonucleotide adducts using ion-pair reversed phase LC-ESI-MS/MS and GenoMass

software. Vaneet Sharma, James Glick Paul Vouros, American Society for Mass Spectrometry, Vancouver, Canada, 2012

CHAPTER 4

GenoMass software: a tool based on electrospray ionization tandem mass

spectrometry for sequencing modified Oligonucleotide

GenoMass software: a tool based on electrospray ionization tandem mass spectrometry for characterization

and sequencing of oligonucleotide adducts

Vaneet k Sharma, James Glick, Qing Liao, Chang Shen and Paul Vouros

J. Mass Spectrom., (2012), 47: 490–501 (cover article)

To develop a tandem mass spectrometry (MS/MS) based software for the characterization and

sequencing of modified Oligonucleotide

Screening of positional isomers of (+)-anti-BPDE adducted Oligonucleotide using GenoMass

Identification and sequencing of positional isomers of AAF adducted 17-mer Oligonucleotide

Data interpretation of AAF adducted 5-mer long Oligonucleotide using GenoMass

Data interpretation of AAF adducted 17- mer long Oligonucleotide using GenoMass

Validation of the GenoMass v3.2 software using AAF adducted Oligonucleotide

Identification of methyl modified CpGs using GenoMass v3.2

Determining the site of (P-S-) linkage in phosphorothioates (S-oligo) mixture

Computational screening of a complex Oligonucleotide mixture

GenoMass v3.2 software

GenoMass software

Enter the criteria to search fragment ions in the data.

When ‘adduct’ is present, enter molecular mass of adduct

and

For fragment ions mass shifts need to be added, i.e. ‘W’

ion fragment enter 79.9 or For ‘(a-B)’ ion fragment enter

161.082

For ‘b’ or ‘y’ fragment enter only molecular mass of

adduct

Singly charged If < 7 mer, doubly charged If > 7mer <

12mer

Graphical User Interface (GUI)

Data converted into masslynx data file format, raw Data

file - load data

If the background needs to be subtracted, load the

background data file & click subtract button

For a known Oligonucleotide, enter the Oligonucleotide

sequence

Isomer types (all combinations possible for A, G, C, T)

The output box lists all the found fragments

Corresponding peak intensity for all listed fragment result

Start button, initiated the data analysis

Plot the data in MassLynx 3.5

5’OH- G(1)G(2)CC -3‘OH

AAF

RT: 5.00 - 30.00 SM: 13G

6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

0

20

40

60

80

100

Re

lativ

e A

bu

nd

an

ce

17.80

15.60

NL:5.46E4

m/z= 1394.55-1395.55 MS 031405_06a

LC-MS/MS

Input file

(Xcalibur)

Mass Lynx 3.5

G(2) C C

OH

Data interpretation of AAF adducted Oligonucleotide using GenoMass software

W3

OH 3’

G(2) C C

AAF 221.09

79.9

AAF

5’OH- G(1)G(2)CC -3‘OH

AAF

Comparison of the

retention time for the

fragment ion obtained in

MassLynx 3.5 and

XCalibur removes the

ambiguity about the origin

of a fragment ion

G2 C C

OH

RT: 5.00 - 30.00 SM: 13G

6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

0

20

40

60

80

100

Re

lativ

e A

bu

nd

an

ce

17.80

15.60

NL:5.46E4

m/z= 1394.55-1395.55 MS 031405_06a

Mass Lynx 3.5

5’OH- G(1)G(2)CC -3‘OH

Data interpretation of AAF adducted Oligonucleotide using GenoMass software

5’OH- G(1)G(2)CC -3‘OH

5’OH- G(1)G(2)CC -3‘OH

W3

OH 3’

G2 C C 79.9

AAF

LC-MS/MS

Input file

(Xcalibur)

AAF

RT: 5.00 - 50.00 SM: 13G

5 10 15 20 25 30 35 40 45 50

Time (min)

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

24.6317.59 NL: 1.45E6


NL: 1.96E6


OH

T T G T A

AAF

5’OH- CCTACCCCTTCC TTGTA -3’OH

W5-

Mass Lynx 3.5

Data interpretation of AAF adducted 17-mer long Oligonucleotide using

GenoMass software

5’OH- CCTACCCCTTCCTTGTA -3’OH

OH

T T T G A

79.9

AAF 221.09

W5-

AAF

LC-MS/MS

Input file

(Xcalibur)

5’OH- CCTACCCCTTCCTTGTA -3’OH

AAF

MS/MS analysis

Automatic mass shifts

for fragment ions

Increase in charge state

(-3 charge state)

MS parameters, 5’

terminal end

Software is made

applicable to RNA/

S-oligos

Plotted in MassLynx

3.5

Oligonucleotide input <

100 mer

For GenoMass v3.2,

the value is -1

User preset value

GenoMass v3.2 software

RT: 5.00 - 30.00 SM: 13G

6 8 10 12 14 16 18 20 22 24 26 28 30

Time (min)

0

20

40

60

80

100

Re

lativ

e A

bu

nd

an

ce

17.80

15.60

NL:5.46E4

m/z= 1394.55-1395.55 MS 031405_06a

G2 C C

OH

AAF

Mass Lynx 3.5

Validation of the GenoMass v3.2 software using

AAF adducted Oligonucleotide

5’OH- G(1)G(2)CC -3‘OH

W3

OH 3’

G2 C C

AAF 221.09

LC-MS/MS

Input file

(Xcalibur)

Comparison of the retention time

for the fragment ion obtained in

MassLynx 3.5 and XCalibur

removes the ambiguity about the

origin of a fragment ion

AAF

5’OH- G(1)G(2)CC -3‘OH

AAF

Computational screening of a complex Oligonucleotide mixture using GenoMass

v3.2

In total, the mixture consisted of more than AAAF, BPDE, and BnzPDE adducted 17-mer long

Oligonucleotide and their corresponding positional isomers

Scenario (i) de novo approach

Presently GenoMass v3.2 is capable of generating isomeric libraries for de novo Oligonucleotide on the

fly (< 7-mer). For oligos >8-mer the calculations become slower and requires high computing power

LC-MS/MS

Input file

(Xcalibur)

5’OH- CCTACCCCTTCCTTGTA- 3’OH

AAF

Mass Lynx 3.5

Scenario (ii) pseudo de novo approach

As a compromise the best of both i.e. de novo and in silico formed local database for the search query was

used to perform the analysis

ATGACCGGAGGCCCCCGCGTCCGCCCCC

CGAGCAATCCAATTGTTTTGCCAACTGGC

CTACCCCTTCCTTGTATAGTCAAGGGCA


v3.2

LC-MS/MS

Input file

(Xcalibur)

5’OH- CCTACCCCTTCCTTGTA- 3’OH

AAF

Mass Lynx 3.5

Scenario (iii) Efficient Computing approach

A ‘targeted’ search was conducted looking for only a “specific Oligonucleotide adduct” from the complex

mixture.

An answer was sought to a specific question, Is this Oligonucleotide adduct present in the complex mixture?


v3.2

LC-MS/MS

Input file

(Xcalibur)

Incorporated the principal dissociation pathways of polyanionic

Oligonucleotide and the corresponding mass shifts in GenoMass software to

perform online tandem mass spectrometry of olignucleotide adducts

A LC-MS/MS based peak assignment software, GenoMass v3.2 has been

developed to perform automated online characterization and sequencing of

modified Oligonucleotide

Chapter 4

Conclusion

GenoMass v3.2 software: An automated tool for the computational data interpretation of the MS/MS spectra of carcinogen

adducted Oligonucleotides, cytosine methyl modified (-CpGs) Oligonucleotide and Phosphorothioates (S-oligo), Vaneet K

Sharma; James Glick, Qing Liao, Chang Shen and Paul Vouros (Manuscript in preparation)

GenoMass: Software tool for high-throughput screening of the LC-MS/MS data to identify the exact location of adducts in

modified Oligonucleotides. Vaneet Sharma; James Glick; Qing Liao; Paul Vouros, American Society for Mass Spectrometry

(ASMS), Denver, USA, 2011

CHAPTER 5


LC-MS/MS data

Oligonucleotide fragments (12-20)

‘transrenal’ DNA

Isolation & purification

Enzymatic digestion

Oligonucleotide

sequencing

Gene identification

Chromatographic separation

[monolithic PS-DVB/ PLOT columns/ chiral

stationary phases]

Higher Resolution Mass Spectrometry

Automated de novo oligonucleotide

sequencing

(GenoMass)

Nucleotide

Basic Local Alignment Search

Tool (BLAST)

Liquid Chromatography - Tandem Mass Spectrometry Methods For

The Analysis Of Isomeric Oligonucleotide Adducts

Conclusions

Development of robust and reliable ion-pair reversed-phase liquid chromatography

electrospray ionization tandem mass spectrometry (IP-RPLC-ESI-MS/MS) method

using monolithic PS-DVB capillary column for the analysis of the isomeric

Oligonucleotide adducts

The ion-pair reversed-phase liquid chromatography electrospray ionization tandem

mass spectrometry (IP-RPLC-ESI-MS/MS) method was used to analyze positional

isomers of carcinogenic adducted Oligonucleotide individually or in mixtures

Site selectivity of different carcinogens for a synthetic Oligonucleotide fragment of

Tp53 gene containing mutational hotspot 157 was determined in order to understand

the relationship between chemical behavior of carcinogens and mutational hotspots

using nanoLC-nanoESI-MS/MS method

A LC-MS/MS based peak assignment software, GenoMass v3.2 has been developed to

perform automated sequencing of modified Oligonucleotide

1) Detection, separation and mapping of sites of base modification in oligonucleotide adducts using ion-pair reversed-phase

nano-HPLC coupled to ion trap mass spectrometry. Vaneet Sharma; James Glick; Paul Vouros, 244th ACS National

Meeting & Exposition - August 19-23, 2012, Philadelphia

2) Separation and sequencing of isomeric oligonucleotide adducts using ion-pair reversed phase LC-ESI-MS/MS and

GenoMass software. Vaneet Sharma; James Glick; Paul Vouros, American Society for Mass Spectrometry (ASMS),

Vancouver, Canada, 2012

3) GenoMass: Software tool for high-throughput screening of the LC-MS/MS data to identify the exact location of adducts in

modified Oligonucleotides. Vaneet Sharma; James Glick; Qing Liao; Paul Vouros, American Society for Mass

Spectrometry (ASMS), Denver, USA, 2011

1) Mass spectrometric based analysis, characterization and applications of circulating cell free DNA isolated from human

body fluids., Vaneet K Sharma, Paul Vouros and James Glick, International Journal of Mass Spectrometry, 2011, 304,

172–183

2) GenoMass software: A tool based on electrospray ionization tandem mass spectrometry for characterization and

sequencing of oligonucleotides adduct., Vaneet K Sharma, James Glick, Qing Liao, Chang Shen and Paul Vouros,

Journal of Mass Spectrometry, 2012, 47(4), 490-501. (Cover article)

3) Reversed-phase ion-pair liquid chromatography electrospray ionization tandem mass spectrometry for separation,

sequencing and mapping of sites of base modification of isomeric oligonucleotide adducts using monolithic column.,

Vaneet K Sharma, James Glick, and Paul Vouros, Journal of chromatography A, 2012, 1245, 65-71

4) The analysis of DNA adducts: the transition from 32P-postlabeling to mass spectrometry, Joshua Klaene, Vaneet K

Sharma, James Glick, and Paul Vouros, Cancers Letters, 2012, (10.1016/j.canlet.2012.08.007)

5) GenoMass v3.2 software: An automated tool for the computational data interpretation of the MS/MS spectra of carcinogen

adducted Oligonucleotides, cytosine methyl modified (-CpGs) Oligonucleotide and Phosphorothioates (S-oligo), Vaneet

K Sharma; James Glick, Qing Liao, Chang Shen and Paul Vouros (Manuscript in preparation)

6) Detection, separation and mapping of sites of base modification in oligonucleotide adducts using ion-pair reversed-phase

nano-HPLC coupled to ion trap mass spectrometry, Vaneet K Sharma; James Glick and Paul Vouros (To be submitted)

Presentations

Publications

Acknowledgements

Prof. Paul vouros

Prof. Penny J. Beuning

Prof. Robert N. Hanson

Prof. Michael Pollastri

Dr. James Glick

Dr Qing Liao

Chang Shen

Dr. Steve Coy, Dr Rojer Kautz

Josh, Rose, Amol, Adam, Kristen

Family & Friends

This work was supported by National Institutes of Health grant

numbers: RO1 CA69390, RO1 CA112231

The Barnett Institute & The Department of Chemistry & Chemical

Biology at Northeastern University

Documents

PhD thesis presentation 2012