Mass Spectrometry of Nucleic Acids

1. Introductiona. Advantages of Mass Spectrometryb. Structures of Nucleotides

2. Fundamentals of Nucleic Acids Analysisa. Ion Formation by MALDIb. Ion Formation by ESI

3. Applications

Bing H. Wang, Ph. D.

MS is Embedded in Modern Drug Discovery Process

MS is Embedded in Modern Drug Discovery Process

Advantages of Mass Spectrometry

•Many advantages compared to gel-based techniques•No interference from secondary structures•Accuracy and specificity

•High information content•Structural information

•Speed•Requiring only seconds for individual analysis • Capable of parallel assays

•Automation

Structures of Nucleotides

OH

OOH

OHOH

OHO

OH

OH

N

N

NH

N

NH2

N

NH

NH2

O

N

NH

NH

N

NH2

O

NH

NH

O

O

CH3

NH

NH

O

O

adenine guanine

cytosine thymine

uracil

ribose 2’-deoxyribose

OO

OHOH

O

O

PO

B

phosphatepentose

purine or pyrimidine base

1'

2'3'

4'

5'

Photo esearchers,Inc./Ken Eward

Ion Formation by MALDI

Science, 279 (1998) 2044

Common Matrices

UV• 3-hydroxypicolinic acid Wu, K. J. et al.. Rapid Commun. Mass Spectrom. 1993, 7, 191.

• 2,4,6-trihydroxyacetophenone Uwe, P. et al. Nucl. Acids Res. 21 (1993) 3191-3196.

• 6-aza-2-thiothymine

Lecchi, P. J. Am. Soc. Mass Spectrom. 6 (1995) 972.

IR• Succinic acid Nordhoff et al. Nucl. Acid Res. 21 (1993) 3347.

• Glycerol

COOH

OH

OH

OHHO

O CH3

NH

O

NH

N

CH3

S

COOHHOOC

OHOH

OH

Zu, L. et al. J. Am. Chem. Soc. 117 (1995) 6048.

Matrix Effect: 3-HPA vs. 2,5-DHB

•15-mer ODN•3-HPA: cleaner spectrum•2,5-DHB: extensive fragmentation

The Effects of Fragmentation of Ion Detection

Source: Bruker Daltonics

•Fragmentation increases the complexity of a mass spectrum.

•In-source Decay (ISD) reduces intact ion signal in linear mode of TOF-MS.

•Post-source Decay (PSD) reduces intact ion signal in reflector mode of TOF-MS.

•On the other hand, ISD and PSD give sequence information.

Factors Influencing Ion Fragmentation

•Matrix

•Acidic matrices (e.g., DHB) promote fragmentation

•Nucleotide structure•the strength of a nucleotide linkage correlates inversely to its gas phase basicity (G>A, C > T).•RNA has higher stability.

Mechanism of Fragmentation

•Loss of nucleobases lead to strand scission

Nomenclature of Product Ions

McLuckey et al. J. Am. Soc. Mass Spectrom. 1992, 3, 60-70

d(TGTT)

1

2

1: (M-G)+, no H/D, ∆m/z =1512: (M-G)+, with H/D, ∆m/z =155

Proof of the Linkage between Protonation and Base Loss

Gross, J. et. al. J. Am. Soc. Mass Spectrom. 1998, 9, 866-878.

Gross, J. et. al. J. Am. Soc. Mass Spectrom. 1998, 9, 866-878.

Guanine Loss Initiated by Deuterium Ion Attachment

Reducing Fragmentation through Structural Modification

Tang, W. at. Al. Anal. Chem. 69 (1997) 302

O

OH

OH C

O

OH

OH

OH

C

sugar modification

ODN = d(C*T3)5-9

Reducing Fragmentation with IR Laser

Laser: 2.94 μm. Matrix: glycerol

•A: 21-nt oligodeoxynucleotide

•B &C: plasmid DNA restriction

digest (2180-nt = 673 kD)

•D: 1206-nt RNA transcript

•Accuracy better than 1%

•Subfemtomole detection limit

Berkenkamp, S. et al. Science, 281 (1998) 260-262.

Duplex Ion Formation by UV MALDI

•With 6-aza-2-thiothymine (ATT) duplex of 12 - 70 bp have been detected

Kirpekar, F. at el. Anal Chem. 71 (1999) 2334-2339.)

•Duplex ions generated by UV MALDI undergo extensive fragmentation

•Duplex ions generated by UV MALDI do not survive ion reflector

•3-HPA is a denaturant

Duplex Ion Formation by IR MALDI

Counter ions are needed to stabilize duplex

Duplex ions are sensitive to laser fluence

Kirpekar, F. at el. Anal Chem. 71 (1999) 2334-2339.)

Detection of Metal Complex

•Ions of cisplatin-DNA complex generated by MALDI •Complex stability is wavelength and matrix dependant •The complex provides information on binding site when combined with enzyme digestion

Costello, C. E. et al. Int. J. Mass Spectrom. Ion Proc. 132 (1994) 239-249.

UV/sinapinic acid

IR/succinic acid

Effect of Salts

•Oligonucleotides have a strong tendency to form salt adducts•High concentration of salts suppresses ionization•Adduct formation reduces signal intensity and mass resolution, while increasing spectra complexity

Gilar, M. et. al. J. Chromatogr. A 921 (2001) 3.

50 mM NaCl 5 mM NaCl

0.5 mM NaCl 5 μM NaCl

Common Ways of Desalting

•Ethanol precipitation•Free float cation-exchange resin •Dialysis •Microconcentrator•Reverse-phase HPLC (RP-HPLC)•Solid phase extraction cartridge (SPEC)

•controlled process•high-throughput

Comparison of Some Desalting Methods

Hornshaw, M. et al. ABI & Millipore

Desalt and Sample Preparation Using Robot

Example of Sample Preparation

1. Prepare a matrix solution consisting of 50 g/L 3-HPA and 40 mM diammonium citrate in 50:50 water/acetonitrile.

2. Condition a 5 mg Oasis Cartridge (Waters, Co.) with 2 mL 70:30 acetonitrile/water.

3. Dilute 10 μL of DNA sample in 0.5 mL TEAA buffer (0.1 M, pH 7.0). Load the solution onto the cartridge.

4. Wash the cartridge with 2 mL of TEAA buffer (0.1 M, pH 7.0).

5. Elute DNA sample with 10 μL of the matrix solution by centrifugation.

6. Apply 1-2 μL of the solution to a MALDI target.

•Advantages of Sample Miniaturization

•Increased sensitivity •Improved sample homogeneity•Increased number of samples per target

~ 600 um

•Miniaturized Sample Preparation

•Use of Piezoelectric Nanopipet to deposit nL amounts of sample; subfemtomole sensitivity for oligonucleotides achieved

•Anchored Target

Improve Sensitivity and Reproducibility through Sample Miniaturization

Oligonucleotide Samples on Anchored Target

Other Factors Contributing to Loss of Sensitivity in MALDI-TOMS

Smith et al, Anal. Chem. 2003, 75, 5944-5952.

Ion Formation by ESI

Source: J. Chem. Ed., 73:4, 1996

Factors Affecting ESI Mass Spectra Quality

• Desalting reduces adduct formation. Approaches used to desalt for MALDI-MS are applicable for ESI-MS.

before

after

desalting by ethanol precipitation

Stults, J. T. et. al. RCMS 5 (1991)359

Factors Affecting ESI Mass Spectra Quality

ethanol precipitation

5mM piperidine

2.5 mM imidazole and piperidine

Greig, M. et al. RCMS 9 (1995) 97

•Organic solvent10-50% methanol, isopropanol, or acetonitrile

•Organic additiveTriethylamine, piperidine,

imidazole

•pH >= 7.0 favored.

26-mer PO: 5’-dTGAGTCAGACGCATCGTCGTCATGG-3’

Factors Affecting ESI-MS Quality

• Polarity

– Negative mode typically gives higher sensitivity

– Positive ions require the presence of ammonium or

nitrogen containing bases

• Desolvation conditions

– Flow rate

– Heating

– Nozzle-Skimmer voltage

Characteristics of ESI-MS of Nucleic Acids •Ions are usually multiply charged

•making large ions more amenable to quadrupole, ion trap, and FTMS.

•improving structural accessibility by MSn (n>2).

•‘Soft’ ionization

•DNA over 100MDa observed

Charge States Are Dependent on Solution Composition

d(T)18 in (a) 80% ACN, (b) 80%ACN/25-mM piperidine/25-mM imidazole, (c) 80% AN/25-mM piperidine/25-mM imidazole/2.5-M acetic acid, and (d) 80% ACN/25mM piperidine/25-mM imidazole/2.5-M formic acid.

Smith, R.D. et al. JASMS 1996, 7, 697-706

Charge State Reduction Simplifies Spectrum Interpretation

A mixture of d(T)18, d(A)6, and d(C)12

Smith, R.D. et al. J Am Soc Mass Spectrom 1996, 7, 697-706

ESI-MS of Non-covalent Complex

•Duplex as small as 8-bp observable•Duplex identity confirmed by MS/MS •stability is size dependant•Buffer: 10 mM ammonium acetate/bicarbonate/citrate, pH=7.5 to 8.5

Ganem, B. et. al. Tetra. Lett. 34 (1993) 1445

Bayer, E. et. al. Anal. Chem. 66 (1994) 3858

Complex of DNA Duplex and Small Molecules

Gale, D. C., et. al. J. Am. Chem. Soc. 116 (1994) 6027

12-mer: 5’-dCGCAAATTTGCG-3’Dm: Distamycin A12M: SS; Δ: DS(Δ+ 1 D): DS/Dm=1:1 (Δ+ 2 D): DS/Dm =1:2

In 10 mM ammonium acetate / ammonium citrate, pH = 8.3

•Results consistent with NMR

•Solution stoichiometry preserved by ESI

without Dm

5 μM Dm

20 μM Dm

Applications

• Location of modification site

• Antisense oligonuleotide sequencing

• Infectious agents identification

• High-throughput diagnostics

• Drug discovery

Location of Modification Site

Piels, U. et. al. Nucl. Acids. Res. 21(1993) 3191.

•X = 2’-O-methyl adenosine

•modification has little effect on SVP

•CSP digestion is stopped by the modification, revealing the site of modification

Sequencing: Antisense Oligonucleotide

•Problems in sequencing antisense oligonucleotides•Antisense oligonucleotides are modified to be nuclease resistant•Not directly amenable to Sanger sequencing•Polymerase may not work well for some modifications•No information on modification by Gel-electrophoresis

PS

O

O

OPO

O

O

O

PO PS

Sequencing of Phosphorothioate by ISD

•Sequence informative ions consist of a, d, and w ionsWang, B.H. et al. Int J. Mass Spectrom Ion Proc. 169/170 (1997) 331-350.

5’-CTCTCGCACCCATCTCTCTCCTTCT-3’

PS

O

O

OPO

O

O

O

PS Residue Mass (Da)

A 329.2C 305.2G 345.2T 320.2

a21

w4

PO PS

a22

w3

MS-based Diagnostic Techniques

(Sequenom)

(ABI)

(Third Wave Tech.)

Detection of Mutation in CFTR exon 10

Braun, A. et. al. Clinical Chem. 43 (1997) 1151

+ddTTP +ddCTP

a and b, homozygous wild-type; c and d, heterozygote wild-type/ F508; e and f, homozygous F508; g and h, compound heterozygote I507/ F508; i and k, heterozygote wild-type/I506S.

+ddTTP

+ddCTP

Identification of Emerging Infectious Agents

• Newly emergent infectious diseases are global public health problem.

– SARS, avian influenza (H5N1), Dengue, etc.

• The number of microbes pathogenic to human is large

– More than 1400 species known

– 175 species contribute to infectious diseases

• Single agent test is cost prohibitive

• Broad-range PCR combined with amplicon base composition analysis by mass spectrometry provides an answer

Base Composition of PCR Amplicon Can be Determined by FTMS

Sannes-Lowery et al., Trends Anal. Chem. , 2000, 19:491-491.

Measurement of SARS Coronavirus

Sampath, R. et al. PloS One 2007, 5, e489.

Sampath, R. et al. PloS One 2007, 5, e489.

HT Drug Screening

Source: www.isip.com

Hofstadler et . al, Mass Spectrom. Rev. 2005, 24, 265-285.

HT Screening: RNA-antibiotics

Source: www.isip.com

Parallel Screening of Multi-ligands against Multi-targets

•High resolving power of FTMS allows the deconvolution of complex spectra

•Multicomponent screening reduces the number of assays

•Multicomponent screening reduces inter assay variances

•Quantitative information such as binding constants can be obtained

Griffey RH et al. PNAS 96(1999) 10129-10133

Drug Discovery: Mechanism of Drug Action

Kloster, M. et. al. Biochemistry, 38 (1999)14731.

Pt

NH3

NH3

ClNH2

NH2

PtNH2

NH2

NH2

NH2Pt

NH3

ClNH3

BBR3464

•Charged trinuclear platinum antitumor cpd.•More potent than cisplatin.•Active against xenografts resistant to ciplatin.•Due to different mode of DNA binding?•Major findings

•BBR3464 preferentially binds to single stranded DNA and RNA (based on gel)•Both mono- and bifunctional substituion occurs on SS DNA, the extent of which is sequence dependent

•Conclusion•intrastrand crosslinks may be an important mechanism for BBR3464.

Oligo 1: 5’-CAGCGTGCGCCATCCTTCCC-3’Oligo 2: 5’-GGGAAGGATGGCGCACGCTG-3’

Questions

1. Why do we need to desalt the samples?2. What is the evidence that gas phase

fragmentation of oligonucleotide involves protonation of nucleobases?

3. Can the charge states of oligonucleotide ions be controlled?

4. What’s the advantage of ESI over MALDI in the non-covalent complex study at the moment?

5. What are the desirable attributes of a mass spectrometer used for HT drug screening?