mass spectrometry of nucleic acids 1.introduction a.advantages of mass spectrometry b.structures of...
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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.
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MS is Embedded in Modern Drug Discovery Process
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MS is Embedded in Modern Drug Discovery Process
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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
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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
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Ion Formation by MALDI
Science, 279 (1998) 2044
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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
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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
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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.
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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.
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Mechanism of Fragmentation
•Loss of nucleobases lead to strand scission
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Nomenclature of Product Ions
McLuckey et al. J. Am. Soc. Mass Spectrom. 1992, 3, 60-70
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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.
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Gross, J. et. al. J. Am. Soc. Mass Spectrom. 1998, 9, 866-878.
Guanine Loss Initiated by Deuterium Ion Attachment
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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
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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.
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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
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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.)
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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
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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
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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
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Comparison of Some Desalting Methods
Hornshaw, M. et al. ABI & Millipore
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Desalt and Sample Preparation Using Robot
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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.
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•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
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Oligonucleotide Samples on Anchored Target
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Other Factors Contributing to Loss of Sensitivity in MALDI-TOMS
Smith et al, Anal. Chem. 2003, 75, 5944-5952.
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Ion Formation by ESI
Source: J. Chem. Ed., 73:4, 1996
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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
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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’
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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
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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
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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
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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
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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
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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
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Applications
• Location of modification site
• Antisense oligonuleotide sequencing
• Infectious agents identification
• High-throughput diagnostics
• Drug discovery
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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
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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
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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
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MS-based Diagnostic Techniques
(Sequenom)
(ABI)
(Third Wave Tech.)
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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
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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
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Base Composition of PCR Amplicon Can be Determined by FTMS
Sannes-Lowery et al., Trends Anal. Chem. , 2000, 19:491-491.
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Measurement of SARS Coronavirus
Sampath, R. et al. PloS One 2007, 5, e489.
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Sampath, R. et al. PloS One 2007, 5, e489.
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HT Drug Screening
Source: www.isip.com
Hofstadler et . al, Mass Spectrom. Rev. 2005, 24, 265-285.
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HT Screening: RNA-antibiotics
Source: www.isip.com
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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
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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’
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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?