Transcript Mass Spectrometry of Oligonucleotides and Nucleic Acids

Mass Spectrometry of Nucleic Acids
Bing H. Wang, Ph. D.
1. Introduction
a. Advantages of Mass Spectrometry
b. Structures of Nucleotides
2. Fundamentals of Nucleic Acids Analysis
a. Ion Formation by MALDI
b. Ion Formation by ESI
3. Applications
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
purine or pyrimidine base
O
NH2
O
O
P
B
O
N
N
NH
N
5'
O
O
NH
N
NH2
1'
4'
pentose
phosphate
NH
N
3'
OH
adenine
guanine
2'
OH
O
NH2
H3C
NH
N
OH
HO
O
HO
OH
O
NH
O
cytosine
OH
OH
NH
O
thymine
OH
O
ribose
2’-deoxyribose
Photo esearchers,Inc./Ken Eward
NH
NH
uracil
O
Ion Formation by MALDI
Science, 279 (1998) 2044
Common Matrices
UV
•
OH
3-hydroxypicolinic acid
Wu, K. J. et al.. Rapid Commun. Mass Spectrom. 1993, 7, 191.
COOH
O
•
2,4,6-trihydroxyacetophenone
CH3
HO
OH
Uwe, P. et al. Nucl. Acids Res. 21 (1993) 3191-3196.
OH
•
6-aza-2-thiothymine
O
Lecchi, P. J. Am. Soc. Mass Spectrom. 6 (1995) 972.
CH3
HN
S
N
H
N
IR
•
Succinic acid
Nordhoff et al. Nucl. Acid Res. 21 (1993) 3347.
COOH
HOOC
OH
•
Glycerol
HO
OH
Matrix Effect: 3-HPA vs. 2,5-DHB
•15-mer ODN
•3-HPA: cleaner spectrum
•2,5-DHB: extensive fragmentation
Zu, L. et al. J. Am. Chem. Soc. 117 (1995) 6048.
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
Proof of the Linkage between Protonation and Base Loss
1
2
d(TGTT)
1: (M-G)+, no H/D, ∆m/z =151
2: (M-G)+, with H/D, ∆m/z =155
Gross, J. et. al. J. Am. Soc. Mass Spectrom. 1998, 9, 866-878.
Guanine Loss Initiated by Deuterium Ion Attachment
Gross, J. et. al. J. Am. Soc. Mass Spectrom. 1998, 9, 866-878.
Reducing Fragmentation through Structural Modification
C
HO
O
OH
sugar
modification
C
HO
O
OH
OH
ODN = d(C*T3)5-9
Tang, W. at. Al. Anal. Chem. 69 (1997) 302
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
•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
Kirpekar, F. at el. Anal Chem. 71 (1999) 2334-2339.)
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
UV/sinapinic acid
IR/succinic acid
Costello, C. E. et al. Int. J. Mass Spectrom. Ion Proc. 132 (1994) 239-249.
Effect of Salts
50 mM NaCl
0.5 mM NaCl
5 mM NaCl
5 μM NaCl
•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.
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.
2.
3.
4.
5.
6.
Prepare a matrix solution consisting of 50 g/L 3-HPA and 40
mM diammonium citrate in 50:50 water/acetonitrile.
Condition a 5 mg Oasis Cartridge (Waters, Co.) with 2 mL
70:30 acetonitrile/water.
Dilute 10 μL of DNA sample in 0.5 mL TEAA buffer (0.1 M,
pH 7.0). Load the solution onto the cartridge.
Wash the cartridge with 2 mL of TEAA buffer (0.1 M, pH
7.0).
Elute DNA sample with 10 μL of the matrix solution by
centrifugation.
Apply 1-2 μL of the solution to a MALDI target.
Improve Sensitivity and Reproducibility through
Sample Miniaturization
•Advantages of Sample Miniaturization
•Increased sensitivity
•Improved sample homogeneity
•Increased number of samples per target
•Miniaturized Sample Preparation
•Use of Piezoelectric Nanopipet to deposit nL amounts of
sample; subfemtomole sensitivity for oligonucleotides achieved
•Anchored Target
~ 600 um
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
before
after
• Desalting reduces adduct
formation. Approaches
used to desalt for
MALDI-MS are
applicable for ESI-MS.
Stults, J. T. et. al. RCMS 5 (1991)359
desalting by ethanol precipitation
Factors Affecting ESI Mass Spectra Quality
•Organic solvent
10-50% methanol, isopropanol,
or acetonitrile
•Organic additive
Triethylamine, piperidine,
imidazole
ethanol precipitation
5mM piperidine
•pH >= 7.0 favored.
2.5 mM imidazole and piperidine
26-mer PO: 5’-dTGAGTCAGACGCATCGTCGTCATGG-3’
Greig, M. et al. RCMS 9 (1995) 97
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
Bayer, E. et. al. Anal. Chem. 66 (1994) 3858
Ganem, B. et. al. Tetra. Lett. 34 (1993) 1445
•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
Complex of DNA Duplex and Small Molecules
without Dm
5 μM Dm
12-mer: 5’-dCGCAAATTTGCG-3’
Dm: Distamycin A
12M: 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
20 μM Dm
•Solution stoichiometry preserved
by ESI
Gale, D. C., et. al. J. Am. Chem. Soc. 116 (1994) 6027
Applications
• Location of modification site
• Antisense oligonuleotide sequencing
• Infectious agents identification
• High-throughput diagnostics
• Drug discovery
Location of Modification Site
•X = 2’-O-methyl adenosine
•modification has little effect on SVP
•CSP digestion is stopped by the
modification, revealing the site of
modification
Piels, U. et. al. Nucl. Acids. Res. 21(1993) 3191.
Sequencing: Antisense Oligonucleotide
O
O P
O
PO
O
O
S P
O
O
PS
•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
Sequencing of Phosphorothioate by ISD
a21 a22
5’-CTCTCGCACCCATCTCTCTCCTTCT-3’
w4 w3
O
O P
O
PO
O
O
S P
O
O
PS
PS Residue Mass (Da)
A
C
G
T
•Sequence informative ions consist of a, d,
and w ions
Wang, B.H. et al. Int J. Mass Spectrom Ion Proc. 169/170 (1997) 331-350.
329.2
305.2
345.2
320.2
MS-based Diagnostic Techniques
(Sequenom)
(ABI)
(Third Wave Tech.)
Detection of Mutation in CFTR exon 10
+ddTTP
+ddCTP
+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.
Braun, A. et. al. Clinical Chem. 43 (1997) 1151
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
BBR3464
NH3
Pt
NH2
NH3
Cl
NH2
NH2
Pt
NH2
H2N
NH2
NH3
Pt
NH3
Cl
•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’
Kloster, M. et. al. Biochemistry, 38 (1999)14731
.
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?