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Mass Spectrometry of Nucleic Acids 1.Introduction a.Advantages of Mass Spectrometry b.Structures of Nucleotides 2.Fundamentals of Nucleic Acids Analysis.

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Presentation on theme: "Mass Spectrometry of Nucleic Acids 1.Introduction a.Advantages of Mass Spectrometry b.Structures of Nucleotides 2.Fundamentals of Nucleic Acids Analysis."— Presentation transcript:

1 Mass Spectrometry of Nucleic Acids 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 Bing H. Wang, Ph. D.

2 MS is Embedded in Modern Drug Discovery Process

3

4 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

5 Structures of Nucleotides adenine guanine cytosinethymine uracil ribose2’-deoxyribose O O OHOH O O PO B phosphate pentose purine or pyrimidine base 1' 2' 3' 4' 5' Photo esearchers,Inc./Ken Eward

6 Ion Formation by MALDI Science, 279 (1998) 2044

7 Common Matrices UV 3-hydroxypicolinic acid Wu, K. J. et al.. Rapid Commun. Mass Spectrom. 1993, 7, ,4,6-trihydroxyacetophenone Uwe, P. et al. Nucl. Acids Res. 21 (1993) aza-2-thiothymine Lecchi, P. J. Am. Soc. Mass Spectrom. 6 (1995) 972. IR Succinic acid Nordhoff et al. Nucl. Acid Res. 21 (1993) Glycerol

8 Zu, L. et al. J. Am. Chem. Soc. 117 (1995) Matrix Effect: 3-HPA vs. 2,5-DHB 15-mer ODN 3-HPA: cleaner spectrum 2,5-DHB: extensive fragmentation

9 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.

10 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.

11 Mechanism of Fragmentation Loss of nucleobases lead to strand scission

12 Nomenclature of Product Ions McLuckey et al. J. Am. Soc. Mass Spectrom. 1992, 3, 60-70

13 d(TGTT) 1 2 1: (M-G)+, no H/D, ∆m/z =151 2: (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,

14 Guanine Loss Initiated by Deuterium Ion Attachment

15 Reducing Fragmentation through Structural Modification Tang, W. at. Al. Anal. Chem. 69 (1997) 302 sugar modification ODN = d(C*T 3 ) 5-9

16 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)

17 Duplex Ion Formation by UV MALDI With 6-aza-2-thiothymine (ATT) duplex of bp have been detected Kirpekar, F. at el. Anal Chem. 71 (1999) ) 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

18 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) )

19 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) UV/sinapinic acid IR/succinic acid

20 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) mM NaCl 5 mM NaCl 0.5 mM NaCl 5 μM NaCl

21 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

22 Comparison of Some Desalting Methods Hornshaw, M. et al. ABI & Millipore

23 Desalt and Sample Preparation Using Robot

24 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.

25 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

26 Oligonucleotide Samples on Anchored Target

27 Other Factors Contributing to Loss of Sensitivity in MALDI-TOMS Smith et al, Anal. Chem. 2003, 75,

28 Ion Formation by ESI Source: J. Chem. Ed., 73:4, 1996

29 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

30 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 solvent  10-50% methanol, isopropanol, or acetonitrile Organic additive  Triethylamine, piperidine, imidazole pH >= 7.0 favored. 26-mer PO: 5’-dTGAGTCAGACGCATCGTCGTCATGG-3’

31 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

32 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 MS n (n>2). ‘Soft’ ionization DNA over 100MDa observed

33 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,

34 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,

35 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

36 Complex of DNA Duplex and Small Molecules Gale, D. C., et. al. J. Am. Chem. Soc. 116 (1994) 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 Solution stoichiometry preserved by ESI without Dm 5 μM Dm 20 μM Dm

37 Applications Location of modification site Antisense oligonuleotide sequencing Infectious agents identification High-throughput diagnostics Drug discovery

38 Location of Modification Site Piels, U. et. al. Nucl. Acids. Res. 21(1993) X = 2’-O-methyl adenosine modification has little effect on SVP CSP digestion is stopped by the modification, revealing the site of modification

39 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 PO PS

40 Sequencing of Phosphorothioate by ISD Sequence informative ions consist of a, d, and w ions Wang, B.H. et al. Int J. Mass Spectrom Ion Proc. 169/170 (1997) ’-CTCTCGCACCCATCTCTCTCCTTCT-3’ PS Residue Mass (Da) A329.2 C305.2 G345.2 T320.2 a 21 w4w4 PO PS a 22 w3w3

41 MS-based Diagnostic Techniques (Sequenom) (ABI) (Third Wave Tech.)

42 Detection of Mutation in CFTR exon 10 Braun, A. et. al. Clinical Chem. 43 (1997) 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

43 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

44 Base Composition of PCR Amplicon Can be Determined by FTMS Sannes-Lowery et al., Trends Anal. Chem., 2000, 19:

45 Measurement of SARS Coronavirus Sampath, R. et al. PloS One 2007, 5, e489.

46

47 HT Drug Screening Source: Hofstadler et. al, Mass Spectrom. Rev. 2005, 24,

48 HT Screening: RNA-antibiotics Source:

49 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)

50 Drug Discovery: Mechanism of Drug Action Kloster, M. et. al. Biochemistry, 38 (1999) 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’

51 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?


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