Xueheng Cheng

Charge-state reduction with improved signal intensity of oligonucleotides in electrospray ionization mass spectrometry

The shift of charge states of oligonucleotide negative ions formed in electrospray ionization mass spectrometry to higher mass-to-charge ratio has been accomplished by addition of organic acids and bases to the solution to be electrosprayed. The use of acetic acid or formic acid combined with piperidine and imidazole effectively reduced charge states. Signal intensity and stability were enhanced greatly when the infused solution contained a high percentage of acetonitrile. In addition, the cocktail that contained imidazole, piperidine, and acetic acid in 80% acetonitrile not only reduced charge states, but also substantially suppressed Na adduction. Several oligonucleotides that varied in base composition and length were investigated, and studies of mixtures showed a significant reduction in spectral complexity.

Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry

The binding stoichiometry of gene V protein from bacteriophage f1 to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution. Addition of oligonucleotides resulted in formation of protein-oligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry. The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of protein-oligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful too] in the study of protein-DNA interactions.

Specific metal-oligonucleotide binding studied by high resolution tandem mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) was used to study the binding of metal ions to two oligonucleotides, d(pGCTTGCATp) and d(TTGGCCCTCCTT). Collision-induced dissociation (CID) of the metal-oligonucleotide complex revealed that metal ions preferentially bound to the central thymine region of the sequence. The most probable binding sites were the phosphodiester backbone since the sum of the maximum number of charge addition from the metal ions and the charge state of the whole complex was found to be equal to the number of ionizable protons on the DNA backbone. Although site-specific and sequence-specific binding was observed for all three of the metal ions studied, the binding specificity of UO2(2+) ions was significantly greater than for Mg2+ and Na+. These experiments demonstrate that ESI-MS/MS can be applied to study the binding of metal ions and their complexes to oligonucleotides, providing not only information on the number of metal ions binding to the oligonucleotide, but also information related to the binding site(s) and binding specificity.

Binding specificity of post-activated neocarzinostatin chromophore drug-bulged DNA complex studied using electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was employed to characterize the binding specificity of a bulged 22-mer DNA hairpin with a post-activated neocarzinostatin chromophore (NCS-Chrom) having two similar forms, where 2a has an H in a location for which 2b has it replaced by a CH2OH group. Specific binding of 2a to the bulged 22-mer DNA was observed whereas little binding was detected for 2a to non-bulged DNA 19-mer and 12-mer duplexes. The stoichiometry of the 22-mer DNA complex with 2a was determined to be predominantly 1:1. Substitution of hydrogen in 2a for the hydroxymethylene group in 2b dramatically reduced the binding strength to the 22-mer DNA. Little complex formation was observed for 2b and 22-mer DNA based upon the ESI-MS data, consistent with earlier fluorescence studies. The results indicate that ESI-MS can be a sensitive technique for probing conformational specificity in studies of biomolecular binding.

Electrospray ionization with high performance fourier transform ion cyclotron resonance mass spectrometry for the study of noncovalent biomolecular complexes

Publisher Summary This chapter describes the feasibility of mass spectrometric analysis of structurally specific noncovalent biomolecular complexes and highlights the advantages of electrospray ionization (ESI)-Fourier transform ion cyclotron resonance (FTICR). Electrospray ionization is effective for various classes of biomolecules. The ESI process generates highly charged micro droplets that upon desolvation, yield (cationic or anionic) intact molecular species. Any compound that can exist as an ion in solution is amenable to ionization by electrospray. ESI-mass spectroscopy (MS) is applied to a wide range of biologically relevant compounds, including proteins and peptides, glycoproteins, oligonucleotides, glycolipids, and a number of metabolic intermediates. ESI is well suited for the combination with FTICR for the study of large molecules as the m/z range of ions produced by ESI overlaps with the m/z range where FTICR provides unparalleled mass accuracy and mass resolving power.

Comparison of 3′,5′- and 2′,5′-linked DNA duplex stabilities by electrospray ionization mass spectrometry

Two mutually complementary strands of 2′,5′-linked DNA form a unique noncovalent complex observed by electrospray ionization mass spectrometry; by the latter method, the double-stranded state of a 2′,5′-linked DNA duplex is less stable than the natural DNA duplex of identical base composition.

The Role of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry in Biological Research — New Developments and Applications

The symbiotic relationship between advances in ionization methods and the improved performance of mass spectrometric instrumentation and its computer control has been key to the growth of mass spectrometry. The new capabilities for the ionization of large molecules introduced in 1988 have, in effect, resulted in a situation where the demands for improved sensitivity, resolution, effective tandem MS capabilities for structural studies, and the online combination with separation methods, are greater than ever and effectively open-ended. This situation coincides with a period in which mass spectrometric hardware has begun a transition from a dominance by quadrupole and sector mass analyzer technologies to other technologies that previously were rarely used for “real world” bioanalytical applications (i. e., time-of-flight mass spectrometers and ion trapping methods).