Analysis of nucleic acids by FTICR MS

Hofstadler, Steven A.; Sannes‐Lowery, Kristin A.; Hannis, James C.

doi:10.1002/mas.20016

Cited by 92 publications

(92 citation statements)

References 103 publications

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“…This soft ionization technique can transfer noncovalent assemblies into the gasphase while preserving their solution-phase association state. 48-50 For this reason, it can be employed to determine the composition, stoichiometry, and binding affinity of large macromolecular complexes consisting of proteins, DNA, RNA, small molecule ligands, and other components (reviewed in ref.s [51][52][53]. Taking advantage of this characteristic, we have recently employed ESI-FTMS to determine the NC binding modes with short single-stranded DNA oligonucleotides 54 and with the hairpins SL2, SL3, and SL4, 55 which are present with SL1 in the packaging signal of viral RNA.…”

Section: Introductionmentioning

confidence: 99%

Dissecting the Protein–RNA and RNA–RNA Interactions in the Nucleocapsid-mediated Dimerization and Isomerization of HIV-1 Stemloop 1

Hagan¹,

Fabris²

2007

Journal of Molecular Biology

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The specific binding of HIV-1 nucleocapsid protein (NC) to the different forms assumed in vitro by the stemloop 1 (Lai variant) of the genome's packaging signal has been investigated using electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The simultaneous observation of protein-RNA and RNA-RNA interactions in solution has provided direct information about the role of NC in the two-step model of RNA dimerization and isomerization. In particular, two distinct binding sites have been identified on the monomeric stemloop structure, corresponding to the apical loop and stem-bulge motifs. These sites share similar binding affinities that are intermediate between those of stemloop 3 (SL3) and the putative stemloop 4 (SL4) of the packaging signal. Binding to the apical loop, which contains the dimerization initiation site (DIS), competes directly with the annealing of self-complementary sequences to form a metastable kissing-loop (KL) dimer. In contrast, binding to the stem-bulge affects indirectly the monomer-dimer equilibrium by promoting the rearrangement of KL into the more stable extended duplex (ED) conformer. This process is mediated by the duplex-melting activity of NC, which destabilizes the intramolecular base pairs surrounding the KL stem-bulges and enables their exchange to form the inter-strand pairs that define the ED structure. In this conformer, high-affinity binding takes place at stem-bulge sites that are identical to those present in the monomeric and KL forms. In this case, however, the NC-induced 'breathing' does not result in dissociation of the double-stranded structure because of the large number of intermolecular base pairs. The different binding modes manifested by conformer-specific mutants have shown that NC can also provide low affinity interactions with the bulged-out adenines flanking the DIS region of the ED conformer, thus supporting the hypothesis that these exposed nucleotides may constitute 'base-grips' for protein contacts during the late stages of the viral lifecycle.

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Section: Introductionmentioning

confidence: 99%

Dissecting the Protein–RNA and RNA–RNA Interactions in the Nucleocapsid-mediated Dimerization and Isomerization of HIV-1 Stemloop 1

Hagan¹,

Fabris²

2007

Journal of Molecular Biology

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“…There is a constant demand for powerful and rapid analytical methods to determine the structure of nucleic acids and their constituents. With the development of "soft" desorption/ionization techniques, matrix-assisted laser desorption/ionization (MALDI) [2,3] and electrospray ionization (ESI) [4,5] methods, mass spectrometry has become an indispensable, quick, and reliable tool for the analysis of oligonucleotides [6]. However, simple mass measurement provides little structural information for confirmation of the sequence for unknown natural and synthetic oligonucleotides.…”

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confidence: 99%

A mechanistic study of the electron capture dissociation of oligonucleotides

Chan

Choy

Chan

et al. 2009

J. Am. Soc. Mass Spectrom.

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Electron capture dissociation (ECD) of a series of custom-synthesized oligonucleotide pentamers was performed in a Fourier-transform mass spectrometer with a conventional filamenttype electron gun. Dissociation of oligonucleotide ions by electron capture generates primarily w/d-type and z/a-type ions with and without the loss of a nucleobase fragment ions. Minor yields of radical [z/a ϩ H]· fragment ions were also observed in many cases. It is interesting to note that some nucleoside-like fragment ions and protonated nucleobase ions (except thymine-related nucleobases and nucleoside-like fragments) were observed in most ECD spectra. The formation of these low-mass fragment ions was tentatively attributed to the secondary fragmentation of the radical S tructural characterization of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) is important, as DNA and RNA play important roles in many biochemical processes. Even with the completion of the human genome project, the importance of studying nucleic acid remains. For instance, a large region of DNA requires re-sequencing [1] to allow the detection and characterization of nucleotide mutation. There is a constant demand for powerful and rapid analytical methods to determine the structure of nucleic acids and their constituents. With the development of "soft" desorption/ionization techniques, matrix-assisted laser desorption/ionization (MALDI) [2,3] and electrospray ionization (ESI) [4,5] methods, mass spectrometry has become an indispensable, quick, and reliable tool for the analysis of oligonucleotides [6]. However, simple mass measurement provides little structural information for confirmation of the sequence for unknown natural and synthetic oligonucleotides. Much research effort has recently been devoted to the development of various tandem mass spectrometry (MS/MS) methods for structural elucidation of biomolecules. The critical event in tandem mass spectrometry of biomolecules is the activation of the selected precursor ions to induce unimolecular dissociation. Common ion activation methods used for analysis of biomolecules include lowand high-energy collision-induced dissociation (CID) [7, 8], surface-induced dissociation (SID) [9], infrared multiphoton dissociation (IRMPD) [10], and blackbody infrared radiative dissociation (BIRD) [11]. However, fragment ions observed in the tandem mass spectra using these ion activation methods mostly originate from cleavages of weak bonds [12].A relatively new ion activation method, electron capture dissociation (ECD) [13] has demonstrated several interesting features. For example, for protein-type biomolecules, ECD leads predominantly to the dissociation of the N-C ␣ linkage even in the presence of labile post-translational modifications [14,15]. A combination of ECD and other dissociation methods, such as IRMPD and CID, has been shown to provide complementary information for unambiguous identification of the type and the position of several important protein posttranslational modifications [16 -18] and for de novo s...

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“…Structural interpretation is greatly simplified for linear (noncyclic) molecules, as cleavage of only one bond is necessary to produce two pieces of the molecule. Rearranged product ions that complicate mass spectral interpretation [31] are usually minimal for MS/MS of linear proteins [37][38][39] and DNA/RNA [5,[12][13][14], and the extent of secondary fragmentation can be reduced by lowering the dissociation power or removing the products from further activation [40]. The primary product ions of linear proteins are complementary fragment ion pairs, each fragment containing one terminus of the molecule; thus if two product ion masses differ by the mass of an amino acid, there is a strong possibility that this residue is located that distance in mass from a terminus, although this could also arise by a coincidence in mass values of products containing different termini.…”

Section: Top-down Methodologymentioning

confidence: 99%

“…For protein and DNA/RNA identification and characterization by MS, two complementary strategies have evolved during the past 15 years: the "bottom-up" [1][2][3][4][5][6] and "top-down" [1,5,[7][8][9][10][11][12][13][14][15] approaches. In the most commonly used bottom-up approach, the mixture of large biomolecules of interest is first digested in solution, and then their combined small (generally <3 kDa) products are analyzed by MS and MS/MS to yield molecular weight (M r ) and fragment mass values, respectively.…”

Section: Introductionmentioning

confidence: 99%

Top-down identification and characterization of biomolecules by mass spectrometry

Breuker

Jin

Han

et al. 2008

J. Am. Soc. Mass Spectrom.

112

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The most widely used modern mass spectrometers face severe performance limitations with molecules larger than a few kDa. For far larger biomolecules, a common practice has been to break these up chemically or enzymatically into fragments that are sufficiently small for the instrumentation available. With its many sophisticated recent enhancements, this "bottom-up" approach has proved highly valuable, such as for the rapid, routine identification and quantitation of DNA-predicted proteins in complex mixtures. Characterization of smaller molecules, however, has always measured the mass of the molecule and then that of its fragments. This "top-down" approach has been made possible for direct analysis of large biomolecules by the uniquely high (>10 5 ) mass resolving power and accuracy (~1 ppm) of the Fourier-transform mass spectrometer. For complex mixtures, isolation of a single component's molecular ions for MS/MS not only gives biomolecule identifications of far higher reliability, but directly characterizes sequence errors and posttranslational modifications. Protein sizes amenable for current MS/MS instrumentation are increased by a "middle-down" approach in which limited proteolysis forms large (e.g., 10 kDa) polypeptides that are then subjected to the top-down approach, or by "prefolding dissociation". The latter, which extends characterization to proteins >200 kDa, was made possible by greater understanding of how molecular ion tertiary structure evolves in the gas phase.

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Analysis of nucleic acids by FTICR MS

Cited by 92 publications

References 103 publications

Dissecting the Protein–RNA and RNA–RNA Interactions in the Nucleocapsid-mediated Dimerization and Isomerization of HIV-1 Stemloop 1

Dissecting the Protein–RNA and RNA–RNA Interactions in the Nucleocapsid-mediated Dimerization and Isomerization of HIV-1 Stemloop 1

A mechanistic study of the electron capture dissociation of oligonucleotides

Top-down identification and characterization of biomolecules by mass spectrometry

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