Journal
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
Volume 17, Issue 11, Pages 1570-1581Publisher
AMER CHEMICAL SOC
DOI: 10.1016/j.jasms.2006.06.002
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Funding
- NIGMS NIH HHS [R01-GM643208] Funding Source: Medline
- Direct For Mathematical & Physical Scien
- Division Of Chemistry [1202641] Funding Source: National Science Foundation
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This report illustrates the approaches employed to investigate critical aspects of the activity of crosslinking reagents toward nucleic acid substrates, which should be evaluated to identify candidate probes for mass spectrometric 3D (MS3D) investigations of biomolecules and macromolecular complexes. Representative members of different classes of bifunctional reagents were taken into consideration, including bikethoxal and phenyl-diglyoxal [bis-(1,2-dicarbonyls)], cisplatin (coordinative binding agents), chlorambucil and nitrogen mustard [bis-(2-chdoroethyl)amines], and sym-triazine trichloride (triazines). Nanospray-Fourier transform mass spectrometry (FTMS) was applied without desalting or separation procedures to characterize the covalent products obtained by probing dinucleotide and trinucleotide substrates under a variety of experimental conditions in vitro. The carefully controlled composition of these substrates enabled us to obtain valid comparisons of probe activity toward individual nucleotides and evaluate possible base-specific effects, including the stability of the different adducts in solution under the selected reaction conditions. The gas-phase behavior of the observed products was investigated using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) to obtain valuable information for guiding the design of sequencing experiments and helping the data interpretation. Structured RNA substrates, such as HIV-1 stemloop 1, were finally employed to investigate the structural determinant of adduct formation and highlight the different nature of the spatial information provided by the various candidate probes.
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