Top-Down Characterization of Nucleic Acids Modified by Structural Probes Using High-Resolution Tandem Mass Spectrometry and Automated Data Interpretation

Abstract

A top-down approach based on sustained off-resonance irradiation collision-induced dissociation (SORI-CID) has been implemented on an electrospray ionization (ESI) Fourier transform mass spectrometer (FTMS) to characterize nucleic acid substrates modified by structural probes. Solvent accessibility reagents, such as dimethyl sulfate (DMS), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT), and beta-ethoxy-alpha-ketobutyraldehyde (kethoxal, KT) are widely employed to reveal the position of single- vs double-stranded regions and obtain the footprint of bound proteins onto nucleic acids structures. Established methods require end-labeling of the nucleic acid constructs, probe-specific chemistry to produce strand cleavage at the modified nucleotides, and analysis by polyacrylamide gel electrophoresis to determine the position of the susceptible sites. However, these labor-intensive procedures can be avoided when mass spectrometry is used to identify the probe-induced modifications from their characteristic mass signatures. In particular, ESI-FTMS can be directly employed to monitor the conditions of probe application to avoid excessive alkylation, which could induce unwanted distortion or defolding of the substrate of interest. The sequence position of the covalent modifications can be subsequently obtained from classic tandem techniques, which allow for the analysis of individual target adducts present in complex reaction mixtures with no need for separation techniques. Selection and activation by SORI-CID has been employed to reveal the position of adducts in nucleic acid substrates in excess of 6 kDa. The stability of the different covalent modifications under SORI-CID conditions was investigated. Multiple stages of isolation and activation were employed in MS(n)() experiments to obtain the desired sequence information whenever the adduct stability was not particularly favorable, and SORI-CID induced the facile loss of the modified base. A new program called MS2Links was developed for the automated reduction and interpretation of fragmentation data obtained from modified nucleic acids. Based on an algorithm that searches for plausible isotopic patterns, the data reduction module is capable of discriminating legitimate signals from noise spikes of comparable intensity. The fragment identification module calculates the monoisotopic mass of ion products expected from a certain sequence and user-defined covalent modifications, which are finally matched with the signals selected by the data reduction program. Considering that MS2Links can generate similar fragment libraries for peptides and their covalent conjugates with other peptides or nucleic acids, this program provides an integrated platform for the structural investigation of protein-nucleic acid complexes based on cross-linking strategies and top-down ESI-FTMS.

Mapping noncovalent ligand binding to stemloop domains of the HIV-1 packaging signal by tandem mass spectrometry

“…All data were acquired in broadband mode and were processed using XMASS 7.0.8 (Bruker). Data reduction and analysis were performed with the aid of MS2Links [51] and the Mongo Oligo Mass Calculator version 2.05 made …”

“…Tandem mass spectrometry has been extensively used to identify the sequence of oligonucleotide samples [42][43][44][45][46] and characterize their covalent modifications [47][48][49][50][51] based on characteristic ions series produced by fragmentation of the nucleic acid backbone. In contrast, gas-phase activation of small ligand-nucleic acid complexes has been more actively pursued to investigate their stability rather than to map the actual binding sites [52][53][54][55][56].…”

Nested Arg-specific bifunctional crosslinkers for MS-based structural analysis of proteins and protein assemblies

“…Applied to PDG and BDG reaction mixtures, each protease provided series of hydrolytic peptides, which afforded excellent sequence coverage and allowed for the characterization of probe adducts. The identification of conjugated products was completed by using LINKS [68], a software package designed to support the analysis of crosslinked biomolecules. As summarized in Table 2, the results indicated that the crosslinkers were capable of modifying all available sites, although with different outcomes.…”

“…Probe-to-substrate ratios should always be kept as low as possible to reduce crosslinking yields and to minimize the risk of detecting sparsely populated conformational states [25,74]. Titration schemes in which the amount of probe is increased in stepwise fashion can effectively differentiate primary modification sites from secondary sites that are exposed only as a result of conformational changes induced by initial alkylation [68]. Yield and position of crosslinked products should be also evaluated by repeating probing reactions under a wide variety of environmental conditions that influence substrate conformation [25].…”

“…Each analysis was performed a minimum of three times. Theoretical mass lists of digestion products were generated using the PeptideMass software available in the ExPASy suite [67], or the LINKS algorithm that was specifically developed to aid the characterization of crosslinked biomolecules ( [68] and refs. therein).…”