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

The combination of chemical probing and high-resolution mass spectrometry constitutes a powerful alternative for the structural elucidation of biomolecules possessing unfavorable size, solubility, and flexibility. We have developed nested Arg-specific bifunctional crosslinkers to obtain complementary information to typical Cys- and Lys-specific reagents available on the market. The structures of 1,4-phenyl-diglyoxal (PDG) and 4,4'-biphenyl-diglyoxal (BDG) include two identical 1,2-dicarbonyl functions capable of reacting with the guanido group of Arg residues in proteins, as well as the base-pairing face of guanine in nucleic acids. The reactive functions are separated by modular spacers consisting of one or two benzene rings, which confer greater rigidity to the crosslinker structure than it is afforded by typical aliphatic spacers. Analysis by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has shown that the probes provide both mono- and bifunctional products with model protein substrates, which are stabilized by the formation of diester derivatives in the presence of borate buffer. The identification of crosslinked sites was accomplished by employing complementary proteolytic procedures and peptide mapping by ESI-FTICR. The results showed excellent correlation with the solvent accessibility and structural context of susceptible residues, and highlighted the significance of possible dynamic effects in determining the outcome of crosslinking reactions. The application of nested reagents with different spacing has provided a new tool for experimentally recognizing flexible regions that may be involved in prominent dynamics in solution. The development of new bifunctional crosslinkers with diverse target specificity and different bridging spans is expected to facilitate the structure elucidation of progressively larger biomolecular assemblies by increasing the number and diversity of spatial constraints available for triangulating the position of crosslinked structures in the three dimensions.

Toward building a database of bifunctional probes for the MS3D investigation of nucleic acids structures.

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-chloroethyl)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.