Fig. 3.

Sequence analysis of peptide-RNA (oligo)nucleotide cross-links derived from human spliceosomal complex U1 SnRNP specific SmB/B' peptide CILQDGR (positions 19–25). A, MS/MS spectrum of m/z 646.8510³⁺ in the presence of DTT shows the peptide cross-linked to the trinucleotide AAU with an additional mass of 152 Da. The fragment nominally corresponding to the [M+H]⁺¹ of the unmodified peptide is annotated as p⁺. All signals corresponding to RNA and RNA fragments and shifting of peptide signal fragment signals by different RNA fragments via 152 Da mass are annotated and highlighted by bold letters according to the nomenclature by McLuckey et al. (61). Marker ions of the adenine base and nucleotide with loss of water are annotated as A' and [y_r1-(y_r-1)] respectively. Similarly, uracil base and uracil nucleotide with the loss of water are annotated as U' and (y_r2-y_r1) respectively. Shifting in signal of uracil nucleobase (U') with 152 Da is also depicted. B, MS/MS spectrum of m/z 972.7957²⁺ in the presence of d₁₀-DTT shows the peptide cross-linked to the trinucleotide AAU with the additional mass of 158 Da. Signals in the high m/z range correspond to shifting of mass of peptide shifted peptide fragment masses by different RNA fragments along with 158 Da mass, and are annotated as in spectrum A. The difference of 6 Da is clearly observed between the two spectra in the high m/z region. C, Proposed structure for peptide-DTT-uracil cross-link formation. Upon UV irradiation at 254 nm, DTT can form covalent bonds with uracil and the thiol group of cysteine.