FIGURE 3.

Splicing of long peptides predominantly occurs via cis-splicing. (A) Splicing of a mixture of ¹⁵N-Gly–labeled (indicated in bold) and unlabeled precursor peptides through cis-splicing or through random splicing results in differently labeled spliced products and therefore in different isotope patterns that can be distinguished by LC-MS. (B) Left panel, Mass spectrum (black trace) and the corresponding experimental isotope pattern (red bars) of [YLGD][KLGSV] resulting from the digestion of a mixture of ¹⁵N-labeled and unlabeled precursor YLGD-SY-KLGSV. Right panel, Comparison of the experimental isotope pattern with theoretical isotope patterns for cis- and random splicing. (C) Mass spectra (black traces) and the corresponding experimental isotope pattern (blue bars) of [GSSFTIMTD][GLASYKIFKIEKGKV], resulting from the digestion of a mixture of ¹⁵N-Gly–labeled (indicated in bold) and unlabeled precursor GSSFTIMTD-GPSD-GLASYKIFKIEKGKV for different time periods. (D) Mass spectrum (black trace) of a 1:1 mixture of single-isotope incubation samples, in which the labeled and ¹⁵N₂-labeled splicing products are present in equal amounts, compared with the theoretical isotope pattern for cis-splicing (gray bars). (E) Comparison of the experimental isotope patterns (blue bars) determined in (C) with theoretical isotope patterns for cis- and random splicing.