Figure 3. ¹⁵N-labeling reveals the interaction of Pif1 with the N-terminus of Rim1.

¹⁴N and ¹⁵N-labeled Rim1 proteins were mixed with Pif1 and cross-linking using SDA was performed. De novo sequencing followed by matching to a sequence database using PEAKS Studio platform revealed Pif1 peptide cross-linked to the Rim1 N-terminal peptide, MDFSK. A, B, The N-terminal peptide of Rim1 has 6 nitrogen atoms, resulting in a visible isotopic doublet in MS spectra. The isotopic doublet shown is the sum of MS spectra over 2 min elution window. C, Sequence coverage map, annotated by PEAKS: MDFSK sequences does not contribute ions to the cross-linked spectra, and when treated as a non-specific modification results in multiple hits clustered around AIEDENE region, indicate by “#” (¹⁴N), and by “X” (¹⁵N). Oxidation of methionine is labeled with shaded “o”. D,E Two high scoring MSMS spectra are shown: ¹⁵N-labeled and ¹⁴N-labeled, as indicated. Masses of y20, y18, and y17[2+] fragments are indicated. F, The sequence of Pif1 isoform used in this work, with the peptide cross-linked to MDFSK highlighted.