Figure 2. Identification of Pif1-Rim1 interaction site using SDA cross-linking.

The charge-deconvoluted tandem MS/MS spectrum obtained using HCD fragmentation from the precursor at 643.078 m/z is shown. Mass deviation from the theoretical mass was 4.83 ppm. The precursor retention time was 29 min, which is close to the expected value, based on the hydrophobicity of the cross-linked peptides. Mass deviation of the fragments was within 20 ppm, on average. Possibility of one C¹³ atom was allowed in calculations of the mass deviation. The most intense ions and the ions indicative of cross-link fragmentation were annotated by StavroX, xComb, and manually. Fragments contacting cross-links are shown in bold. Fragments used to identify the cross-linked residue on Rim1 are marked with (**). For the details of StavroX and xComb annotations see Supplemental Figure 4. Stavro score was 109, with the 5% false discovery rate corresponding to the score above 68, according to the Stavro decoy analysis (Supplemental Figure 4, C). Greek letters α and β correspond to the cross-linked peptides from Rim1 and Pif1, respectively. Fragments related to the loss of the cross-link are marked with asterisk (*). Letter ‘M’ notes precursor-related ions