Figure 2.

Mass spectrometric detection of biotin accessibility of Ycg1. (A) Schematic presentation of biotin labeling of non-SMC proteins with sulfo-NHS biotin. (B) LC–MS/MS identification of sulfo-NHS-biotin-labeled peptide. Biotinylated lysines were detected through their increased m/z value. The y-ion series includes fragments that contain the C-terminus of the intact peptide. Note the large mass increment between y3 and y4 corresponding to the biotinylated lysine. (C) Identification of protein–protein and protein–DNA interactions within the non-SMC subunits. (D) Ycg1 lysines that were labeled identically in Ycs4-Brn1-Ycs4 (Ycs4-BY) and Ycs4-BY-DNA complexes. Tick marks and labels indicate every other lysine in the protein. Lysines in forest green color signify the identically labeled lysines in the presence and absence of DNA. Lighter green-colored lysines represent identically unlabeled but detected lysines in both the complexes. (E) Differentially labeled Ycg1 lysines in Ycs4-BY and Ycs4-BY-DNA complexes. Ruby color denotes lysines labeled in the absence of DNA but unlabeled in the presence of DNA or with the biotinylation level reduced by more than 80%. Salmon color denotes lysines labeled in the absence of DNA but undetected in the presence of DNA. Warm pink lysines are the differentially detectable lysines that are inaccessible to biotinylation.