Fig. 6: Identification and quantification of potential interaction sites of Imh1-C213 by isotope-labeled crosslinkers and MS.
From: Glycerol mediates crosstalk between metabolism and trafficking through the golgin Imh1
a, Schematic representation of the workflow of crosslinking MS analysis with or without glycerol treatment. The purified His-tagged Imh1-C213 fragment (C-terminal 699–911 residues) was first preincubated with or without 0.1 M glycerol and then crosslinked with equimolar amounts of d0 or d4 BS3. The crosslinking step was repeated with an exchange of labels. Equal amounts of light (d0) and heavy (d4) crosslinked proteins were pooled and subjected to trypsin digestion and LC–MS/MS analysis. The crosslinked peptides were identified and quantified using the pLink3 software. The detailed procedure is described in Methods. b, Map of potential crosslinked sites in His–Imh1-C213 dimer in the absence or presence of 0.1 M glycerol. A total of 13 crosslinked peptide pairs, consistently detected all across six experiments, each supported by at least one spectrum match with a pLink3 spectrum score > 0.85 in every experiment, are presented. The crosslinked lysine residues are represented by connecting lines in different colors on the basis of quantified ratios (glycerol to control). Six crosslinks displayed consistent regulatory trends in at least three of the six quantified ratios, with mean glycerol-to-control ratios of >1.05 or <0.95. Specifically, crosslinks with increased abundance after glycerol treatment are represented by red lines (solid for ratios ≥ 1.1; dashed for ratios between 1.1 and 1.05), while those with decreased abundance are shown in blue (solid for ratios ≤ 0.90; dashed for ratios between 0.90 and 0.95). Crosslinks that do not meet these criteria are displayed in gray. Potential hydrophobic regions in the amino acid sequence are highlighted in yellow. The crosslinked sites and their peptide sequences in the map are listed in Supplementary Table 1. The MS data were deposited to the ProteomeXchange consortium through the PRIDE partner repository with the dataset identifier PXD061125.
