Journal: Nucleic Acids Research

Article Title: Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes

doi: 10.1093/nar/gkv1366

Figure Lengend Snippet: Prediction and validation of Mtr2-CH anchored cross-links on the Mex67-PrA/Mtr2-CH complex. ( A ) Mex67-PrA and Mex67-PrA/Mtr2-CH complexes were purified under low salt (lanes 1–8) or high salt conditions (lanes 9 and 10). Samples were taken before (lanes 1, 3, 5, 7, 9) and after cross-linking (lanes 3, 8, 10), or prior to trypsin digest in control samples (lanes 2, 6). Proteins cross-linked to Mtr2-CH were resolved by western blotting and visualized by anti-His antibody (lanes 5–10). Anti-His antibody cross-reactive bands are indicated with an asterisk (*). Mex67-PrA was visualized with peroxidase anti-peroxidase antibody (lanes 1–10). A Mex67-Mex67 dimer in the cross-linked control sample (Lane 4) is denoted with Δ. Cross-linked proteins are inferred by molecular weight of bands and listed in Tables and . ( B ) MS/MS spectra of the validated cross-link between C186 of Mtr2-CH and Mex67 K120. Peptides and fragment ions derived from Mex67 or Mtr2 shown in green and blue, respectively. ( C ) Close-up view of XWalk-predicted cross-links between the cysteine of the Mtr2-CH (C186; orange) and nearby lysines on Mex67 (PDB ID: 4WWU.PDB) . Predicted and confirmed crosslinks are depicted in red (T119, K120, (K343, K348), and predicted cross-links that were not identified in yellow (K191). The lines are the solvent accessible surface distances (SASD) measurements, which correspond to the length of the shortest path between Mtr2 (C186) and nearby lysines, where the path leads through solvent occupied space without penetrating the protein surface. ( D ) Representation of a Mex67:Mtr2 tetramer complex obtained recently (PDB ID: 4WWU.PDB) , to which the cross-linking anchoring CH-tag (dark grey) containing the cross-linker anchoring cysteine (orange) has been added at the C-terminal of Mtr2 in I-TASSER . Mex67 molecules are coloured light and dark-green, Mtr2 in cyan and blue. Identified cross-links within Mex67and Mtr2 are depicted in red.

Article Snippet: His-tagged proteins were detected using a mouse anti-His antibody (ABM), followed by an HRP-conjugated anti-mouse antibody (GE Healthcare).

Techniques: Purification, Western Blot, Molecular Weight, Tandem Mass Spectroscopy, Derivative Assay