Quantitative chemical proteomics approach to identify post-translational modification-mediated protein-protein interactions

Abstract

Post-translational modifications (PTMs) (e.g., acetylation, methylation, and phosphorylation) play crucial roles in regulating the diverse protein-protein interactions involved in essentially every cellular process. While significant progress has been made to detect PTMs, profiling protein-protein interactions mediated by these PTMs remains a challenge. Here, we report a method that combines a photo-cross-linking strategy with stable isotope labeling in cell culture (SILAC)-based quantitative mass spectrometry to identify PTM-dependent protein-protein interactions. To develop and apply this approach, we focused on trimethylated lysine-4 at the histone H3 N-terminus (H3K4Me(3)), a PTM linked to actively transcribed gene promoters. Our approach identified proteins previously known to recognize this modification and MORC3 as a new protein that binds H3M4Me(3). This study indicates that our cross-linking-assisted and SILAC-based protein identification (CLASPI) approach can be used to profile protein-protein interactions mediated by PTMs, such as lysine methylation.

Figure 1

Schematic for the CLASPI strategy to profile H3K4Me₃ binding partners in whole-cell proteomes.

Figure 2

Profiling of H3K4Me3 readers by CLASPI. (a) A 2D-plot showing the Log2 values of the SILAC ratios (L/H) of each identified protein for the ‘forward’ (x axis) and ‘reverse’ (y axis) experiments. The H3K4Me₃-binding and unmodified H3 (H3K4Me0)-interacting partners are indicated in the top right and bottom left quadrants, respectively. Representative MS spectra of peptide from (b) non-specific binders (HSP90A), (c) H3K4Me3 binders (ING2) and (d) H3K4Me0 binders (BHC80). The ‘light’ and ‘heavy’ peptide isotopes are indicated by blue and red dots, respectively.

Figure 3

Characterization of CLASPI-identified histone methylation ‘readers’. Isothermal titration calorimetry measurements for the binding affinities of (a) SPIN1 and (b) MORC3 for the indicated histone H3 peptides. (c) A summary of dissociation constants (K_d) of SPIN1 and MORC3 for histone peptides. The values are the average of two independent measurements ± standard deviation.