Degronomics: Mapping the Interacting Peptidome of a Ubiquitin Ligase Using an Integrative Mass Spectrometry Strategy

Abstract

Human cells make use of hundreds of unique ubiquitin E3 ligases to ensure proteome fidelity and control cellular functions by promoting protein degradation. These processes require exquisite selectivity, but the individual roles of most E3s remain poorly characterized in part due to the challenges associated with identifying, quantifying, and validating substrates for each E3. We report an integrative mass spectrometry (MS) strategy for characterizing protein fragments that interact with KLHDC2, a human E3 that recognizes the extreme C-terminus of substrates. Using a combination of native MS, native top-down MS, MS of destabilized samples, and liquid chromatography MS, we identified and quantified a near complete fraction of the KLHDC2-binding peptidome in E. coli cells. This degronome includes peptides that originate from a variety of proteins. Although all identified protein fragments are terminated by diglycine or glycylalanine, the preceding amino acids are diverse. These results significantly expand our understanding of the sequences that can be recognized by KLHDC2, which provides insight into the potential substrates of this E3 in humans. We anticipate that this integrative MS strategy could be leveraged more broadly to characterize the degronomes of other E3 ligase substrate receptors, including those that adhere to the more common N-end rule for substrate recognition. Therefore, this work advances "degronomics," i.e., identifying, quantifying, and validating functional E3:peptide interactions in order to determine the individual roles of each E3.

Figure 1.

Native mass spectrum of KLHDC2 expressed in E. coli cells. In addition to features expected for apo KLHDC2, additional features corresponding to ions that are approximately 400–1500 Da larger in mass relative to the apo protein are also observed. These features are attributed to KLHDC2:peptide complexes. Intensities below 3200 m/z are increased tenfold to aid in visualization.

Figure 2.

Native top-down MS of KLHDC2:peptide complexes. (A) Complexes were ionized and subjected to collision-induced dissociation (CID) at the atmospheric-pressure interface of the mass spectrometer, resulting in the release of peptide ions that are labeled using the IDs in Table 1. Intensities are increased fivefold to aid in visualization. Additional ions are annotated in Figure S2. (B) Ion F was isolated and subjected to CID in the trap collision cell of the instrument. CID fragments (labeled using the scheme of Roepstorff and Fohlman) were used to assign ion F to ASDEGEVIVFGG, which is a fragment of KLHDC2. (C) Synthesized ASDEGEVIVFGG was resuspended in aqueous 200 mM ammonium acetate, ionized using electrospray, and subjected to CID in the trap collision cell of the same instrument. The fragmentation spectra in B and C are nearly identical, which corroborates the assignment of peptide F to ASDEGEVIVFGG.

Figure 3.

Integrative MS for degronomics. Native MS enabled the direct observation and mass determination of E3:peptide complexes that were purified from cells. Native top-down MS provided primary amino acid sequences for a subset of the bound peptides. Complementary MS-based approaches, including destabilized-sample MS² and LC-MS² provided additional peptide identifications. Together, these methods provide an integrative strategy to identify and quantify protein fragments interacting with an E3 in cells that could be used to study E3:degron interactions more broadly.

Figure 4.

Identification and quantification of interacting peptides. (A) Native mass spectrum of the 12+ KLHDC2:peptide complex ions, plotted using a false mass axis that is relative to apo KLHDC2. Vertical bars are plotted using the masses of the interacting peptides that had a 1% or greater relative abundance and are labeled using the IDs in Table 1. For comparison, this region is plotted with all peptide IDs as a function of m/z in Figure S5. (B) The total residual (Equation 1) of the experiment (black trace) and the sum of modelled components (blue trace) was minimized by optimizing the relative intensity of each component. The contribution from each KLHDC2:peptide complex is represented using a different color. (C) The relative abundances of the interacting peptides determined using this approach for the 11+, 12+, and 13+ ions.

Figure 5.

The peptide F (ASDEGEVIVFGG) was synthesized and added at various concentrations to the sample of KLHDC2 expressed in E. coli cells. (A-E) The intensity of the feature for the KLHDC2:F complex increases with peptide concentration, which is consistent with the intensities in the native mass spectra depending on the abundances of the corresponding complexes in solution. (F) The relative abundances of apo KLHDC2 and the KLHDC2:peptide complexes were determined for the spectra shown in A to E using the same method used for Figure 4. (G) The abundance of the KLHDC2:F complex relative to apo KLHDC2 and all KLHDC2:peptide complexes as function of the concentration of additional ASDEGEVIVFGG after it was spiked into the sample.

Figure 6.

Recognition of KLHDC2 protein fragments. (A) The sequences near the six internal diglycines of KLHDC2 are shown. The colored regions represent internal peptides that were identified using integrative MS. (B) These internal fragments are highlighted on the structure of KLHDC2 and correspond by color to the sequences in panel A (diglycines represented as spheres). (C) The peptide ASDEGEVIVFGG binds to KLHDC2 with an IC₅₀ value of 5.3 nM. (D) LOGO plot of all peptides in Table 1.