Peptide level immunoaffinity enrichment enhances ubiquitination site identification on individual proteins

Abstract

Ubiquitination is a process that involves the covalent attachment of the 76-residue ubiquitin protein through its C-terminal di-glycine (GG) to lysine (K) residues on substrate proteins. This post-translational modification elicits a wide range of functional consequences including targeting proteins for proteasomal degradation, altering subcellular trafficking events, and facilitating protein-protein interactions. A number of methods exist for identifying the sites of ubiquitination on proteins of interest, including site-directed mutagenesis and affinity-purification mass spectrometry (AP-MS). Recent publications have also highlighted the use of peptide-level immunoaffinity enrichment of K-GG modified peptides from whole cell lysates for global characterization of ubiquitination sites. Here we investigated the utility of this technique for focused mapping of ubiquitination sites on individual proteins. For a series of membrane-associated and cytoplasmic substrates including erbB-2 (HER2), Dishevelled-2 (DVL2), and T cell receptor α (TCRα), we observed that K-GG peptide immunoaffinity enrichment consistently yielded additional ubiquitination sites beyond those identified in protein level AP-MS experiments. To assess this quantitatively, SILAC-labeled lysates were prepared and used to compare the abundances of individual K-GG peptides from samples prepared in parallel. Consistently, K-GG peptide immunoaffinity enrichment yielded greater than fourfold higher levels of modified peptides than AP-MS approaches. Using this approach, we went on to characterize inducible ubiquitination on multiple members of the T-cell receptor complex that are functionally affected by endoplasmic reticulum (ER) stress. Together, these data demonstrate the utility of immunoaffinity peptide enrichment for single protein ubiquitination site analysis and provide insights into the ubiquitination of HER2, DVL2, and proteins in the T-cell receptor complex.

Fig. 1.

Methods to map ubiquitination sites. A, Schematic of the gel-based method for identifying ubiquitination sites on proteins of interest (POI). Proteins are immunoprecipitated using an immunoaffinity tag or with an antibody specific for a POI followed by SDS-PAGE analysis. Anti-ubiquitin Western blots are used to confirm the location of ubiquitinated POI and Coomassie stained gel bands are cut accordingly. In-gel tryptic digestions are performed, peptides are extracted, and then analyzed by LC-MS/MS. This example shows a representative Western blot and Coomassie from the HA-tagged TCRα studies. B, In solution digestion method on a POI. This is the same as (A) except trypsin digestions are performed immediately post-IP (in the presence of the IP antibody) and no SDS-PAGE analysis is performed. C, Schematic of the K-GG peptide immunoaffinity enrichment method for mapping ubiquitination sites on a POI. Whole cell lysates are digested with trypsin and the resultant peptides are immunoprecipitated with an antibody against the K-GG remnant left on ubiquitinated lysine residues. Peptides are eluted and analyzed by LC-MS/MS.

Fig. 2.

Ubiquitination site mapping on DVL2, HER2 and TCRα. Coverage reports obtained for (A) DVL2 using the in-solution digestion method, (B) HER2, and (C) TCRα using the gel-based method of ubiquitination site mapping. Representative spectra for ubiquitinated peptides from (D) DVL2, (E) HER2, and (F) TCRα using the K-GG peptide immunoaffinity enrichment method.

Fig. 3.

K-GG peptide immunoaffinity enrichment reveals ubiquitination sites on the kinase domain of HER2. A, The main structure of the kinase domain is labeled in yellow and lysine residues identified as ubiquitin carriers are labeled in blue in the left panel. The structure is rotated 180 degrees in the right panel. B, Same as A except the structure is shown in the context of the HER2 kinase domain homo-dimer. One monomer is labeled in yellow and one monomer is labeled in green for clarity.

Fig. 4.

K-GG peptide immunoaffinity enrichment out-performs conventional methods of ubiquitination site mapping on single proteins. A, Cells grown in heavy media were lysed and 10 mg of protein was subjected to the K-GG peptide immunoaffinity enrichment method; cells grown in light media were lysed and 10 mg of protein was subjected to the gel-based method. Peptides eluted from each method were reconstituted separately in equal volumes. Samples were combined at a 1:1 ratio and examined by LC-MS/MS to compare the relative abundance of ubiquitinated peptides from our protein of interest isolated from each method. An example of a representative ubiquitinated peptide from (B) HER2 or (C) TCRα is shown. The top left panel shows the total ion chromatogram followed by the extracted ion chromatograms for ions corresponding to the heavy and light peptides (light ion on top, heavy ion on bottom). The middle panel is a narrow range full-MS scan depicting the relative intensity of the SILAC pair and the MS/MS spectrum for the ubiquitinated peptide is on the left panel. Heavy ions are labeled in green and light ions are labeled in red. b- and y- fragment ions are denoted by blue and red respectively. N.L. is the normalized level of the base peak for each ion.

Fig. 5.

K-GG peptide immunoaffinity enrichment identifies ubiquitination sites on endogenous T-cell receptor components during ER stress. Jurkat T-cells were untreated, treated with tunicamycin, treated with MG132 or treated with a combination of tunicamycin and MG132. 35 mg of cellular lysate for each condition was digested with trypsin and subjected to K-GG peptide immunoaffinity enrichment. Area under the curve was quantified using the VistaQuant algorithm in XQuant mode. Protein coverage is represented by blue lines and individual ubiquitination sites for TCRα (A), TCRβ (B), or TCRζ (C) are represented as red circles on a linearized version of the protein. The area of the circle indicates the relative abundance among conditions on a logarithmic scale.