Protein ubiquitination is a common post-translational modification that affects several cellular processes such as protein degradation, protein localization, DNA repair, transcriptional regulation, cell cycle regulation, apoptosis and DNA repair. Some methods of studying the ubiquitin proteome, known as the ubiquitinome, take a global approach that relies on mass spectrometry-based proteomics, while other strategies, such as immunoblotting, target specific proteins of interest.
Research involving ubiquitination has been building over the last decade, and some researchers are now beginning to shift their focus from the ubiquitination of individual proteins to the identification of protein complexes that can be regulated through ubiquitination. Related to this goal, Schwertman et al. recently published a study involving a new method of purifying endogenously ubiquitinated protein complexes for mass spectrometry (MS) using immunoaffinity purification.1 This research group initially tested three different methods of isolating and purifying ubiquitinated proteins. Method one used a high-affinity ubiquitin trap agarose–TUBEs, which are based on ubiquitin-binding domains that bind only polyubiquitinated conjugates. The second method employed UbiQapture-Q matrix, a high-affinity ubiquitin trap. UbiQapture-Q is also based on ubiquitin-binding domains; however, it is able to bind both monoubiquitinated and polyubiquitinated proteins. The third method utilized the anti-ubiquitin antibody FK2. In the initial studies, the FK2 method was the most efficient method of isolating endogenously ubiquitinated protein complexes and recognizing monoubiquitinated and polyubiquitinated conjugates. Out of the three methods analyzed, the FK2 method also resulted in the highest yield of ubiquitinated proteins.
Experiments using the FK2 antibody were performed on HeLa and XP2OS cells that were cultured, washed and later incubated with the FK2 beads. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed and followed by in-gel trypsin digests in preparation for MS. Nanoflow liquid chromatography–tandem mass spectrometry (LC–MS/MS) was performed on an 1100 Series capillary LC system (Agilent Technologies) coupled to an LTQ Orbitrap XL mass spectrometer (Thermo Scientific).
Immunoblotting and silver staining made it possible to visualize the total protein levels in the experimental samples. The MS analysis of two independently executed FK2 immunoprecipitations identified 296 FK2-specific proteins in both experiments. Pathway analyses, via Ingenuity Pathway Analysis and Gene Ontology-annotation enrichment, confirmed the isolation of both ubiquitinated and ubiquitination-related proteins, and revealed that FK2-specific proteins were residing in protein complexes, rather than in random protein sets. Ubiquitinated proteins were identified from 32 canonical pathways, and analysis of protein networks revealed that 53% of the FK2-specific proteins interacted with at least one other protein within the FK2-specific protein group. The results revealed that this immunoaffinity purification method has the ability to isolate endogenously ubiquitinated proteins and to identify interacting proteins.
This new method to isolate and purify ubiquitinated protein complexes using FK2 immunoprecipitations can also be combined with other quantitative techniques to identify changes in the ubiquitinome in response to environmental stresses, disease states and drug therapies. In a previous publication, Schwertman et al. applied the same strategy — using FK2 immunoprecipitations, combined with SILAC-based proteomics — to investigate transcription-coupled repair in UV-sensitive syndrome, and found UV-stimulated scaffold protein A as the causative gene for UV-sensitive syndrome.2 Taken together, both studies from Schwertman et al. add value to this method of analyzing ubiquitinated protein complexes.
1. Schwertman, P., et al. (2013, June 3) “An immunoaffinity purification method for the proteomic analysis of ubiquitinated protein complexes,” Analytical Biochemistry, pii: S0003-2697(13)00251-0, doi: 10.1016/j.ab.2013.05.020 [Epub ahead of print].
2. Schwertman, P., et al. (2012) “UV-sensitive syndrome protein UVSSA recruits USP7 to regulate transcription-coupled repair,” Nature Genetics, 44 (pp. 598–602).
Post Author: Emily Humphreys. As a biology undergraduate at the University of Utah, Emily balanced a heavy class schedule while working long hours in a lab studying eye development. Following graduation, she became involved in infectious disease and aging research involving SNPS.
While she enjoyed the thrill of research, Emily has since traded bench work for science journalism.
And has been a regular contributor to Accelerating Science since 2012.