Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Post-translational modification plays a crucial role in regulating many cellular functions. One such modification is ubiquitination, which helps control protein turnover, cell cycling, apoptosis and DNA repair.
In ubiquitination, the 8-kDa ubiquitin (Ub) peptide is coupled to lysine residues of target proteins, singly or as a “poly-Ub” chain. Isolating ubiquitinated proteins is usually accomplished via affinity purification. Here are your options.
Antibodies and binding domains
According to Anthony Couvillon, a development scientist at Cell Signaling Technology (CST) there are four main types of tools available for studying protein ubiquitination. One is an antibody against the ubiquitin peptide, which binds free and linked Ub and can be used in conjunction with additional antibodies for downstream target analysis. “[For] profiling pathway-specific changes in ubiquitination on a low-to-medium throughput scale, antibodies or non-antibody affinity reagents could be used to first isolate a specific subset of the total cellular ubiquitinated proteins, prior to western blot using target-specific antibodies,” says Couvillon.
In addition to antibodies against total ubiquitin, CST offers antibodies that are specific for target proteins that have been ubiquitinated at a specific site as well as antibodies that detect only polyubiquitin chains with particular linkages (such as an antibody to “K63-linkage specific polyubiquitin”). In addition, it offers an antibody that can be used for enriching ubiquitinated peptides (as opposed to intact proteins; see below).
An alternative strategy uses Ub-binding domains to purify ubiquitinated proteins. Ub-binding domains are non-antibody affinity reagents that are easy to produce for affinity purification. However, they vary in their binding characteristics. “Variability of reagent specificity and selectivity means that multiple reagents must be used to enrich for all ubiquitinated proteins,” Couvillon says.
One commercial option is MultiDsk, an affinity resin studded with five Ub-binding domains available from Abcam.
The UbiQapture®-Q Kit from Enzo Life Sciences also isolates Ub proteins using Ub-binding domains. The kit uses a high-binding-affinity matrix to isolate Ub proteins from cell extracts and tissue lysates. Subsequent western blot or other analysis of the captured Ub proteins can be performed with antibodies to ubiquitin (included) or antibodies to your protein of interest.
According to Courtney Noah, senior marketing manager at Enzo Life Sciences, one advantage of the UbiQapture-Q Kit is that its matrix-binding properties “permit complete isolation of a full range of ubiquitin-protein conjugates from a specific lysate.”
Affinity tags
Another protein-enrichment strategy is overexpression of target proteins and/or ubiquitin that have been labeled with affinity tags, such as polyhistidine residues (6xHis), FLAG or streptavidin. Following overexpression of the tagged proteins, enrichment is fairly straightforward, because protocols for purifying proteins with these kinds of tags are already well established.
Such tools—with a twist—are used by Petro Starokadomskyy, a research scientist in the departments of molecular biology and internal medicine at University of Texas Southwestern Medical Center. Using a widely used protocol called tandem affinity purification as a guide, he created a variation known as bimolecular affinity purification (BAP) [1].
BAP provides a useful strategy to enrich for specific, target Ub proteins, Starokadomskyy says. In this method, he attaches an affinity tag to a target protein (the protein that will become ubiquitinated), and then attaches a different affinity tag to recombinant ubiquitin. After overexpression of both proteins in cultured cells and ubiquitination, the protein can be purified using both tags, resulting in a highly enriched sample. The method also “allows selective purification of its ubiquitin-modified forms with co-precipitation of their interacting co-factors,” says Starokadomskyy.
Though useful and widely adopted, overexpression also poses potential problems. According to Blagoy Blagoev, professor in the department of biochemistry and molecular biology at the University of Southern Denmark, overexpressing ubiquitin leads to higher levels of free ubiquitin in cells. When this occurs, “proteins start to undergo unregulated ubiquitination,” he says. “For example, proteins become ubiquitinated on different sites than their usual, physiological sites, [and] proteins that are usually not ubiquitinated are now being ubiquitinated.” Another complication of ubiquitin overexpression is that endogenous ubiquitin is still present. “Therefore, only a fraction of cellular [ubiquitin] contains the tagged ubiquitin,” Blagoev says. “Only this fraction is targeted for enrichment and subsequent analyses.”
To address these problems, Blagoev developed a system called StUbEx (stable tagged ubiquitin exchange) [2]. Using this system, he replaces endogenous ubiquitin with recombinant ubiquitin labeled with the 6xHis and FLAG affinity tags, rather than overexpressing the tagged ubiquitin. Consequently, virtually all the ubiquitin expressed by cells is tagged and usable for enrichment. Importantly, the tagged ubiquitin expressed in the StUbEx system is maintained at normal levels in cells, suggesting that overexpression artifacts are minimized or eliminated.
Enriching at the peptide level
The StUbEx system, the BAP method and commercial affinity tools can all be used in conjunction with enrichment of ubiquitinated protein fragments at the peptide level. Here, “the most common method used is enrichment with an antibody against the Gly-Gly remnant that is obtained after tryptic digestion,” says Sonja Hess, director of the proteome exploration laboratory in the Beckman Institute at the California Institute of Technology and author of a recent review on the topic [3].
Trypsin cleaves ubiquitin groups from the purified target protein, leaving a diglycine signature at the site of ubiquitin attachment. According to Starokadomskyy, the residues correspond to a mass shift of 114 Daltons, which he uses to identify ubiquitinylation sites on target proteins in a mass spectrometer. Alternatively, researchers can detect ubiquitination sites by Western blotting using tagged antibodies.
“The highest number of ubiquitination sites are usually detected on the peptide level,” says Hess, “but it is currently not possible to say what type of ubiquitination was present at each site.” That's because the ubiquitin itself is gone following trypsiniztation. Enrichment at the protein level, she says, can yield “more mechanistic insights, such as whether the protein is mono- or polyubiquitinated.”
One commercial implementation of this enrichment strategy is CST’s PTMScan® proteomics platform. By using the diglycine antibody, the approach enables identification of “thousands of ubiquitinated sites on hundreds of proteins in a single sample,” Couvillon says.
Many researchers who focus on ubiquitination eventually use several or all of these enrichment strategies. The smartest route is to use the one that best matches your experimental goals—such as whether ubiquitination affects the actions of drugs. Hess has used all these enrichment methods but currently uses mainly “peptide-level enrichment of fractionated samples, because they give us the highest number of Gly-Gly sites,” she says. “This allows us to test whether certain drug treatments have an effect on specific proteins.”
References
[1] Starokadomskyy, P, and Burstein, E, “Bimolecular affinity purification: A variation of TAP with multiple applications,” Methods Mol Biol, 1177: 193–209, 2014. [PubMed ID: 24943324]
[2] Akimov, V, et al., “StUbEx: Stable Tagged Ubiquitin Exchange System for the global investigation of cellular ubiquitination,” J Proteome Res, 13: 4192–4204, 2014. [PubMed ID: 25093938]
[3] Porras-Yakushi, TR, and Hess S, “Recent advances in defining the ubiquitylome,” Expert Rev Proteomics, 11:477–90, 2014. [PubMed ID: 24961939]
Image: Lysine-48-linked diubiquitin, by Wikimedia Commons user "Rogerdodd." Source: Wikimedia Commons.