Detecting Post-Translational Modifications
Post-translational modifications (PTMs) are one of the fastest-growing areas of molecular biological research. Detecting post-translational modifications, knowing how they work, influence the proteome, and regulate the genome will greatly improve our understanding of both genetics and epigenetics. Currently, more than 300 types of PTMs have been discovered, but the number of those that have been examined at the proteome level is much smaller.
The most commonly studied post-translational modifications are phosphorylation and acetylation, but others include ubiquitination, SUMOylation, and glycosylation. Check out our A-Z of post-translational modifications for more information on the different PTMs found!
Like any scientific work, moving from theory to laboratory practice can be the tricky part. There are many methods for detecting post-translational modifications. These methods vary greatly by the ease of use, sophistication (and cost!), as well as delivery of results.
Western Blotting for Detecting Post-Translational Modifications
This mainstay of the biochemistry laboratory has been used to detect proteins for decades and has been particularly useful for identifying PTMs.
Gel electrophoresis followed by Western blotting for specific proteins can sometimes be used to detect a shift in mobility. This is a useful technique when you have no idea what kind of modification to expect, as many modifications may cause a shift (such as ubiquitin ladders or a phosphorylation shift).
Specific antibodies have been raised to detect protein phosphorylation, methylation, acetylation, and ubiquitination. Researchers can use these modification-specific antibodies either on purified, enriched unmodified protein or specific for a PTM of the protein.
In-gel reagents can also be used to detect PTMs via Western blotting. One technique involves colloidal Coomassie staining, after which the proteins are partially transferred to a suitable membrane. PTMs are then immunodetected using fluorescence tagging. Other staining techniques include silver or fluorescent protein.
One drawback to western blotting is that the technique may not be specific enough to identify an individual protein change, and not all proteins have adequate antibodies available for detection.
Immunoprecipitation With Post-Translational Modification Affinity Beads
Immunoprecipitation (IP) is another laboratory workhorse and can be used to detect PTMs. With IP, it’s possible to enrich for low-abundance PTMs. Antibodies that are attached to a solid surface or resin will bind to the PTM (or target protein) exclusively, and isolate the proteins. Proteins can then be analysed further by other methods like Western blotting or Mass spectrometry (MS).
Detecting Post-Translational Modifications Using Mass Spectrometry
MS can detect nearly all PTMs and can also be used to identify unknown PTMs. Covalent modifications in proteins affect the molecular weight of modified amino acids, so the differences in mass can be detected by MS.
Some of the pros of MS are that it is very sensitive, can determine PTMs in complex matrices, and can point to the site of the PTM in the protein molecule. The technique can be used to more precisely analyse protein fragments separated by Western blot, IP, and other separation methods.
One MS method in particular, tandem MS (MS/MS), can determine the amino acid sequence in a modified protein segment, and compare it to an unmodified protein.
MS is an unbiased approach for searching for PTMs as it does not require specific antibodies to detect proteins. However, it still may not detect a target protein, depending on the abundance and availability of the protein in solution. In addition, MS is expensive, requires access to the instrument, and requires expertise to run samples and analyse the massive amounts of data produced.
In Vitro Assays for Detecting Post-Translational Modification
With this technique, purified or in vitro targeted proteins are examined to see if modifications occurred through a PTM. The purified protein is mixed with specific enzymes, substrate, other factors, and energy sources in a test tube. Once incubated, Western blotting is used to analyse the sample.
This technique is useful for determining if a protein can be modified, especially by phosphorylation, ubiquitination, and SUMOylation, among others. It can also be used to determine what regulatory enzymes are needed for PTM of the protein to occur. However, in vitro assays require purified or translated target proteins, as well as any enzymes required by a specific assay. As these substances are not tested at physiological concentrations it’s possible to generate false-positive results that do not exist in nature.
Post-Translational Modification Staining of Acrylamide Gels
Directly staining proteins in an acrylamide gel is another technique for the detection of PTMs. Certain stains can identify phosphorylation, glycosylation, or other signs of modification, and multiple stains can be used to identify different proteins in the same sample.
Some stains are compatible with MS and Western blotting for further analysis.
The advantage of staining is that no antibody is necessary, so proteins for which good antibodies are not available can be identified. However, this technique is not as specific as an antibody test and can detect changes (like phosphorylation) that might not be related to a PTM.
Choose Your Weapon — Carefully
For detecting post-translational modifications, any method will produce solid results. But what works for you will depend on several factors that vary widely among detection methods: cost, available antibodies (or whether you use an antibody at all), a target, or no target! Combinations of different methods can work well, too.
What methods are you using? How well are they working for you? Share your experiences with us in the comments.
Resources:
- ThermoFisher Scientific. Protein Modification and Fusion Tag Stains. Accessed 9 October 2020.
- Alpha Laboratories. Phos-tag™. Accessed 9 October 2020
- ROCKLAND™ Antibodies and Assays. Post-Translational Modification (PTM) Antibodies. Accessed 9 October 2020.
- Wikipedia. Post-translational modification. Accessed 9 October 2020.
- Cytoskeleton Inc. Post-Translational Modification Detection Techniques. Accessed 9 October 2020.
- ThermoFisher Scientific. Overview of Post-Translational Modifications (PTMs). Accessed 9 October 2020.
- Larsen MR, et al. Analysis of posttranslational modifications of proteins by tandem mass spectrometry. BioTechniques 2006 40:6, 790-798.
- Uversky, V.N. (2013), Posttranslational Modification. in Brenner’s Encyclopedia of Genetics (Second Edition). Editors Maloy, S and Hughes K. pp. 425-30. doi: 10.1016/B978-0-12-374984-0.01203-1