Validating PTMs

Mass spectrometry (MS)-proteomics in combination with the plethora of PTM prediction tools now available make it quite simple to gain insight into whether or not a protein may be modified by a particular PTM. For more information about mass spectrometry based PTM proteomics click here.  Once a potentially interesting PTM for a target protein has been identified by these approaches, what’s the next step?

For cell and molecular biologists there is a critical step that occurs when taking a target identified from informatics data and investigating it in their biological system. This essential step is validation.  Why is it so critical? In-depth investigation of an unexplored PTM of a target protein hinges on whether or not the modified target can be effectively identified in the investigator’s biological system. Utilizing sub-optimal approaches may result in the inability to detect the modified protein; thus, leading to a premature end to potentially novel discoveries. 

As an example, the PTM, ubiquitin, was first shown to interact with microtubule networks several decades ago in 1988 ^[19]. However, at that time the data suggested that ubiquitin was modifying microtubule-associated proteins but not tubulin itself ^[19], which may have played a role in the nearly two decade dormancy in the  ubiquitinated tubulin field.  Unfortunately, these findings were based on western blotting data performed without any enrichment techniques ^[19].  With the advancement in PTM detection techniques, there are now several papers that have successfully identified ubiquitinated tubulin; furthermore, these studies link tubulin ubiquitination with microtubule dynamics and function ^[20-23]. To learn more about the importance of tubulin ubiquitination see our recent newsletter. This example highlights how vital it is to use optimal approaches and tools when validating whether or not a protein is post-translationally modified.   

Our PTM Detection Techniques page provides a summary of enrichment techniques and validation approaches like PTM IP, protein IP, and PTM and/or protein overexpression.  Furthermore, Cytoskeleton offers comprehensive PTM detection kits that have been optimized for maximal detection of endogenous PTMs ^[24]. A recent publication utilized these kits to examine the endogenous PTM states of proteins in the EGFR-Ras-c-Fos axis ^[24].

Points to Consider When Investigating Functional Importance of Target Protein PTMs 

The following sections will discuss critical points to consider when investigating PTMs such as dealing with transient changes, the importance of studying PTMs endogenously, how to minimize false negative and positive results, as well as, the need to consider investigating PTM crosstalk.

For technical tips on studying target protein PTMs using enrichment strategies click here.

Want to see a peer-reviewed video protocol ^[25] for endogenous PTM enrichment and detection? Click here.

Studying transient and dynamic PTM changes

An overarching problem with studying PTMs is their transient nature which often leads to low stoichiometry (< 1-5%) ^[26-28]. While an impediment towards their investigation, these qualities also make them key regulatory agents or switches for rapid biological functions in cells.

A common approach to overcome this barrier is to enrich for the PTM of interest for a target protein.  Enrichment can be achieved by two paths: 1. Enrich for the PTM using affinity matrices and probing for the protein of interest or 2. Enrich for the protein using affinity matrices and probing for the PTM.  Click here for specific tips for success when performing these enrichment strategies.  We recommend a combination of these approaches to properly validate that a protein is modified by a target PTM; however, as a first approach enriching for the PTM may be a more successful strategy.  

The robust Signal-Seeker tools can detection PTMs as low as 17 molecules per cell ^[24]. The ability to detect these small, but potentially meaningful changes are critical, as measuring endogenous changes of PTMs are necessary to understand the physiologic relevance of the modification as discussed below.

Investigate PTMs endogenously and physiologically

Another approach to circumvent low levels of PTM modified proteins is to exogenously overexpress a PTM, target protein, and in many cases both. This approach will provide insight into whether or not a target protein is modified by a PTM of interest; however, there are cases where false interactions between an overexpressed protein or PTM may occur ^[29].

Another problem with overexpression systems is that they are often used for follow-up physiologic studies of the modified target. For example, a study investigating Ras di-ubiquitination (di-Ub) using overexpression systems determined that the modification, while important for Ras activation, was not regulated by upstream EGFR signaling ^[30]. Contrarily, endogenous Ras di-Ub was examined using Cytoskeleton’s Ubiquitin Detection Kit, and robust, dynamic changes in di-Ub of Ras occurred in response to EGF stimulation ^[24]. It is likely that the overexpression system masked physiologic changes in di-Ub Ras. These data highlight the importance of understanding the limitations of specific tools, as well as identifying robust tools that effectively capture endogenous, physiologic changes in a target protein’s PTM profile.

The Signal-Seeker PTM Detection toolkits have been used to effectively measure the endogenous level of multiple PTMs for many target proteins ^[24,31,32], and all of these studies can be found on our validation data page.

Minimize false positive, false negative data

Negative detection of a PTM for a target protein can be due to a multitude of reasons both biologically and technically; therefore, a negative result does not definitively prove that the protein is incapable of being modified. For example, a particular target protein may only be modified under specific biological conditions in a specific cell type, which may not have been examined in the available literature. Often it is only a very limited pool of researchers that have studied any given protein of interest (POI), and therefore have the expertise and insight to know what experimental system, conditions, and timelines are necessary to study their POI.  A set of tools that empower these researchers to investigate their proteins PTM state would greatly facilitate PTM studies.

Another factor that may produce false positive or false negative data is protein–protein interactions that are not disrupted during cell lysis.  Therefore, it is recommended to utilize stringent buffers that are compatible with the affinity reagent.   Cytoskeleton has developed a unique denaturing lysis buffer system that is compatible with all of our PTM detection systems to minimize the potential for protein-protein interference.

 As with all enrichment techniques that utilize affinity matrices the possibility for non-specific binding to PTM affinity matrices is a possibility that may produce false positive data.  Thus, specific IgG control beads will help to delineate specific from non-specific interactions.  Cytoskeleton offers matrix-matched control beads for all of our PTM affinity matrices as having the right controls will simplify data interpretation. 

It is noteworthy to consider the potential for false negative detection of endogenous PTM changes with IP-pan-PTM methodologies. For example, the protein dihydrolipoamide dehydrogenase (DLD) is acetylated at potentially 22 different lysine residues. Thus, one prominent Ac site on DLD may mask small, but meaningful changes that occur at alternative lysine residues on the protein. This hypothetical example highlights the importance of investigating PTMs using multiple approaches, as site-directed mutagenesis would be beneficial for these types of analysis. Additionally, it further enhances the call for additional PTM tools that enable site specific investigation; for example, site specific PTM antibodies.             

Approaches to investigate PTM crosstalk

Common crosstalk paradigms have emerged ^[33,34], such as phosphorylation being a precursor to target protein Ub ^[35,36]. Another pattern of crosstalk exists between Ac and Ub where these modifications compete to regulate the stability of the protein ^[37,38]. Ac has been shown to stabilize target proteins ^[39], while poly-Ub is well characterized as a mark for proteasomal degradation ^[40]. Both Ac and Ub primarily modify lysine residues, and many proteins have been identified to be both acetylated and ubiquitinated on the same lysine residue, providing further evidence of negative crosstalk between these PTMs. 

                Signal-Seeker PTM detection tools have the ability to investigate multiple PTMs of interest, which allows investigators to gain insight into potential PTM crosstalk. This is possible because all of the affinity matrices used to capture the PTMs of interest were designed to work with a universal lysis buffer as reported previously ^[25], which enables more meaningful comparisons when investigating crosstalk between PTMs.            

For more specific information about Signal-Seeker™ post-translational modification detection tools please view our product pages, resource links, or contact technical support.