eBook Chapter 2: Proteoform Discovery and Validation Techniques for the Molecular Biologist

Brief Overview

Post-translational modifications (PTMs) are enzymatically-regulated, covalent modifications that change a protein’s structure. These alterations to functional groups on a protein can alter its stability, spatial localization, and binding partners; ultimately, regulating its function (1). Any given protein is likely to be modified by a number of different PTMs, and each specific PTM primes the protein to perform a unique and dynamic function in the cell. Therefore, it is reasonable to hypothesize that PTMs are an omnipresent mechanism regulating many diverse functions of any given protein.

In support of this hypothesis, PTMs such as ubiquitination, glycosylation, acetylation, SUMOylation, and phosphorylation have been shown to regulate nearly every biological process through control of signal transduction, protein turnover, protein recognition, and localization (2, 3). These PTMs and others vastly increase the proteome complexity by orders of magnitude, because each post-translationally modified protein represents a unique protein-form (proteoform) (4-6). Importantly, characterization of specific proteoforms has resulted in great insight into the target protein’s function (see article 5 for specific examples), and a significant number are being actively pursued as promising therapeutic targets and/or biomarkers (see article 1 for further insight). For example, a particularly promising therapeutic targets the p53 proteoform, MDM2 ubiquitinated p53, which has been shown to be deregulated in a wide variety of cancers (7).

Unravelling mechanistic aspects of PTM proteoforms via molecular biology approaches is technically very challenging. With the exception of phosphoproteins, histones and several medically relevant proteins such as p53 and Tau, there are few proteoform specific antibodies in the PTM toolbox, necessitating the use of more indirect analytical techniques. The remainder of this article addresses key technical issues with studying PTM proteoforms as well as useful discovery and validation techniques utilized to overcome them.

Technical Issues When Studying PTMs

  • Low Stoichiometry
    • Proteoforms tend to be sub-stoichiometric; any one proteoform accounts for less than 1-5% of a protein’s total population at any given time making them difficult to detect
  • Highly Dynamic
    • Proteoforms are dynamic, often appearing transiently in response to a specific signal
  • Cell or Tissue Specificity
    • Proteoforms can be cell or tissue type specific
  • Sample Preparation: Gain or loss of PTMs
    • PTM profiles can be positively and negatively altered during sample processing
  • Utilizing Proper Controls
    • Key controls are critical for interpreting results

             The preceding list comprises some of the major technical issues that make detecting PTM proteoforms challenging (see article 5 for specific examples). The following are commonly used techniques that molecular biologists use to investigate novel proteoforms of their target protein.

Enrichment of PTM Proteoforms

            Enrichment of PTM proteoforms is necessary due to stoichiometry issues (8-10), and is often achieved by immunoprecipitation (IP) techniques. Enrichment with IP utilizes antibodies, protein-binding domains, metal ion affinity, and others specific binding molecules that are attached to a solid support matrix (such as agarose resin). These affinity matrices bind to the target protein or PTM, while non-targeted proteins in the complex lysate are not captured and removed through wash steps. The enriched PTM proteoforms are then detached and isolated from the support matrix using a concentrated volume of elution buffer. The isolated population is analyzed in downstream applications like western blotting or mass spectrometry to determine if a target protein is post-translationally modified.

Two approaches are possible for endogenous PTM proteoform enrichment; one approach uses an affinity matrix that recognizes the target protein, while the other uses an affinity matrix specific for the PTM of interest. An affinity matrix against the target protein of interest may immunoprecipitate potentially all proteoforms of that specific protein, while IP with a PTM-specific affinity matrix may immunoprecipitate nearly all proteoforms modified by that PTM.

Western Blotting Analysis

Western blotting is a standard molecular biology approach used to identify a specific protein of interest from a complex protein mixture or lysate. In the case of PTM proteoform analysis, the enriched sample is mixed with SDS, separated to specific regions in an acrylamide gel based on size (SDS-PAGE), transferred to a blotting membrane, and specifically identified by antibody recognition.

Figure 1A shows an example where ubiquitinated EGFR was enriched with a ubiquitin affinity reagent, and visualized with an EGFR antibody. The western blot results show unambiguous identification of ubiquitinated EGFR. These data are supported by the loss in the ubiquitinated EGFR band when the deubiquitinase inhibitor, NEM, was removed from the lysis buffer; thus allowing deubiquitination to occur (Figure 1A). The reciprocal experiment was performed using an EGFR antibody for IP enrichment. The enriched proteins were then visualized by probing with a pan-ubiquitin antibody. Figure 1B shows minimal ubiquitinated EGFR was detected with this approach. One possible explanation for this stark difference could be due to the bound-ubiquitin protein blocking the EGFR antibody recognition site (Figure 1C and D). Previous research has shown that some PTM modifications may block the antibody binding site on a target protein and prevent interaction with the antibody; thus, producing a false negative result (11).

Figure 1: Detection of EGFR ubiquitination

Serum-restricted A431 cells were either unstimulated (-) or stimulated with EGF (+) for 15 minutes prior to lysis with BlastR lysis buffer with or without NEM. WCL was analyzed for EGFR levels (Input). (A). Lysates were incubated with UBA01 ubiquitin affinity beads and analyzed for ubiquitinated EGFR with an anti-EGFR antibody. (B). Lysates were incubated with anti-EGFR antibody and protein-G beads. Captured proteins were analyzed for ubiquitinated EGFR with an anti-Ub-HRP conjugated antibody.

Mass Spectrometry Analysis

            Mass spectrometry (MS) is an analytical method that can identify a PTM proteoform based on its mass to charge ratio. Two distinct MS approaches are used to investigate PTM proteoforms, bottom-up and top-down analysis (which are described further in article 4 of this eBook). Benefits of mass spectrometry are its potentially unbiased approach, PTM site specificity, and independence from antibodies for detection. Technical challenges when utilizing MS include protein abundance bias (12), method sensitivity(13), and methodological expertise, such as, sample preparation, digestion strategies, fractionation approaches, and other considerations (14). Investigating PTM proteoforms with MS requires both a comprehensive understanding of the biological model and technical expertise with the instrumentation/analysis; thus, we have found that successful PTM proteoform investigation with this method requires strong collaboration between molecular biologists and MS experts.

Overexpression and Mutagenesis

            Overexpression is a well-established system where a plasmid of a tagged version of a target protein is transfected into cells, which usually results in high expression levels. The expressed protein normally contains a tag that is recognized by a well characterized antibody (ex. His). The increased expression and optimized enrichment antibody improves the chance of identifying the PTM proteoform, and is commonly analyzed by western blot. The overexpression system is particularly important when a proteoform is challenging to study endogenously; because it allows investigators to control expression, localization, and other factors through mutagenesis approaches. Importantly, mutagenesis approaches enable site specific PTM investigation and characterization. Due to the overexpressed nature, any critical identification should be validated by additional methods, and chapter 5 of this eBook provides examples where overexpression studies were performed during PTM proteoform characterization.

in vitro: Biochemical Assay

            Biochemical assays utilize purified or in vitro translated versions of a target protein to determine if it can be modified by a specific PTM. The purified protein is added to a test tube with specific enzymes (e.g. E1, E2, E3 ubiquitin ligase) and the appropriate substrate (e.g. ubiquitin), co-factors, and energy sources. After incubation, the sample is then analyzed by western blot analysis. It is important to note that in vitro biochemical analysis is not available for all types of PTMs; however, it is routinely performed to investigate phosphorylation (15), ubiquitination (16), SUMOylation (17) and other PTM modifications. A limiting step in performing in vitro biochemical assays is obtaining purified versions of the target protein and modifying enzymes (18).

Proximity Ligation Assay

            Proximity ligation assay (PLA) is a novel immunoassay technology that can be used to study protein interactions and PTMs. PLA is unique in its ability to identify proteoforms in fixed tissues and cells (19). The principle of PLA-PTM works by utilizing two antibodies; one targets a PTM of interest while the other binds a specific protein of interest (20). The initial steps are similar to standard immunofluorescence staining where the primary antibodies bind the epitopes of interest, and the secondary antibodies recognize their respective primary antibodies.  The difference with PLA is that these secondary antibodies have short DNA strands covalently attached to them (these antibody-DNA complexes are called PLA probes). If the two PLA probes are in close enough proximity, presumably because the two antibodies are bound to a proteoform, they will form circularized DNA. PCR amplification of circularized DNA is performed and fluorescently labeled complementary DNA probes are added for visualization. Due to the significant DNA amplification, which can be up to several hundred-fold, the fluorescent signal from very few molecules will be visible by microscopy.

Specialized Techniques

            An emerging technology that will have profound effects on the PTM field is genetic code expansion. This technology allows researchers to specifically add a PTM of interest, homogenously onto a target protein without a requirement for the specific modifying enzyme (21). Having homogenous purified versions of the PTM proteoform will be critical for structural studies, in vitro functional studies, and others. There are numerous other techniques available that are outside the scope of this article, such as, NMR spectroscopy for site specificity and structural insight of proteoforms (22), thermal dissociation assays for detection of enzymatic PTM removal (23), and Nanopore technology for label free PTM detection (24). Additionally, for each specific PTM there are highly specialized methods that have been developed. For example, investigators are using Hotspot Thermal Profiling of native proteins in live cells to identify which site-specific phosphorylation events alter protein stability (25). Other investigators are utilizing novel ubiquitin clipping mechanisms to gain insight into poylubiquitin chains and architecture (26). Furthermore, novel approaches have been developed to quantitate kinetic parameters of regulatory enzymes such as lysine acetyl transferases using reverse phase HPLC (27).

Conclusions

Due to a PTM proteoform’s biological nature, i.e. post-translationally modified proteins are present at low stoichiometric levels, identification of a specific proteoform often goes undetected via standard protein detection methods like western blotting and immunofluorescence staining. Table 1 highlights the pros and cons of several enrichment and detection approaches that may be used for preliminary investigation. The PTM-specific IP approach is particularly beneficial as kits are available in this format, which allows investigators to bypass extensive optimization issues. A recent review identified Signal-Seeker kits as a novel tool to investigate PTMs of target proteins (28), and more information is provided in Article 3 of this eBook. Better tools and approaches to efficiently detect these important modifications will undoubtedly facilitate the PTM proteoform discovery and validation process, and when used in combination with other PTM investigation tools will allow for successful functional characterization of a PTM proteoform.

Table 1: Pros and Cons of PTM enrichment and detection strategies


Related Products & Resources

Signal-Seeker™ Ubiquitination Detection Kit (30 assay)


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