eBook Chapter 3: Signal-Seeker PTM Discovery Toolkits: Utilities and Applications

Overview of the Importance of PTMs

Dysregulation of post-translational modifications (PTMs) have been implicated in the progression of diseases like cancer, metabolic diseases, neurologic diseases, and others (1-4). Therapies such as tyrosine kinase inhibitors, specifically target PTM regulatory mechanisms. Emerging therapeutics that target the acetylation (Ac), SUMOylation 2/3 (SUMO 2/3), and ubiquitination (Ub) pathways are in development and demonstrate the relevance of PTMs as critical physiological and pathological regulators (see article 1 for more information).

            The characterization of proteins such as p53, epidermal growth factor receptor (EGFR), and histones has unveiled the essential role that PTMs play in regulating a proteins function to effectively orchestrate cellular events (5-7). PTM regulation is not restricted to this small subset of proteins; contrarily, evidence from proteomic analyses suggests greater than 70% of proteins are phosphorylated or ubiquitinated at some point during their “life cycle” (8).

           Identification of a novel PTM for a target protein, defining its physiologic role, and studying its potential cross-talk with other PTMs is still a challenging process. Article 2 of this eBook highlights tools and approaches for investigating PTMs, and provides tips and suggestions for the molecular biologist to utilize when performing initial discovery studies. Here we provide additional insight into Signal-Seeker kits, a PTM discovery tool, which enables investigators to rapidly identify if their protein of interest is endogenously modified by one or more of these critical PTMs in their biological system.

            Importantly, every Signal-Seeker kit is a comprehensive immunoprecipitation (IP) system that utilizes optimized reagents including a universal lysis system, validated IP and elution buffers, and finely-tuned affinity matrices to simplify and accelerate the discovery process. Figure 1 highlights the general workflow for utilizing this system. Below we describe three applications where investigators utilized these toolkits to determine if their target protein was post-translationally modified.

SUMOylation of Viral Protein APH-2

           Human T lymphotropic virus type 2 (HTLV-2) is an asymptomatic virus that shares similar genomic organization to the HTLV-1 virus, which has been linked to several diseases including cancer (9). Both viruses have anti-sense proteins but while the HTLV-1 antisense protein, HBZ, plays a significant role in virus induced pathogenicity such as cell transformation (10) the HTLV-2 antisense protein, APH-2 does not play a role in cell proliferation or transformation. Interestingly, several studies have shown that the APH-2 protein is highly unstable with a half-life of 20-30 minutes, and may explain its minimal role in HTLV-2 infection (11, 12).  Comparative studies were performed to decipher critical and functional differences between the two anti-sense proteins to better understand their roles in viral pathogenicity.

           There is a growing body of knowledge whereby viruses hijack the cellular SUMOylation machinery to aide in propagation (13, 14). Since SUMOylation can affect protein stability, Dubuisson et al. sought to decipher if this PTM altered APH-2 instability. A critical first step was the discovery that APH-2 is endogenously modified by SUMO 2/3, which was determined using the Signal-Seeker toolkit (15). Additional molecular studies determined that SUMO modification localized APH2 to PML nuclear bodies for degradation. Interestingly, alteration of SUMOylation had little effect on HBZ expression.  This study highlights the importance of PTMs like SUMOylation in virus regulation and supports ongoing strategies to utilize the SUMO molecular machinery to potentially combat viral infection (15).

Mono-Ubiquitination of Immune Checkpoint Protein PD-L1

           Cancer cells have evolved mechanisms to suppress the host’s immune system through cell surface expression of checkpoint inhibitors, like the programmed cell death ligand 1 (PD-L1) protein, as a key mechanism for cancer progression (16). PD-L1 is overexpressed in many different cancers(17), and has garnered significant attention as a key target for anti-cancer therapies. Several antibody-based drugs targeting the PD-L1/PD-1 axis have been FDA approved (18); still, ongoing research aims to determine why only some PD-L1–positive tumors respond to treatment (19). A better understanding of the mechanisms regulating PD-L1 may result in more efficacious therapeutics.

           While the transcriptional regulatory mechanisms of PD-L1 expression have been reported previously (20), it was difficult to find reports regarding PTM of this protein. Using the Signal-Seeker toolkits, the authors examined PD-L1's levels of tyrosine phosphorylation, ubiquitination, acetylation, and SUMOylation in A431 cells treated with epidermal growth factor (EGF) (21). This study led to the novel identification of PD-L1 modified tyrosine phosphorylation, acetylation, and mono- and multi-ubiquitination (Figure 2).

Figure 1: Workflow of Signal-Seeker PTM identification kits

Diagram depicting steps performed in order to obtain PTM profiles for a target protein

           The investigators utilized these toolkits to examine the functional role of PD-L1 ubiquitination and determined, through temporal studies, that mono- and multi-ubiquitination preceded an EGF-stimulated increase in total PD-L1 protein expression (21). Pharmacological inhibition of the EGFR activation further demonstrated that mono- and multi-ubiquitination of PD-L1 relies on EGFR activation.  Importantly, inhibition of ubiquitin E1 activating enzyme blocked EGF induced increases in total PD-L1 protein, revealing a potential mechanistic role for mono-ubiquitinated PD-L1 in the regulation of total PD-L1 protein levels. Ultimately, these studies suggest that regulatory PTM mechanisms of PD-L1 may be important for regulating its expression and function for immune homeostasis.

Figure 2: Schematic of PD-L1 PTMs

Figure adapted from Horita et al. 2017. Neoplasia(21). Model: Profile of PD-L1 post-translational modifications and their roles in regulating PD-L1 protein levels..

PTM characterization of the EGFR Signaling Pathway

           The third example highlights the use of Signal-Seeker toolkits to examine the PTM profile changes in the well-studied EGFR–rat sarcoma (Ras)–c-Fos axis in response to EGFR stimulation. This pathway was selected for several reasons: (i) the level of endogenous, non-EGF stimulated target proteins spans a range from abundant to low level expression (EGFR > Ras > c-Fos), this gave some indication of the dynamic range of the toolkits, (ii) selected protein targets represent transmembrane (EGFR), cytoplasmic/membrane bound (Ras), and nuclear (c-Fos) proteins, which gave an indication of the efficiency of the toolkits to detect protein targets from a comprehensive range of cellular compartments, and (iii) multiple reports of PTM proteoforms for this set of proteins are available in the literature, which gave an indication of the reliability of the toolkits (22).

           While tyrosine phosphorylation (pY) of this pathway is well-characterized, investigation of other PTMs like Ac, SUMO 2/3, and Ub in the same biological system using a single lysis system has not been previously reported. 11 of the possible 12 proteoforms were identified using the Signal-Seeker system, and correlates with all 10 of the previously identified proteoforms, while also identifying an unreported proteoform, acetylated c-Fos (22).

           The Signal-Seeker toolkits enabled investigation of the PTM status of proteins in various cellular compartments that ranged from low to high abundance. The dynamic and endogenous levels of these PTMs were investigated in a single lysis system, providing insight into potential crosstalk between these four PTMs in response to physiologic stimulants like EGF. Collectively, the data suggests that these toolkits provide a simple approach for effective investigation of established and novel PTMs for any target protein.


            Often, it is only a very limited pool of researchers that have studied any given target protein, and therefore have the expertise and insight to know what experimental system, conditions, and timelines are necessary to study their target protein. A set of validated tools that empower these researchers to effectively determine it their proteins is modified in their specific experimental model, without the need to develop specialized methods should greatly facilitate PTM discovery. We highlighted three examples where Signal-Seeker tools identified endogenous PTMs of the target proteins. While Signal-Seeker toolkits were developed based on conventional IP principles, they are far from a standard IP reagent. The comprehensive, highly refined, and optimized nature of the toolkits, akin to QIAGEN MAXI prep kits for plasmid purification, make them simple yet powerful PTM discovery tools that are perfect for non-PTM experts.

Related Products & Resources

Signal-Seeker™ Ubiquitination Detection Kit (30 assay)


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