Abstract
Identification of a novel post-translational modification (PTM) for a target protein, defining its physiologic role and studying its potential cross-talk with other PTMs is a challenging process. A set of highly sensitive tools termed as Signal-Seeker kits was developed, which enables rapid and simple detection of PTMs on any target protein. The methodology for these tools utilizes affinity purification of modified proteins from a cell or tissue lysate, and immunoblot analysis. These tools utilize a single lysis system that is effective at identifying endogenous, dynamic PTM changes, as well as the potential cross-talk between PTMs. As a proof-of-concept experiment, the acetylation (Ac), tyrosine phosphorylation (pY), SUMOylation 2/3, and ubiquitination (Ub) profiles of the epidermal growth factor (EGF) receptor (EGFR)–Ras–c-Fos axis were examined in response to EGF stimulation. All ten previously identified PTMs of this signaling axis were confirmed using these tools, and it also identified Ac as a novel modification of c-Fos. This axis in the EGF/EGFR signaling pathway was chosen because it is a well-established signaling pathway with proteins localized in the membrane, cytoplasmic, and nuclear compartments that ranged in abundance from 4.18 × 108 (EGFR) to 1.35 × 104 (c-Fos) molecules per A431 cell. These tools enabled the identification of low abundance PTMs, such as c-Fos Ac, at 17 molecules per cell. These studies highlight how pervasive PTMs are, and how stimulants like EGF induce multiple PTM changes on downstream signaling axis. Identification of endogenous changes and potential cross-talk between multiple PTMs for a target protein or signaling axis will provide regulatory mechanistic insights to investigators.
Highlights
The mammalian proteome has been estimated to contain multiple millions of unique proteoforms [1,2]
Effective identification of novel post-translational modification (PTM) proteoforms and potential regulatory mechanisms In the present study, the Signal-Seeker PTM detection system was used to identify pY, Ub, SUMO 2/3, and Ac profiles for three target proteins in the epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) signaling pathway, which resulted in the confirmation of ten previously identified proteoforms as well as identification of c-Fos Ac
SUMO 2/3 modification of Ras was not detected, which corresponds with zero publications or reports on this proteoform in the literature
Summary
The mammalian proteome has been estimated to contain multiple millions of unique proteoforms [1,2]. This level of complexity is derived from a relatively simple genome 25,000 genes), a transcriptome which increases the potential protein footprint to 100,000, and protein post-translational modifications (PTMs) which account for the vast increase in proteome complexity and an almost limitless potential for functional diversity [3,4,5]. Modifications include tyrosine phosphorylation (pY), ubiquitination (Ub), small ubiquitin-like modifier 2/3 (SUMOylation 2/3 (SUMO 2/3)), and acetylation (Ac), in addition to many others [6,7,8,9]. For human health and disease, misregulation of PTMs has been implicated in the progression of diseases like cancer, heart failure, neurologic, and metabolic diseases [11,12,13,14,15]; several emerging therapeutics targetting the Ac, Ub, and SUMOylation pathways serve to demonstrate the therapeutic potential of PTM targets [16,17,18]
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