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Epitome Team Develops Way to Measure Site-Specific Phosphorylation Proteins

Scientists from Epitome Biosystems have developed an approach that they say can for the first time quantitatively measure site-specific phosphorylation events and total proteins in a single sample.
The approach, a multiplexed, peptide-based sandwich immunoassay, represents “a new paradigm for standardized protein and phosphorylation analysis,” and can overcome the obstacles found in other methods, they write in their study. They added that the method “will further elucidate pathway dynamics and their potential role in disease pathogenesis and modulation by kinase inhibitors.”
The study, which appears in the March edition of Analytical Biochemistry, describes a technology that is contained in Epitome’s EpiTag assays run on Luminex’s xMAP platform. Many of the targets described in the paper have also been commercialized via Epitome’s collaboration with EMD Chemicals.
In the article, the researchers write that while much work is being done in the area of kinase-inhibitor development to address intracellular signaling dysfunction, such an approach has been met with a high failure rate, prompting a need for more comprehensive systems strategies to drug discovery and development on the protein level.
“In response, phosphoproteomics is moving to the forefront of signal transduction research,” they say in the article.
DNA microarrays for measuring RNA expression have become a basic tool for
studying biological systems as indirect measurements of protein expression, but, according to the authors, they have not proven to be a particularly effective method because protein levels do not consistently correspond to mRNA expression. More importantly, they add, gene transcript profiling “is not informative about critical post-translational modifications that regulate protein function.”

“Prolonged EGFR phosphorylation may result in additional cellular events outside the direct activation of MAPK pathway, and we currently are applying our epitope tag-based approach to generate broad phosphoprotein profiling arrays to explore these tissues.”

At the same time, while antibody array-based technologies can have greater utility, technical challenges to such methods exist, including a shortage of suitable antibodies with the specificity needed for multiplex analysis; a shortage in protein standards that meet the requirements needed for quantitative analyses; and “the lack of standardized assay conditions with inherently different properties.”
One approach, sandwich assays, have been used to address one issue inherent in proteins — undefined binding epitopes in most antibodies that make it challenging to create specific immunoassays in a multiplex format. But while sandwich assays increase specificity, they still have problems measuring several complex proteins simultaneously and so “cannot quantitatively measure total protein and post-translational modifications for a target protein in the same assay,” the researchers say.
Instead, they propose a peptide-based sandwich immunoassay. While such an approach had been formulated earlier, the ability of anti-peptide antibodies to detect native protein structure has been compromised because of the “inaccessibility of the peptide epitopes in the context of the native protein structure,” according to the researchers.
The approach described in Analytical Biochemistry, however, reduces inherent protein conformational issues, allowing researchers to analyze biological samples on the peptide level. Their method started with a bioinformatics platform in which an in silico analysis of the human proteome is done “to identify continuous linear sequences, or ‘epitope tags,’ in proteins that are unique within the proteome,” the authors say.
They scanned the Ensembl human proteome to identify linear amino acid sequences between eight to 12 amino acids in length and unique across the proteome. They then choose epitope tags based on predicted ease of synthesis, solubility, potential antigenicity, and spacing.
Using the epitope tags, the researchers then designed synthetic peptide immunogens and developed specific antibodies that can detect and bind predefined linear peptide sequences. By digesting proteins in a sample with specific proteolytic enzyme, selected peptide sequences were made accessible to the antibodies.
Further, “by designing antibody pairs using epitope tags that reside on the same proteolytically cleaved peptide fragment, targets can be captured and detected in sandwich immunoassays with increased specificity,” the researchers say. The approach generates high-affinity antibodies with preselected isotopes, “allowing standardized conditions for assaying peptides instead of whole proteins in multiplex.”
Total protein measurement can be achieved by generating antibodies to two unique epitope tags on the same peptide fragment of a protein, forming a sandwich. “For site-specific, post-translational-modification detection, the capture antibodies that are generated bind unique epitope tags flanking the PTM sites on the same fragment and combined with a motif or PTP-specific detection antibody,” according to the article.
Using standard peptide and phosphopeptide standards, quantification was done by “interpolating protein concentration from standard curves,” the authors say. To test their claim, they then quantified time-dependent changes in total and phosphorylated epidermal growth factor receptor, MEK1, MEK2, ERK1, and ERK2 in multiplex in epidermal-growth factor-stimulated A431 human cancer cells.      
Cells were serum-starved for 24 hours before being stimulated with 100 ng/ml of EGF in the presence or absence of the MEK1- and MEK2-specific inhibitor SL327. Lysates were prepared and measurements made in multiplex for total protein and phosphorylation targets. For the first two hours, total EGFR protein remained “relatively constant” following EGF stimulation at about 10 nanomoles.
The scientists detected activated EGFR within four minutes of EGF stimulation, and said it increased to about 93 percent of total EGFR being phosphorylated four hours after EGF stimulation. Phosphorylated EGFR continued to increase although a slight decrease in total EGFR protein was detected after two hours.
The researchers also reported that pretreatment with SL327 had no effect on the amount of total or EGF-induced phosphorylation of EGFR at early time-points, “but did result in slightly elevated levels of phosphorylated EGFR between 32 and 128 minutes,” they said. “Western blot analyses confirmed the general EGFR activation trends.”
They also measured the activation of downstream targets of the EGFR-MAPK pathway. While total amounts of MEK1 and MEK2 in A431 cells did not change “appreciably” during the course of the experiment, the amount of the two MAPK kinases increased after EGF treatment, and “the data correlated well with results from immunoblots using commercial phosphospecific antibodies,” the authors say.
Amounts of total ERK1 and ERK2 protein also remained similar during EGF stimulation of A431 cell, which support the involvement of negative feedback mechanisms, and not protein degradation, in the deactivation of MAPKKs and MAPKs.
Further “immunoblotting with a commercial anti-phospho ERK1/ERK2 antibody confirmed the time-dependent phosphorylation of both ERK1 and ERK2 in response to EGF treatment.”
In total, their method allowed them to detect total and multiple PTMs on the same protein with the same sample in a quantitative and site-specific manner, they say. 

“Due to the unique peptide-based strategy, these assays are the first to quantify total and phosphoyrlated EGFR and downstream targets MEK1, MEK2, ERK1, and ERK2 from a single source, at the same time, with site-specific phosphorylation measurements using a robust immunoassay approach,” the researchers say. “Prolonged EGFR phosphorylation may result in additional cellular events outside the direct activation of MAPK pathway, and we currently are applying our epitope tag-based approach to generate broad phosphoprotein profiling arrays to explore these tissues.”

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