Site-Specific Phosphorylation Profiling

Site-specific tyrosyl phosphorylation is a key mediator of intracellular communication and represents a rapid readout of the cell-signaling network’s response to drugs or other stimuli. For this reason, proteins involved in these signaling responses have become major targets for therapeutic intervention. The complexity of these responses, however, requires powerful tools that can monitor information flow through the signaling network and identify important biomarkers associated with disease or drug responses.

Epitome Biosystems developed quantitative phosphorylation-profiling chips that broadly survey the signaling network. The company’s approach provides site-specific phosphorylation data, utilizes quantitative reference standards, is multiplexed, and accommodates parallel processing of multiple samples.

EpiTag™ technology translates protein-sequence information into functioning assays in a predictable manner. It uses computational algorithms to identify unique, linear peptide sequences (EpiTag™ sequences) in proteins found only once in the entire proteome. EpiTag™ antibodies are then generated against synthetic peptides bearing the EpiTag sequences.

Unlike traditional immunoassays, the EpiTag approach involves digesting proteins in the sample prior to analysis. As a result, the peptide sequence being targeted is liberated and accessible for antibody binding. Assay specificity is ensured using two antibodies in a sandwich-immunoassay format. An EpiTag antibody is used to capture the targeted peptide fragment, and a second pan-phospho-specific antibody is used to detect the phosphorylation state of a particular site on the fragment (Figure 1).

The EpiTag™ assay platform measures peptide fragments as surrogates for proteins, making it possible to develop accurate reference standards using synthetic peptides. The approach allows for simultaneous measurement of multiple phosphorylation sites on the protein as well as multiple sites of phosphorylation across different protein targets.

Profiling Chips

The Ti-Tyr™ Profiling Chip is a tyrosine-phosphorylation focused product based on the EpiTag technology. Tyrosine phosphorylation is measured quantitatively at a site-specific level after proteolytic fragmentation of samples. The Ti-Tyr chip allows for multiplexed analysis across more than 75 phosphotyrosine sites on 62 different proteins using a planar microarray.

The Ti-Tyr chip covers a wide range of content including receptors (e.g., EGFR and HER2), adapter and scaffold proteins (e.g., IRS1 and Crk), intracellular mediators (e.g., Erk1 and Raf-1), and transcription factors (e.g., c-Jun and STAT3). The chip consists of specific EpiTag capture antibodies in four replicate subarrays in each of 16 array chambers (Figure 2). A single chip can be used to analyze 16 different samples including reference standards and controls, or four chips can be mounted in a carrier and treated similar to a microtiter plate.

Standards, controls, and samples are added to the chips containing EpiTag capture antibodies. After incubation, tyrosine phosphorylation is detected by incubating with a fluorescently labeled pan-phosphotyrosine detection antibody, followed by signal acquisition using a fluorescent slide scanner.

Cell-Signaling Network Dynamics

EGF-stimulated A431 cell lysates were analyzed using the Ti-Tyr chip. Cells were grown to 80% confluence, serum starved for 24 hours, and treated with EGF (100 ng/mL) alone or EGF after pretreatment with SL327 (a MEK inhibitor) for eight minutes. Lysates were harvested, digested, and analyzed on Ti-Tyr™ Chips. The concentration of each target was determined by interpolation from standard curves.

The signaling proteins in the EGFR pathway exhibited increased phosphorylation upon stimulation. With addition of the MEK inhibitor, downstream targets of MEK did not show increased phosphorylation (Erk1/2 and STAT3), while upstream effectors (EGFR and Shc) were phosphorylated following EGF stimulation (Figure 3). A wide dynamic range in phosphorylation responses was observed.

Phospho-EGFR was greater than 700 pM, whereas the low-abundant phospho-STAT3 was detected at less than 2 pM. MEK was not measured in this experiment; the relevant phosphorylation sites on MEK are not tyrosines. Its inhibition, however, was clearly demonstrated via other targets on the Ti-Tyr chip.

EpiTag™ technology provides a platform for targeted, quantitative protein measurements. The approach provides a predictable, streamlined path for assay development and enables rapid content generation. EpiTag’s specificity supports multiplexed measurements of hundreds of phosphorylation sites and may be applied more broadly for analysis of other post-translational modifications including threonine phosphorylation, serine phosphorylation, acetylation, and glycosylation. Epitome’s custom assay services provide researchers access to EpiTag technology to meet specific assay development needs.

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Tim Nadler, Ph.D., is director of product development, and Ed Machuga is vp, sales and marketing, at Epitome Biosystems. Web: www.epitomebiosystems.com. Phone: (781) 478-1422. E-mail: emachuga@epitomebiosystems.com.