Understanding Intracellular Phosphorylation and its Importance in Identifying Cancer Biomarkers

Phosphorylation is one of the most prevalent intracellular protein modifications and plays an important role in processes including cell differentiation, proliferation, and migration.1 Through phosphorylation, cells regulate growth, apoptosis, cell cycle progression, and signal transduction. As such, the ubiquitous, constant addition and removal of phosphate from proteins serves as a specific on/off switch for individual cell operations, and for the cell as a whole.2 Any disruption to these processes can wreak havoc on the cell. In fact, mutations in the proteins that regulate phosphorylation have been discovered to be a factor in a range of cancers. Thus, determining the extent and locations of phosphorylation is critical to understanding biochemical pathways in cells, particularly how cellular activity is activated or suppressed.2

As a result, researchers are keen on having efficient, accurate methods to measure intracellular phosphorylation, which can help gain insight into active signaling pathways in the cells, as well as targets for therapeutic intervention.

Traditional Techniques for Measuring Phosphoproteins

Currently, there are a number of methods used to detect and measure phosphoproteins, including:

  • Phosphoflow: Over the last decade, phosphoflow cytometry has grown exponentially. It allows for the analysis of phosphorylation events and cell surface markers simultaneously, which enables researchers to resolve complex biochemical signaling networks in cell populations. Phosphoflow cytometry can be used in a number of areas including antigenic stimulation and microbial challenges, high-throughput, and high-content drug discovery, as well as the characterization of signaling pathways in both normal and disease-altered immune responses.3 However, there are challenges with this method, as it’s limited by the number of analytes that can be measured (3-5) and the ability to create panels with low spectral overlap.
  • Mass Spectrometry: Mass spectrometry (MS)based proteomics has emerged as the preferred technology for protein identification, characterization and quantification.4 Even still, MS analysis of protein phosphorylation is still far from being routine, and often has unsatisfying results due to being limited by the ability to differentiate the weaker signals of less abundant phosphoproteins from strong signal of highly abundant non-phosphorylated proteins.
  • Multi-Analyte profiling (microplate and membrane based): Mass spectrometric techniques can provide comprehensive parallel analysis of peptide sequences and phosphorylation, however, these methods can also be labor-intensive, and comprehensive phosphorylation analysis may not be needed if particular pathways are of primary interest. This has led to researchers measuring protein phosphorylation of multiple analytes simultaneously. In general, these methods use phospho-specific antibodies and include microplate-based and membrane-based detection formats. The benefit of these assays is that throughput capability is greatly enhanced by bypassing the need for running multiple individual Western blots or traditional ELISA-based assays. These techniques are also known for providing more data while requiring very little sample volume. However, protein profiling assays are typically seen as being less sensitive than their more traditional counterparts due to potential antibody cross-reactivity.5 Despite measuring a large number of proteins, they cannot quantify at the single-cell level and with low sensitivity.
  • Enzyme-Linked Immunosorbent Assay (ELISA): This protocol uses a capture antibody specific for the desired protein, independent of the phosphorylation state. The addition of a target protein allows it to bind to the antibody-coated plate. A detection antibody is then added that specifically binds to the phosphorylation site of the target protein. The results are analyzed by colorimetric or fluorometric detection.6 However, this method is limited by the number of phosphoproteins that can be measured and is both time and labor intensive.
  • Western Blots: As one of the more established techniques, this method is commonly used to measure phosphoproteins. Yet despite it being used for decades, traditional western blotting is typically considered time consuming with many labor intensive, manual steps — from running a gel, to blot transfer, blocking, antibody incubations, washing, and detection.2
  • Kinase Activity Assays: Kinases are one of the most important and well studied groups of enzymes. They catalyze the transfer of phosphate from ATP to a variety of cellular substrates and many cellular processes, including cell signaling, cell division and growth, development, differentiation, and cell death, are coordinated by kinases. Under normal conditions, kinase activities are tightly regulated. However, when they are dysregulated, disruption is caused to intracellular signaling networks which can lead to diseases such as cancer and inflammation. Thus, kinases are an important target for drug discovery and development. Kinase assays work by measuring the activity of the kinase. However, these assays provide a limited picture of the signaling pathway, and tend to be time and labor intensive.7,8

Understanding Phosphoproteins in Precision Medicine for Cancer

Detecting protein biomarkers early is key for cancer prognosis and disease prevention. Recent advancements in proteomics have helped scientists to better study and understand changes in proteins, including post-translational modifications, such as phosphorylation. By understanding and identifying changes in proteins, important cellular pathways can be discovered, which, in turn, helps make the search for cancer biomarkers a little easier.9

However, as discussed above, many of the traditional methods used to measure protein activity are only able to detect a limited number of proteins at one time and tend to be labor intensive, requiring large amounts of sample. The protein samples may be denatured or separated by charge in these methods, thus destroying information about the target proteins.  In addition, they might not provide the sensitivity and robustness required as these techniques typically provide bulk cell analysis and measure average protein levels within the sample. For scientists to better identify the right biomarkers and targets, and understand the full pathway map, they need to be able to study and measure protein activity at the single cell level.9

A Single-Cell Intracellular Proteome Solution

To address many of the challenges associated with existing techniques, we have developed a breakthrough alternative: the IsoPlexis Single-Cell Intracellular Proteome Solution, which allows users to analyze signaling cascades of many phosphoproteins directly from a single cell, across thousands of cells in parallel.

In this method, live cells are lysed directly on the chip, providing real-time insight into intracellular pathways, disease progression, and mechanisms of therapy resistance. In addition, by using proteomic barcode technology, 15+ analytes per cell and up to 1,500 cells per chip, can be measured. The data is automatically analyzed with IsoSpeak software for instant analysis and publication-ready figures.

The leap over existing technologies is the Single-Cell Intracellular Proteome Solution’s ability to quantify and highly multiplex 15+ intracellular analytes simultaneously from each cell, and thus detect critical protein to protein interactions and signaling networks in rare cells and cell subsets.


Protein phosphorylation is an essential component of a cell biologists’ repertoire for understanding intracellular factors underlying cellular activities6 – therefore it’s critical for researchers to have high quality and reliable tools.

As a critical post-translational modification, phosphorylation regulates a wide range of cellular activities. However, abnormal phosphorylation is implicated in many diseases. The method researchers choose when analyzing phosphorylation will vary depending on many factors including the specific area of research as well as availability of equipment and/or reagents.

Fortunately, methods continue to improve, enabling researchers to effectively measure and detect phosphoproteins so they can fully understand mechanisms behind patient resistance and advance oncology as a whole.

What can the Single-Cell Intracellular Proteome solution do for your research? Talk with an expert now.


  1. Steen, H., et al. (2005, October 4). Phosphorylation Analysis by Mass Spectrometry. MCP Papers in Press. https://www.mcponline.org/article/S1535-9476(20)31514-0/fulltext
  2. DePalma, A. (2018, May 29). Detecting Phosphorylation. Biocompare. https://www.biocompare.com/Editorial-Articles/349977-Detecting-Phosphorylation/.
  3. Bushnell, T. (2021, March 11). How To Do Phospho-Flow Cytometry. Cheeky Scientist. https://expert.cheekyscientist.com/how-to-do-phospho-flow-cytometry/
  4. Wu, S., et al. (2010, June). Development and application of ‘phosphoflow’ as a tool for immunomonitoring. Expert review of vaccines. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2933839/
  5. Methods for Detecting Protein Phosphorylation. R&D Systems. https://www.rndsystems.com/resources/articles/methods-detecting-protein-phosphorylation
  6. Protein Phosphorylation. Cell Biolabs. https://www.cellbiolabs.com/news/protein-phosphorylation
  7. Kinase Activity Assays. Promega. https://www.promega.com/products/cell-signaling/kinase-assays-and-kinase-biology/#related-resources
  8. How Does a Biochemical Kinase Assay Work? BellBrook Labs. http://www.bellbrooklabs.com/how-does-a-biochemical-kinase-assay-work/
  9. The Scientist Creative Services Team in Collaboration with IsoPlexis. Advancing Cancer Biomarker Detection with Single Cell Proteomics. The Scientist Magazine. https://www.the-scientist.com/research-products-blog/single-cell-intracellular-proteome-isoplexis-68832
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