Insights

What is Phosphoproteomics?

Ryan Smith - 1 November 2021

Phosphorylation is one of the most important post-translational modifications in a biological system, playing a key role in cell cycle regulation, growth, and apoptosis.

Phosphorylation is one of the most important post-translational modifications in a biological system, playing a key role in cell cycle regulation, growth, and apoptosis. Phosphoproteomics concerns itself with the study of this modification, but why is it so important, how do we measure it, and what can we do with this information?

Cells respond to external and internal perturbations by way of signalling networks; a common molecular event for the transmission of these signals is protein phosphorylation – which in essence is the addition of a phosphoryl group to a protein. Protein enzymes—specifically protein kinases—are the catalysts of this modification and can become activated by other kinases or even by the kinase itself, forming complex cascades of kinase signalling and providing the mechanism for cell signalling pathways. Complicating matters further is the existence of phosphatases, enzymes responsible for dephosphorylation, meaning phosphorylation is a reversible post-translational modification. Kinases and phosphatases work in concert to regulate phosphorylation and together play a critical role in cell signalling pathways. The stability of these signalling pathways is linked to the healthy metabolism of cells and controls cellular events such as cell growth, proliferation, and differentiation. It follows, rather intuitively, that the corruption and dysregulation of these signalling pathways is almost always implicit in causing or sustaining diseases such as cancer. Phosphoproteomics as it extends into revealing novel cell signalling insights is of great importance to biological research and has direct implications in the clinic, for both diagnosis and treatment of disease patients.

The abundance of phosphorylated proteins in a biological sample is—by definition—directly linked to the activity of the protein kinases in the sample. The ability to measure the abundance of phosphorylated proteins on a global scale should therefore be an alluring prospect for cell signalling researchers. Mass spectrometry is—and has been for many years—the workhorse of proteomics experiments, allowing researchers to profile the proteome of complex biological samples. Advances in instrumentation, sample preparation strategies, and bioinformatics have paved the way for experiments in the sub-discipline of mass spectrometry-based phosphoproteomics. In such experiments, researchers will digest proteins in a biological sample and then enrich phosphorylated peptides.  The resulting mix of phosphopeptides can then be identified and quantified, revealing the abundance of specific phosphorylated residues which are known substrates of protein kinases. Indeed, astute applications of such methods have allowed researchers to probe further; elucidating putative kinase-phosphosite relationships and kinase networking to deconvolute network topologies and reveal novel insights in cancer research.

Many challenges in phosphoproteomics remain, such as determining kinase-substrate specificity, phosphosite localisation, and extensive functional characterisation of the phosphoproteome, to name a few. However, what is clear is that as the field continues to rapidly expand, the study of phosphoproteomics promises to provide an understanding of how protein kinases are manifested in biological systems, particularly in cancer where protein kinases already offer targets for therapies, with many more inhibitory agents entering clinical trials.

Sources

  • Hijazi, M., Smith, R., Rajeeve, V. et al. Reconstructing kinase network topologies from phosphoproteomics data reveals cancer-associated rewiring. Nat Biotechnol 38, 493–502 (2020). https://doi.org/10.1038/s41587-019-0391-9

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