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Researchers from Utrecht ľ�ϸ���Ӱ�� designed an efficient tool to fish exclusively for newly synthesized proteins, in a vast background of “older” cellular proteins. This work was published in the scientific journal Nature Communications on 26th of June. Such a tool allows them to trace specific cellular responses, for instance to a drug or hormone treatment.

Trillions of proteins make up our bodies and these proteins are continuously made and degraded, in a dynamic balance, depending on cellular needs. Understanding which new proteins will be made is the first step to deciphering cellular responses. Researchers thus want to know which new proteins are made in response to a cellular perturbation, on top of the vast background of “older’ proteins. Conventional methods typically measure a steady-state composite of these opposing processes, which is insufficient to tell if a protein is synthesized more or degraded less.

“This conundrum can now be resolved by our method, which involves adding a chemical tag on all newly synthesized proteins. With this unique tag, and the chemical ligation process we pioneer here, we can specifically monitor protein synthesis that takes place over short durations,” says Wei Wu, assistant professor at the Department of Pharmaceutical Sciences, and co-investigator of this new method named ‘PhosID’.

The Chemistry and the Biology

The chemical moieties ‘azide’ and ‘alkyne’ can react together to form a stable compound that is strong enough to withstand harsh purification conditions. After azide functionalities are metabolically incorporated into freshly synthesized proteins, in living cells, a phosphonate-containing alkyne is ligated subsequently. This finally allows such newly synthesized proteins to be isolated by highly selective metal-ion binding properties of the phosphonate-handle. The novelty of this approach lies in the use of extremely stable phosphonate-handles for analyte retrieval. By applying this methodology, the complete biosynthetic response to inflammation factor interferon gamma was comprehensively mapped.

Multidisciplinary team effort

A diverse mix of talents was instrumental to this achievement. “By evaluating the pros and cons of traditional ‘click’ chemistry methods, we put our diverse expertise together and designed a new enrichment tag, with superior specificity and sensitivity,” says Fleur Kleinpenning, a postdoctoral fellow with expertise in bio-orthogonal ‘click’ chemistry, and first author on this manuscript. Having a resident organic chemist on board in the team also fostered creativity across disciplines, and enabled the chemical tags needed to be designed and made in-house.

“This phosphonate handle was designed to incorporate excellent features that enable strong enrichment, stability and efficient reaction times.  We are also extremely happy that this was achievable via a robust synthetic route, even for non-experts to make use of our technology,” says Barbara Steigenberger, a postdoctoral fellow with expertise in organic synthesis and mass spectrometry.

Assembling existing tools into a very efficient new workflow

This new method boosts detection sensitivity, which also allows quick changes in protein synthesis to become easily measurable. In addition, the enrichment step via the phosphonate-handle offers a unique window to monitor protein synthesis, without the interference of potential degradation. As such, a much clearer view connecting protein induction and cellular behaviour can be revealed.

“As most inventions, our invention combines the best of different worlds that were just not brought together earlier” says Albert Heck, head of the Biomolecular Mass Spectrometry and Proteomics group where this work was conceived and came to fruition.

 “We cleverly combined existing tools in metabolic labelling (biology) and bio-orthogonal chemistry (chemical biology), with IMAC chromatography and mass spectrometry (analytical chemistry), into a new and efficient method to fish for newly synthesized proteins. I am very proud of the team who contributed their expertise in this multidisciplinary effort. As the tool is straightforward to use, I am convinced it will find its way into many research laboratories with interest in cell biology”.

Publication

. Nature Communications, 2020.
Fleur Kleinpenning*, Barbara Steigenberger*, Wei Wu* & Albert J. R. Heck*

*Affiliated with Utrecht ľ�ϸ���Ӱ��