Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

An affinity for new proteins


Metabolic incorporation of an unnatural amino acid allows researchers to rapidly affinity purify newly synthesized proteins from mammalian cell lysates for direct identification by mass spectrometry.


The proteins expressed at any given time in the life cycle of a cell make up its proteomic profile, which is dynamic and ever-changing in response to environmental stimuli. Although mass spectrometry has grown into perhaps the most popular technique for proteomic profiling of different cellular states, it is not always easy to verify which genes are turned on in response to a stimulus because newly synthesized proteins are of course chemically identical to their 'older' siblings.

Hoping to address this problem, Erin Schuman and her colleagues at the California Institute of Technology developed a strategy to directly isolate newly synthesized proteins from cells using the metabolic incorporation of an unnatural amino acid with a bio-orthogonal affinity handle. Using a complementary affinity reagent to isolate the tagged newly synthesized proteins, these 'baby' proteins could be directly identified by mass spectrometry. Improving on the more traditional method of radioactive tracing with [35S]cysteine or [35S]methionine metabolic incorporation, an affinity handle not only allows the detection of newly synthesized proteins but also their isolation for unambiguous identification.

The unnatural amino acid azidohomoalanine (AHA) is an analog of methionine, which the cell's translational machinery will insert in place of methionine if methionine is left out of the mix. The side chain of AHA contains an azide functional group which is not found in nature, but spontaneously reacts with an alkyne (another functional group not found in nature) to form a covalent bond. To pull out the newly synthesized azide-tagged proteins from the total proteomic pool, Schuman and her colleagues developed an alkyne-based affinity reagent containing a Flag antibody epitope and biotin, which forms an extremely tight noncovalent association with the protein avidin. They entitled their labeling strategy 'BONCAT', or 'bioorthogonal noncanonical amino acid tagging'.

As proof of principle, the researchers exposed human embyronic kidney 293 cells to AHA for 2 hours. They used western blotting to verify that AHA was incorporated into a wide range of proteins. They incubated cell lysates with the biotin–Flag–alkyne affinity reagent and used avidin resin to isolate the newly synthesized AHA-tagged proteins. They digested the immobilized proteins on the resin, and subjected the fragments to shotgun mass-spectrometry analysis and database searching (Fig. 1). They successfully identified 1,028 nonredundant peptides, which made up 195 different newly synthesized proteins with a diverse range of functions and biochemistries.

Figure 1: Overview of the BONCAT strategy.

The cell lysate pool containing a 'snapshot' of old and new proteins is incubated with the biotin-Flag-alkyne affinity reagent, which reacts to form a covalent bond to the newly synthesized, azide-labeled proteins. The newly synthesized tagged proteins are pulled out of the lysate with avidin affinity resin and subjected to on-resin digestion and mass spectrometry analysis.

Schuman and her colleagues believe their method provides a unique and simple route to obtaining temporal 'snapshots' of diverse mammalian cellular proteomes. They suggest that BONCAT may be particularly useful for metabolic labeling in post-mitotic cell cultures, in which stable isotope labeling with heavy and light amino acid variants has been challenging.


  1. 1

    Dieterich, D.C . et al. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc. Natl. Acad. Sci. USA 103, 9482–9487 (2006).

    CAS  Article  Google Scholar 

Download references


Rights and permissions

Reprints and Permissions

About this article

Cite this article

Doerr, A. An affinity for new proteins. Nat Methods 3, 584 (2006).

Download citation


Quick links