Upon completion of brain development neurons and glia form a high-performance communication network courtesy of trillions of specialized contact sites. Synaptogenesis, the formation of new synaptic contacts, and long-lasting forms of synaptic plasticity, which are believed to be the molecular substrate for learning and memory, require the synthesis of new proteins and the correct localization of these proteins within the neuron at active synaptic sites.

Despite considerable efforts and the characterization of multiple synaptic proteins, a comprehensive and dynamic characterization of the synaptic proteome during synaptogenesis and synaptic plasticity is still missing. Moreover, the role of glia during neuronal development and processes of synaptic plasticity as well as the identity of the glial proteome are largely unknown.

With the projects of the Neuralomics group we hope to attain a molecular and systemic characterization of both the neuronal and glial proteomes at different stages of development of the hippocampal formation of the rat using the two recently developed techniques BONCAT and FUNCAT. These tools enable the time-resolved dynamic identification and in situ visualization of the subpopulation of newly synthesized, endogenous proteins.

The BONCAT & FUNCAT Technologies

How can one identify and monitor the locale of an entity of proteins that is synthesized during development or as a consequence of a changed environment? To address these questions, BONCAT (bioorthogonal non-canonical amino acid tagging, (2)) was developed at Caltech. This approach enables one to specifically identify the subpopulation of newly synthesized proteins. The core of the technique capitalizes on the manifold potential of small bioorthogonal chemo-selective groups. These groups deliver unique chemical functionality to their target molecules, which can subsequently be tagged with exogenously delivered probes for detection or isolation in a highly selective manner using click chemistry (see Figure 1).

Fig. 1 : Click chemistry reaction scheme.

In the first step of BONCAT (see Figure 2) newly synthesized proteins are labeled using the azide-bearing artificial amino acid Azidohomoalanine (AHA) as a surrogate for methionine, thus endowing the proteins with a novel azide functionality that serves to distinguish them from the pool of pre-existing proteins. Employing copper-catalyzed click chemistry, the reactive azide group of AHA is covalently coupled to an alkyne-bearing affinity tag in the second step of BONCAT. The affinity tag enables the subsequent detection, affinity purification and MS identification of AHA-labelled proteins. The enrichment for newly synthesized proteins subsequent to affinity purification decreases the complexity of the sample, enabling the identification of proteins expressed at low levels. In combination with leucine-based (tenfold deuterated leucine, d10L) mass tagging, candidates are immediately validated as newly synthesized proteins.

Fig. 2 : From Dieterich et al., Nature Protocols 2007: The BONCAT strategy.