Neuroproteomics of the Synapse: Subcellular Quantification of Protein Networks and Signaling Dynamics

Abstract

One of the most fascinating features of the brain is its ability to adapt to its surroundings. Synaptic plasticity, the dynamic mechanism of functional and structural alterations in synaptic strength, is essential for brain functioning and underlies a variety of processes such as learning and memory. Although the molecular mechanisms underlying such rapid plasticity are not fully understood, a consensus exists on the important role of proteins. The study of these neuronal proteins using neuroproteomics has increased rapidly in the last decades, and advancements in MS-based proteomics have broadened our understanding of neuroplasticity exponentially. In this review, we discuss the trends in MS-based neuroproteomics for the study of synaptic protein-protein interactions and protein signaling dynamics, with a focus on sample types, different labeling and enrichment approaches, and data analysis and interpretation. We highlight studies from the last 5 years, with a focus on synapse structure, composition, functioning, or signaling and finally discuss some recent developments that could further advance the field of neuroproteomics.

Keywords: bio-orthogonal amino acids; mass spectrometry; neuroproteomics; protein networks; protein translation; proximity-labeling; synapse.

Graphical abstract

Fig. 1

Sample characteristics in neuroproteomics studies of the synapse.A, contribution of different sample materials. Although earlier studies were mostly performed on brain homogenates, increase in sensitivity of mass spectrometers resulted in a shift toward the use of tissue from specific brain regions and more recently toward more defined and homogeneous primary cultures. B, the most represented organisms in neuroproteomics studies are the rat and mouse. Although both brain homogenates and primary neurons are predominantly derived from rats, largely because of their larger brain size, genetically modified samples are often derived from mice. C, the distribution of the most studied brain regions has not significantly changed during the last decades.

Fig. 2

The relative contribution of specific cellular compartments in studies utilizing subcellular fractionation techniques. Percentages reflect the relative contribution of each subcellular fraction to all published studies. In total, 51% of all neuroproteomics studies that were included in this review make use of subcellular fractions.

Fig. 3

Overview of bio-orthogonal and proximity labeling approaches and prevalence of labels in neuroproteomics studies.A, prevalence of the use of enrichment labels. In the last decade, an increase in labeling under native conditions (i.e., in living cells or even in living organisms) for labeling of specific cellular structures and/or cellular processes such as protein translation can be observed. B, characteristics of the most prominently used proximity labeling constructs. C, workflow of bio-orthogonal labeling experiments. Unnatural amino acids can be genetically introduced using orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase (upper panel) or supplemented to cell culture media or animal chow (lower panel). The unnatural amino acid can contain an enrichable tag, or a heavy isotope, so that nascent proteins can be enriched and subsequently analyzed by MS or heavy isotope–containing proteins can be analyzed simultaneously with existing, natural proteins, after which the ratio between heavy and light proteins can be determined. D, prevalence of the use of quantification labels.

Fig. 4

Post-translational modifications in neuroproteomics studies focused on the synapse.A, only a minority of published studies have analyzed the prevalence of one or more PTMs in their proteomics dataset. Although phosphorylation is the most studied PTM, other PTMs such as N-glycosylation and ubiquitination are gaining interest. B, known phosphorylation sites and supporting evidence from high-throughput (HT) and low-throughput (LT) studies for the top three used model organisms in neuroproteomics studies. The data used in this graph were taken from the PhosphoSitePlus knowledgebase (133). PTMs, post-translational modifications.