Neuroproteomics of the synapse: Subcellular quantification of protein networks and signaling dynamics

The advancement in mass spectrometry-based proteomics now allows for the study of highly dynamic, low abundant neuronal processes in specific cellular compartments such as the synapse. In chapter 2, we discuss the trends in mass spectrometry-based neuroproteomics of the synapse. We focus on choices in sample types, different labeling and enrichment approaches for the study of protein-protein interactions and protein signaling, and data analysis and interpretation. We highlight studies from the last five years and finally discuss some recent advancements that could benefit the advancement of neuroproteomics studies. In chapter 3, we integrated quantitative high-resolution phosphoproteomics with the analyses of newly synthesized proteins via bio-orthogonal amino acids (azidohomoalanine) in a pulsed labeling strategy combined with tandem mass tag label-based quantification in cultured hippocampal neurons stimulated with DHPG, to study mGluR5-induced protein phosphorylation and translation. We identified several kinases with important roles in DHPGmGluR-LTD, which we confirmed using small molecule kinase inhibitors. Furthermore, changes in the AMPA receptor endocytosis pathway in both protein synthesis and protein phosphorylation upon mGluR5 activation were identified, whereby Intersectin-1 was validated as a vital player in this pathway. This study revealed several novel insights into the molecular mechanisms underlying mGluR-LTD and provides a broad view on its molecular basis, which serves as a rich resource for further analyses. In chapter 4 we describe the optimization and validation of APEX2 fused to the epidermal growth factor receptor (EGFR). We show that with this proximity labeling protocol, we can distinguish the subtle alterations in receptor trafficking upon stimulation with either EGF or TGF-α, resulting in receptor degradation and recycling, respectively. We identified and quantified EGFR stable and transient interactions at different time points after stimulation and were able to use bystander proteins to map EGFR subcellular location at each time point. Utilizing the fast and concise biotinylation of proximity proteins by APEX2, we were able to detect slight differences in early signaling kinetics between TGF-α and EGF, thereby increasing our knowledge on receptor tyrosine kinase signaling and differential trafficking. In chapter 5 we continued to use APEX2 and fused it to mGluR5 to study receptor localization bias. We deleted a 25 amino acid sequence in the receptor c-terminal tail, which contains the nuclear localization signal to the inner nuclear membrane (INM) of mGluR5, ΔINM-APEX2, and identified a subset of proteins that were specifically localized to the nuclear fraction of mGluR5. Using siRNA and western blot validation approaches, we confirm the role of these ‘bystander’ proteins in the differential trafficking of mGluR5 nuclear versus plasma membrane pools. We confirm the role of the coatomer I complex for the retrograde transport of nuclear mGluR5 from the Golgi to the endoplasmic reticulum (ER) and identify casein kinase 2 as an INM-mGluR5-specific kinase. Moreover, we used glycoproteomics to study localization-based differential glycosylation of mGluR5.