Development of Targeted Mass Spectrometry-Based Approaches for Quantitation of Proteins Enriched in the Postsynaptic Density (PSD).

Rashaun Wilson,Fumika Sakaue,Tukiet Lam,Angus Nairn,Kenneth Williams,Navin Rauniyar

doi:10.3390/proteomes7020012

Abstract

The postsynaptic density (PSD) is a structural, electron-dense region of excitatory glutamatergic synapses, which is involved in a variety of cellular and signaling processes in neurons. The PSD is comprised of a large network of proteins, many of which have been implicated in a wide variety of neuropsychiatric disorders. Biochemical fractionation combined with mass spectrometry analyses have enabled an in-depth understanding of the protein composition of the PSD. However, the PSD composition may change rapidly in response to stimuli, and robust and reproducible methods to thoroughly quantify changes in protein abundance are warranted. Here, we report on the development of two types of targeted mass spectrometry-based assays for quantitation of PSD-enriched proteins. In total, we quantified 50 PSD proteins in a targeted, parallel reaction monitoring (PRM) assay using heavy-labeled, synthetic internal peptide standards and identified and quantified over 2100 proteins through a pre-determined spectral library using a data-independent acquisition (DIA) approach in PSD fractions isolated from mouse cortical brain tissue.

Highlights

The postsynaptic density (PSD) is an electron-dense region of excitatory glutamatergic synapses located just beneath the postsynaptic membrane
Immunoblot quantitation revealed that the PSD-enriched fraction displayed a higher ratio of PSD-95/GAPDH expression when compared to the P2 fraction (Figure S1B)
It was apparent that the PSD fraction isolated from all biological replicates was enriched for a PSD marker while being depleted of cytosolic and mitochondrial contaminants, indicating that these samples were suitable for mass spectrometry-based quantitation of PSD proteins

Summary

Introduction

The postsynaptic density (PSD) is an electron-dense region of excitatory glutamatergic synapses located just beneath the postsynaptic membrane. The PSD was first discovered by electron microscopy in 1956 [1] and was later found to consist of 30–50 nm-thick, disc-shaped protein structures [2,3]. Within these protein structures are several classes of protein families, many of which are involved in processes such as scaffolding and signal transduction. Each of these families are organized in two different structural layers of the PSD: the core and the pallium [4]. The core is the structural layer located near the postsynaptic membrane, while the pallium is positioned beneath the core and is thought to be more labile.

Methods

Results

Conclusion