Synaptosome proteomics

Abstract

Our knowledge of the complex synaptic proteome and its relationship to physiological or pathological conditions is rapidly expanding. This has been greatly accelerated by the application of various evolving proteomic techniques, enabling more efficient protein resolution, more accurate protein identification, and more comprehensive characterization of proteins undergoing quantitative and qualitative changes. More recently, the combination of the classical subcellular fractionation techniques for the isolation of synaptosomes from the brain with the various proteomic analyses has facilitated this effort. This has resulted from the enrichment of many low abundant proteins comprising the fundamental structure and molecular machinery of brain neurotransmission and neuroplasticity. The analysis of various subproteomes obtained from the synapse, such as synaptic vesicles, synaptic membranes, presynaptic particles, synaptodendrosomes, and postsynaptic densities (PSD) holds great promise for improving our understanding of the temporal and spatial processes that coordinate synaptic proteins in closely related complexes under both normal and diseased states. This chapter will summarize a selection of recent studies that have drawn upon established and emerging proteomic technologies, along with fractionation techniques that are essential to the isolation and analysis of specific synaptic components, in an effort to understand the complexity and plasticity of the synapse proteome.

Figure 1

Electron photomicrograph a synapse (56,000×) illustrating the synaptic knob (S) as it ends on the shaft of a dendrite (D) in the central nervous system. P, postsynaptic density; M, mitochondrion. (From: Review of Medical Physiology, 22nd Edition, The McGraw-Hill Co., 2005)

Figure 2

Electron micrograph of a synaptosome fraction isolated from mouse brain by Ficoll density-gradient centrifugation. SV, synaptic vesicles; M, mitochondria; SJ, synaptic junction with attached PSyD. Bar = 0.5 mm. (From: Schrimpf et al. 2005)

Figure 3

Western blots of several novel members of the PSD. Ten to 20 μg of PSD protein were separated by SDS-PAGE and blotted using a variety of antibodies. W, whole brain extract; Syn, synaptosomal fraction; and PSD, postsynaptic density. Known molecular markers were also blotted to demonstrate the purity of the fractions used for MS/MS. ARP, actin-related protein; BAF-1, barrier to autointegration 1; BAP-37, B cell receptor-associated protein; ARVCF, armadillo-repeat-velo-cardio-facial syndrome protein; ARP2/3sub5, actin-related protein complex 2/3 subunit 5; SCCA-1, squamous cell carcinoma antigen-1, serpin3a; NAP-125, NCK-associated protein 1; LGN-1, leucine-rich repeat LGI family, member 1; DCLK, doublecortin-like kinase/mKIAA0369; ICAp69, islet cell autoantigen protein p69. (From: Jordan et al. 2004).

Figure 4

Diagrammatic illustration of the major pre- and postsynaptic proteins identified by 2-DE/MS and/or shotgun proteomics, and normally expected as constituent in synaptosomal preparations. Blue: the proteins that were identified by 2-DE/MS; Yellow: the proteins that were identified by shotgun proteomics; Green: the proteins that were identified by both 2-DE/MS and shotgun proteomics; and Blank (white): those major constituents expected but not identified. *proteins were identified by shotgun proteomics only after the PTM analysis; **proteins were identified as a complex with other proteins by 2-DE/MS; ^aEAA1, EAA2, and GABA transporter. ^bpost-synaptic proteins. (From: Witzmann et al. 2005)

Figure 5

(A). the relative distribution of phosphoserines (pS), phosphothreonines (pT), and phosphotyrosines (pY). The majority of identified sites were phosphoserines (739) followed by phosphothreonines (79) and lastly phosphotyrosines (three). (B). the number of unique phosphorylation sites as a function of the protein functional class. Proteins involved in adhesion/cytoskeleton represent the most prevalent class followed by proteins involved in adaptor/sorting functions. (From: Trinidad et al. 2006)