Organelle proteomics of rat synaptic proteins: correlation-profiling by isotope-coded affinity tagging in conjunction with liquid chromatography-tandem mass spectrometry to reveal post-synaptic density specific proteins.
Organelle proteomics is the method of choice for global analysis of cellular proteins. However, it is difficult to isolate organelles to homogeneity. Recently, correlation-profiling has been used to filter off the contaminants ad hoc and to disclose the genuine organelle-specific proteins. In the present study, we further extend the method to include subcellular compartments that contain proteins shared by multiple distinct subcellular domains. We performed correlation profiling of proteins contained in synaptic membrane and postsynaptic density (PSD) fractions isolated from rat brain. Proteins were labeled with isotope-coded affinity-tag reagents, digested with trypsin, and resulting peptides were resolved by cation exchange chromatography followed by reversed phase chromatography. Peptides were then subjected to mass spectrometry for quantification and identification. We confirm that the core PSD proteins were enriched in the PSD preparation. Other functional protein groups such as cytoskeleton-associated proteins, protein kinases and phosphatases, signaling components and regulators, as well as proteins involved in energy production partitioned to multiple organelles. When analyzed as groups, they were shown to accumulate to a lesser extent. Mitochondrial proteins and transporters were generally strongly depleted from the PSD fraction confirming that they were contaminants of the PSD preparation. Finally, immunoelectron microscopy was performed on selected proteins to validate the proteomics results, and confirm that synaptophysin that was highly depleted in the PSD preparation is localized in the presynaptic compartment, whereas LASP-1 that was slightly enriched in the PSD preparation is present in the PSD as well as other subdomains within the synapse.
Li
Ka wan
Kw
Department of Molecular and Cellular Neurobiology, Center of Neurogenomics and Cognitive Research, Faculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands. ka.wan.li@falw.vu.nl
Hornshaw
Martin P
MP
van Minnen
Jan
J
Smalla
Karl-Heinz
KH
Gundelfinger
Eckart D
ED
Smit
August B
AB
D000345:Affinity Labels; D000818:Animals; D002458:Cell Fractionation; D002845:Chromatography; D013058:Mass Spectrometry; D008840:Microfilament Proteins / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000032:analysis*; D015388:Organelles / Q000737:chemistry; D016548:Prosencephalon; D040901:Proteomics / Q000379:methods*; D051381:Rats; D013569:Synapses / Q000737:chemistry*; D016708:Synaptophysin / Q000032:analysis
D000345:Affinity Labels; C495806:Lasp1 protein, rat; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D016708:Synaptophysin; C033652:postsynaptic density proteins