Abstract
Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex. A majority of neuropsychiatric illnesses are complex trait disorders, in which multiple etiologic factors converge at the synapse via many signaling pathways. Investigating the protein composition of synaptic microdomains from human patient brain tissues will yield valuable insights into the interactions of risk genes in many disorders. These types of studies in postmortem tissues have been limited by the lack of proper study paradigms. Thus, it is necessary not only to develop strategies to quantify protein and post-translational modifications at the synapse, but also to rigorously validate them for use in postmortem human brain tissues. In this study we describe the development of a liquid chromatography-selected reaction monitoring method, using a stable isotope-labeled neuronal proteome standard prepared from the brain tissue of a stable isotope-labeled mouse, for the multiplexed quantification of target synaptic proteins in mammalian samples. Additionally, we report the use of this method to validate a biochemical approach for the preparation of synaptic microdomain enrichments from human postmortem prefrontal cortex. Our data demonstrate that a targeted mass spectrometry approach with a true neuronal proteome standard facilitates accurate and precise quantification of over 100 synaptic proteins in mammalian samples, with the potential to quantify over 1000 proteins. Using this method, we found that protein enrichments in subcellular fractions prepared from human postmortem brain tissue were strikingly similar to those prepared from fresh mouse brain tissue. These findings demonstrate that biochemical fractionation methods paired with targeted proteomic strategies can be used in human brain tissues, with important implications for the study of neuropsychiatric disease.
Highlights
Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex [1, 2]
Stable isotope labeling by amino acids in cell culture (SILAC)1 can produce only a partially labeled neural proteome [18] and we have found that full expression levels and synaptic architecture of brain tissue are difficult to reproduce in fully labeled synaptic cultures
The recent availability of stable isotope labeling in mammals (SILAM) mouse tissue allows for the generation of a stable isotope labeled neuroproteome within the context of native tissues to serve as an internal standard for the quantification of a vast number of proteins [19]
Summary
Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex [1, 2]. Mass spectrometry (MS)-based proteomic methodologies, paired with biochemical fractionation techniques, have enabled us to begin cataloging the proteomes and signaling of mammalian synaptic microdomains [1, 3,4,5,6,7] and microdomain specific protein complexes (8 –10) These studies have revealed that synaptic architecture and plasticity involve numerous interactions between multiple signaling mechanisms with robust crosstalk and protein interactions [8, 9, 11, 12]. MS-based proteomic methodologies can monitor numerous proteins and post-translational modifications simultaneously, permitting us to examine signaling pathways in the context of many other intracellular molecular events. Bioinformatic analyses indicate that this method has the capability to quantify thousands of additional neuronal proteins in many model systems
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