Application Note 27
Targeted LC-SRM/MS Quantification of Mammalian Synaptic Proteins with Mouse Express® Brain Tissue, a New Isotopically Labeled Proteome Standard
Matthew L. MacDonald,1* Eugene Ciccimaro,2* Ian Blair,1 Chang-Gyu Hahn3
1. Department of Pharmacology, University of Pennsylvania, Philadelphia, PA USA
2. ThermoFisher Scientific, Somerset, NJ USA
3. Department of Psychiatry, University of Pennsylvania, Philadelphia, PA USA
*Primary Authors
Maintenance and reshaping of synaptic connections in the brain underlies the ability to learn and remember. Complex protein machinery at the synapse is constantly seeking out, strengthening and pruning neuronal connections, literally rewiring in response to experience. The process by which neurons accomplish this feat has been termed “neuroplasticity.” Elucidating the molecular mechanics of neuroplasticity is critical to understanding how the human brain interacts with the world around it, as well as how neuropsychiatric diseases assault our memories, thoughts, and motor function.
Trafficking of synaptic proteins is critical to neuroplasticity, and many of these trafficked proteins have been implicated in a broad spectrum of neuropsychiatric disorders.1,2 An in-depth understanding of how these trafficking events function could hold the key to developing new therapeutics. It has become clear that targeted multiplexed quantification of proteins across neuronal subcellular domains is essential to probe these normal and aberrant trafficking events. Mass spectrometry (MS)-based proteomics has emerged as a valuable tool for studying these phenomena. Stable isotope labeling with amino acids in cell culture (SILAC)-based methodologies have been successfully applied to cellular models of many diseases.3 However, this methodology has found limited utility in neuroscience research as complex psychiatric diseases are difficult to model in animals and are impossible to model in culture. Isobaric-labeling methodologies, such as iTRAQ, have proven more useful in assaying trafficking in humans and animal models,4,5 but peptide chemical-tagging methodologies suffer from compression of the quantitative signal and have difficulty repetitively quantifying a targeted subset of peptides. The availability of protein standards from CIL, generated from stable isotope labeling in mammals (SILAM) with L-lysine·2HCl (13C6, 99%) (CLM-2247-H), allows multiplexed targeted quantitative analysis of protein trafficking in brain tissue without the costly and untimely synthesis of individual peptide or protein standards.
In this note, we describe a liquid chromatography-selected reaction monitoring LC-SRM/MS approach for the targeted quantification of synaptic peptides in subcellular fractions of mammalian brain tissue utilizing CIL’s Mouse Express® brain tissue L-lysine (13C6, 97%).This method utilizes membrane preparations from SILAM brain homogenate as an internal standard facilitating the quantification of over 100 proteins across three neuronal subfractions: vesicular, presynaptic and postsynaptic density, isolated from mouse brain tissue.
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