Abstract
In mammals, the suprachiasmatic nucleus (SCN) is the central circadian pacemaker that governs rhythmic fluctuations in behavior and physiology in a 24-hr cycle and synchronizes them to the external environment by daily resetting in response to light. The bilateral SCN is comprised of a mere ~20,000 neurons serving as cellular oscillators, a fact that has, until now, hindered the systematic study of the SCN on a global proteome level. Here we developed a fully automated and integrated proteomics platform, termed AutoProteome system, for an in-depth analysis of the light-responsive proteome of the murine SCN. All requisite steps for a large-scale proteomic study, including preconcentration, buffer exchanging, reduction, alkylation, digestion and online two-dimensional liquid chromatography-tandem MS analysis, are performed automatically on a standard liquid chromatography-MS system. As low as 2 ng of model protein bovine serum albumin and up to 20 μg and 200 μg of SCN proteins can be readily processed and analyzed by this system. From the SCN tissue of a single mouse, we were able to confidently identify 2131 proteins, of which 387 were light-regulated based on a spectral counts quantification approach. Bioinformatics analysis of the light-inducible proteins reveals their diverse distribution in different canonical pathways and their heavy connection in 19 protein interaction networks. The AutoProteome system identified vasopressin-neurophysin 2-copeptin and casein kinase 1 delta, both of which had been previously implicated in clock timing processes, as light-inducible proteins in the SCN. Ras-specific guanine nucleotide-releasing factor 1, ubiquitin protein ligase E3A, and X-linked ubiquitin specific protease 9, none of which had previously been implicated in SCN clock timing processes, were also identified in this study as light-inducible proteins. The AutoProteome system opens a new avenue to systematically explore the proteome-wide events that occur in the SCN, either in response to light or other stimuli, or as a consequence of its intrinsic pacemaker capacity.
Highlights
From the ‡Ottawa Institute of Systems Biology, §Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5, ¶Department of Chemistry, Faculty of Science, University of Ottawa, 10 Maria Curie, Ottawa, Ontario, Canada K1N 6N510
Endogenous peptides secreted from the suprachiasmatic nucleus (SCN) were studied by both in situ solid-phase extraction followed by off-line matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) MS as well as LC-Fourier transform (FT) MS using multiple tissues [21, 22]
The protocol for the proteomic reactor [33] was modified such that dithiotreitol was replaced with TCEP on the proteomic reactor, allowing the protein reduction to be accomplished under acidic conditions, and some of the buffer exchange procedures used in the manual proteomic reactor protocol were eliminated
Summary
Subsequent validation by immunostaining and Western blot analysis demonstrated the light-inducible expression of two key proteins that have previously been implicated in clock timing processes, as well as three additional proteins that have not been linked to circadian rhythms. In order to explore the light-responsive proteome of the SCN, a quantitative proteomic study based on spectral counts was performed following photic stimulation [42,43,44].
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