title: Uncovering the proteome response of the master circadian clock to light using an AutoProteome system.
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.
authors.affiliation: Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5.
authors.forename: Ruijun
authors.initials: R
authors.lastname: Tian
authors.forename: Matias
authors.initials: M
authors.lastname: Alvarez-Saavedra
authors.forename: Hai-Ying M
authors.initials: HY
authors.lastname: Cheng
authors.forename: Daniel
authors.initials: D
authors.lastname: Figeys
mesh_terms: D000818:Animals; D057205:Automation, Laboratory; D002417:Cattle; D002853:Chromatography, Liquid / Q000592:standards; D057906:Circadian Clocks; D015870:Gene Expression / Q000528:radiation effects; D008027:Light; D008297:Male; D053858:Metabolic Networks and Pathways; D051379:Mice; D008810:Mice, Inbred C57BL; D060066:Protein Interaction Maps; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000592:standards; D013493:Suprachiasmatic Nucleus / Q000378:metabolism* / Q000528:radiation effects; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D020543:Proteome; D012710:Serum Albumin, Bovine