Uncovering the Proteome Response of the Master Circadian Clock to Light Using an AutoProteome System

Aug 24, 2011Molecular & cellular proteomics : MCP

Protein changes in the body’s main internal clock in response to light using an automated analysis system

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Abstract

A total of 2131 proteins were confidently identified from the suprachiasmatic nucleus (SCN) tissue of a single mouse.

Of the identified proteins, 387 were found to be light-regulated based on spectral counts quantification.
Bioinformatics analysis revealed diverse distribution of light-inducible proteins across various canonical pathways and 19 protein interaction networks.
The AutoProteome system identified vasopressin-neurophysin 2-copeptin and casein kinase 1 delta, previously linked to clock timing processes, as light-inducible proteins.
Additionally, Ras-specific guanine nucleotide-releasing factor 1, ubiquitin protein ligase E3A, and X-linked ubiquitin specific protease 9 were also identified as light-inducible proteins, despite not being previously associated with SCN clock timing.

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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.

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Uncovering the Proteome Response of the Master Circadian Clock to Light Using an AutoProteome System

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