title: Mass spectrometry-based absolute quantification reveals rhythmic variation of mouse circadian clock proteins.
abstract: Absolute values of protein expression levels in cells are crucial information for understanding cellular biological systems. Precise quantification of proteins can be achieved by liquid chromatography (LC)-mass spectrometry (MS) analysis of enzymatic digests of proteins in the presence of isotope-labeled internal standards. Thus, development of a simple and easy way for the preparation of internal standards is advantageous for the analyses of multiple target proteins, which will allow systems-level studies. Here we describe a method, termed MS-based Quantification By isotope-labeled Cell-free products (MS-QBiC), which provides the simple and high-throughput preparation of internal standards by using a reconstituted cell-free protein synthesis system, and thereby facilitates both multiplexed and sensitive quantification of absolute amounts of target proteins. This method was applied to a systems-level dynamic analysis of mammalian circadian clock proteins, which consist of transcription factors and protein kinases that govern central and peripheral circadian clocks in mammals. Sixteen proteins from 20 selected circadian clock proteins were successfully quantified from mouse liver over a 24-h time series, and 14 proteins had circadian variations. Quantified values were applied to detect internal body time using a previously developed molecular timetable method. The analyses showed that single time-point data from wild-type mice can predict the endogenous state of the circadian clock, whereas data from clock mutant mice are not applicable because of the disappearance of circadian variation.
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, Suita, Osaka 565-0874, Japan;
authors.forename: Ryohei
authors.identifier: 0000-0003-3889-9018
authors.initials: R
authors.lastname: Narumi
authors.affiliation: Laboratory for Cell-Free Protein Synthesis, RIKEN Quantitative Biology Center, Suita, Osaka 565-0874, Japan; yshimizu@riken.jp uedah-tky@umin.ac.jp.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Shimizu
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, Suita, Osaka 565-0874, Japan;
authors.forename: Maki
authors.initials: M
authors.lastname: Ukai-Tadenuma
authors.affiliation: Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan;
authors.forename: Koji L
authors.initials: KL
authors.lastname: Ode
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, Suita, Osaka 565-0874, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan;
authors.forename: Genki N
authors.initials: GN
authors.lastname: Kanda
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, Suita, Osaka 565-0874, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan;
authors.forename: Yuta
authors.initials: Y
authors.lastname: Shinohara
authors.affiliation: Laboratory for Cell-Free Protein Synthesis, RIKEN Quantitative Biology Center, Suita, Osaka 565-0874, Japan;
authors.forename: Aya
authors.initials: A
authors.lastname: Sato
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, Suita, Osaka 565-0874, Japan;
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Matsumoto
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, Suita, Osaka 565-0874, Japan; Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan yshimizu@riken.jp uedah-tky@umin.ac.jp.
authors.forename: Hiroki R
authors.initials: HR
authors.lastname: Ueda
mesh_terms: D000818:Animals; D002940:Circadian Rhythm / Q000502:physiology*; D056925:Circadian Rhythm Signaling Peptides and Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D018345:Mice, Knockout
chemical_list: D056925:Circadian Rhythm Signaling Peptides and Proteins
keywords: absolute quantification; cell-free protein synthesis system; mammalian circadian clock protein; mass spectrometry; targeted proteomics