Mass spectrometry-based absolute quantification reveals rhythmic variation of mouse circadian clock proteins

Author:

Narumi RyoheiORCID,Shimizu Yoshihiro,Ukai-Tadenuma Maki,Ode Koji L.,Kanda Genki N.,Shinohara Yuta,Sato Aya,Matsumoto Katsuhiko,Ueda Hiroki R.

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.

Funder

Japan Society for the Promotion of Science

Core Research for Evolutional Science and Technology, Japan Science and Technology Agency

Ministry of Education, Culture, Sports, Science, and Technology

Publisher

Proceedings of the National Academy of Sciences

Subject

Multidisciplinary

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