Mass spectrometry-based absolute quantification PNAS PLUS reveals rhythmic variation of mouse circadian clock proteins Ryohei Narumia,1, Yoshihiro Shimizub,1,2, Maki Ukai-Tadenumaa, Koji L. Odec, Genki N. Kandaa,d, Yuta Shinoharaa,d, Aya Satob, Katsuhiko Matsumotoa, and Hiroki R. Uedaa,c,d,e,2 aLaboratory for Synthetic Biology, RIKEN Quantitative Biology Center, Suita, Osaka 565-0874, Japan; bLaboratory for Cell-Free Protein Synthesis, RIKEN Quantitative Biology Center, Suita, Osaka 565-0874, Japan; cDepartment of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan; dGraduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan; and eCore Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan Edited by Joseph S. Takahashi, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 26, 2016 (received for review March 7, 2016) Absolute values of protein expression levels in cells are crucial large amounts of isotopically labeled amino acids as substrates. information for understanding cellular biological systems. Precise Difficulties of synthesis vary depending on the peptide sequence. quantification of proteins can be achieved by liquid chromatogra- Due to these features, it is difficult to reduce the cost per peptide, phy (LC)–mass spectrometry (MS) analysis of enzymatic digests of which leads to difficulties in preparing large numbers of peptides proteins in the presence of isotope-labeled internal standards. for the quantification. Multiple peptide production enables not Thus, development of a simple and easy way for the preparation only the multiplexed protein quantification but also highly sensitive of internal standards is advantageous for the analyses of multiple detection and quantification of the proteins of interest by selecting target proteins, which will allow systems-level studies. Here we effective internal standards with good signal/noise ratio from can- describe a method, termed MS-based Quantification By isotope- didate peptides (3). labeled Cell-free products (MS-QBiC), which provides the simple Several methods using a cellular gene-expression system have and high-throughput preparation of internal standards by using been developed in recent years for multiple peptide production. a reconstituted cell-free protein synthesis system, and thereby facil- Multiple peptides can be obtained by the proteolysis of recom- itates both multiplexed and sensitive quantification of absolute binantly expressed full-length proteins of interest (4, 5) or artificial amounts of target proteins. This method was applied to a sys- proteins that compose a set of proteotypic peptides concatenated tems-level dynamic analysis of mammalian circadian clock proteins, with each other (6, 7). These peptides are metabolically labeled which consist of transcription factors and protein kinases that gov- with stable isotopes through Escherichia coli or other cellular ern central and peripheral circadian clocks in mammals. Sixteen pro- gene-expression systems, allowing them to function as internal teins from 20 selected circadian clock proteins were successfully standards. The methods have advantages in that multiple peptides quantified from mouse liver over a 24-h time series, and 14 proteins are produced from a single construct, which can be applied to both SYSTEMS BIOLOGY had circadian variations. Quantified values were applied to detect multiplexed and sensitive quantification. internal body time using a previously developed molecular timeta- A cell-free protein synthesis system is a useful tool for the ble method. The analyses showed that single time-point data from expression of such proteins (3, 5, 6, 8). The cost for expensive stable wild-type mice can predict the endogenous state of the circadian isotope-labeled amino acids can be reduced because the volume for clock, whereas data from clock mutant mice are not applicable be- reaction mixtures is much lower than for culturing media. Protein cause of the disappearance of circadian variation. expression and purification occurs in a high-throughput manner absolute quantification | mass spectrometry | cell-free protein synthesis Significance system | mammalian circadian clock protein | targeted proteomics A method for absolute quantification of proteins for targeted uantitative information on protein expression levels is im- proteomics is developed. It introduces a simple and high- Qportant to define the dynamic state of cells. Accurate mea- throughput synthesis of internal standards for peptide quan- surements of absolute protein abundance in cells can be performed tification and thereby facilitates both multiplexed and sensitive by emerging quantitative proteomics approaches such as selected absolute quantification of proteins. Application of this method reaction monitoring (SRM) or high-resolution mass spectrometry to the systems-level dynamic analysis of core circadian clock (MS) in combination with isotope dilution strategies (1). The proteins and detection of internal body time using quantified methods require a known concentration of internal standards, typ- values of circadian clock proteins is shown. The results dem- ically prepared as the tryptic digests of target proteins, which are onstrate the validity of the developed method in which labeled with isotopically heavy atoms. The standard peptides are quantified values from wild-type mice can predict the endog- combined with samples containing the same peptides, and the enous state of the circadian clock. mixtures are analyzed by MS. Quantities of peptides, which repre- sent the target proteins, can be calculated by comparing ion in- Author contributions: R.N., Y. Shimizu, K.L.O., and H.R.U. designed research; R.N., Y. Shimizu, M.U.-T., K.L.O., G.N.K., Y. Shinohara, A.S., and K.M. performed research; R.N. and Y. Shimizu tensities for isotopically light and heavy peptides. contributed new reagents/analytic tools; R.N., Y. Shimizu, and M.U.-T. analyzed data; The preparation of isotope-labeled peptides plays a major role and R.N., Y. Shimizu, and H.R.U. wrote the paper. in these approaches. The most common way to prepare such The authors declare no conflict of interest. peptides is by absolute quantification, which uses chemical syn- This article is a PNAS Direct Submission. thesis of peptides containing isotopically labeled amino acids (1, 2). 1R.N. and Y. Shimizu contributed equally to this work. It provides not only normal proteotypic peptides, but also modified 2To whom correspondence may be addressed. Email: [email protected] or uedah-tky@ peptides that mimic posttranslational modification. However, the umin.ac.jp. method has several limitations originating from the use of the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. chemical synthesis. It requires individual peptide synthesis using 1073/pnas.1603799113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1603799113 PNAS | Published online May 31, 2016 | E3461–E3467 Downloaded by guest on September 25, 2021 because there is no need for culturing, harvesting, and disrupting Synthesis of MS-QBiC peptide with PURE System cells. Notably, cellular metabolism causes isotope scrambling and DNA template Pu + U dilution, which is a problem where the homogeneity of isotope- FW primer Pr Q Pr Q T + ++ labeled peptides is reduced due to conversion of labeled amino acids RV primers into others, or vice versa (9). This problem can be overcome in a cell- PCR free system by artificial adjustment of the system components (10). DNA Pr U Pu Q T Translation Here we describe a workflow for multiplexed absolute quan- Purification tification of proteins using SRM-based targeted proteomics. The MS-QBiC peptide Pu Q T workflow, termed MS-based Quantification by isotope-labeled M K* K* K* Cell-free products (MS-QBiC), has several features that expand Addition Addition Quantification of MS-QBiC peptide Quantification of target peptide advantages of the internal standard synthesis using a cell-free sys- Light version of Q Biological sample tem. It is based on the use of the PURE system, a reconstituted quantification-tag K Protein + Pu Q T MS-QBiC peptide M K* K* K* + cell-free protein synthesis system (11). Because the PURE system MS-QBiC peptide Pu Q T consists of purified factors and enzymes for E. coli translation Digestion Trypsin M K* K* K* Q Digestion Trypsin machinery, synthesized peptides are rarely challenged by protease Pu K T T K R Peptide M K* Q K* K K K* Peptide T R R R degradation that usually occurs in cell-extract systems. Addition- K* R R K ally, isotope scrambling or dilution is avoided without the need to Q T adjust system components (10). The developed workflow was ap- K K LC-MS LC-MS Q T of MS plied to the absolute quantification of core circadian clock proteins of MS K* K* Signal intensity in mouse livers across the circadian day. To obtain optimal pep- Signal intensity Retention time Retention time tides for the detection and quantification by the SRM-based tar- geted proteomics analysis, we synthesized 120 peptides for 20 Calculation of the absolute amount of target protein circadian clock proteins. All of the peptides were successfully Fig. 1. Development of the MS-QBiC workflow. Schematic description of synthesized from PCR-amplified genes by the PURE system. Dy- the MS-QBiC workflow. A purification tag, a quantification tag,