PNAS PLUS

Mass spectrometry-based absolute quantification
reveals rhythmic variation of mouse circadianclock 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,2aLaboratory 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

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 ofproteins in the presence of isotope-labeled internal standards.
Thus, development of a simple and easy way for the preparationof 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 isotopelabeled 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 absoluteamounts 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 fromwild-type mice can predict the endogenous state of the circadianclock, whereas data from clock mutant mice are not applicable because of the disappearance of circadian variation.

|

large amounts of isotopically labeled amino acids as substrates.
Difficulties of synthesis vary depending on the peptide sequence.
Due to these features, it is difficult to reduce the cost per peptide,which leads to difficulties in preparing large numbers of peptides
for the quantification. Multiple peptide production enables not
only the multiplexed protein quantification but also highly sensitive
detection and quantification of the proteins of interest by selecting
effective internal standards with good signal/noise ratio from candidate peptides (3).
Several methods using a cellular gene-expression system havebeen developed in recent years for multiple peptide production.
Multiple peptides can be obtained by the proteolysis of recombinantly expressed full-length proteins of interest (4, 5) or artificial
proteins that compose a set of proteotypic peptides concatenatedwith each other (6, 7). These peptides are metabolically labeledwith stable isotopes through Escherichia coli or other cellular
gene-expression systems, allowing them to function as internal
standards. The methods have advantages in that multiple peptides
are produced from a single construct, which can be applied to both
multiplexed and sensitive quantification.
A cell-free protein synthesis system is a useful tool for the
expression of such proteins (3, 5, 6, 8). The cost for expensive stable
isotope-labeled amino acids can be reduced because the volume forreaction mixtures is much lower than for culturing media. Protein
expression and purification occurs in a high-throughput manner||

absolute quantification mass spectrometry cell-free protein synthesis
system mammalian circadian clock protein targeted proteomics|

SignificanceA method for absolute quantification of proteins for targeted
proteomics is developed. It introduces a simple and highthroughput synthesis of internal standards for peptide quantification and thereby facilitates both multiplexed and sensitive
absolute quantification of proteins. Application of this method
to the systems-level dynamic analysis of core circadian clock
proteins and detection of internal body time using quantified
values of circadian clock proteins is shown. The results demonstrate the validity of the developed method in whichquantified values from wild-type mice can predict the endogenous state of the circadian clock.

Quantitative information on protein expression levels is important to define the dynamic state of cells. Accurate measurements of absolute protein abundance in cells can be performedby emerging quantitative proteomics approaches such as selectedreaction monitoring (SRM) or high-resolution mass spectrometry(MS) in combination with isotope dilution strategies (1). The
methods require a known concentration of internal standards, typically prepared as the tryptic digests of target proteins, which arelabeled with isotopically heavy atoms. The standard peptides are
combined with samples containing the same peptides, and the
mixtures are analyzed by MS. Quantities of peptides, which represent the target proteins, can be calculated by comparing ion intensities for isotopically light and heavy peptides.
The preparation of isotope-labeled peptides plays a major rolein these approaches. The most common way to prepare such
peptides is by absolute quantification, which uses chemical synthesis of peptides containing isotopically labeled amino acids (1, 2).
It provides not only normal proteotypic peptides, but also modified
peptides that mimic posttranslational modification. However, themethod has several limitations originating from the use of thechemical synthesis. It requires individual peptide synthesis using

www.pnas.org/cgi/doi/10.1073/pnas.1603799113

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
contributed new reagents/analytic tools; R.N., Y. Shimizu, and M.U.-T. analyzed data;
and R.N., Y. Shimizu, and H.R.U. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1

R.N. and Y. Shimizu contributed equally to this work.

2

To whom correspondence may be addressed. Email: yshimizu@riken.jp or uedah-tky@umin.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1603799113/-/DCSupplemental.

PNAS | Published online May 31, 2016 | E3461–E3467

SYSTEMS BIOLOGY

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)

Results
Design of the MS-QBiC Workflow. To develop a simple strategy forthe multiplexed absolute quantification of protein using SRMbased targeted proteomics, we devised the MS-QBiC workflow(Fig. 1). This method takes advantage of the PURE system, areconstituted cell-free protein synthesis system (11) for internal
peptide synthesis without peptide degradation and without isotope scrambling or dilution (10). It is also noteworthy that the
PURE system is suitable for protein or peptide expression fromlinear DNA, which enables the direct addition of the PCRamplified gene into reaction mixtures. Thus, gene preparation,
peptide synthesis, and purification can be performed in a simple
and a high-throughput manner. The whole process can be carriedout in 1 d from one or two DNA primers per peptide.
After examination and validation of the workflow by several
liquid chromatography (LC)-MS analyses (Fig. S1 and SI Results),
the MS-QBiC peptide was designed as shown in Fig. S2. A FLAGtag was selected as a purification tag and the quantification tag
was obtained from the tryptic digest of bovine serum albumin(BSA), which was attached downstream of the FLAG tag and aspacer peptide sequence. The gene for the peptide was cloned into
the plasmid vector comprising a T7 promoter and the 5′-UTR
sequence for the E. coli translation system. The target peptide wasdesigned to be directly attached to the quantification tag by PCR
using the plasmid as a template. The MS-QBiC peptide can bequantified by comparing ion peak intensities of the stable isotopelabeled quantification tag with those of the chemically synthesized
quantification tag (Fig. S2, Lower Left) or the tryptic digest ofcommercially available BSA (Fig. S1D). The quantified MS-QBiC
peptide can be then used to quantify the target peptide by comparing the intensities of stable isotope-labeled target peptide in
MS-QBiC peptide with those of the nonlabeled target peptide in
the sample (Fig. S2, Lower Right).
The workflow was further examined for SRM analysis. Boththe quantification of the MS-QBiC peptide and the data collection
for the development of the SRM method for the target peptide(Fig. S3 A–C) were carried out by the LC-SRM analysis using
mixtures containing digested MS-QBiC peptide and the chemically synthesized quantification tag. The developed SRM method
was then applied to target peptide quantification in mouse liver.
E3462 | www.pnas.org/cgi/doi/10.1073/pnas.1603799113

Synthesis of MS-QBiC peptide with PURE System
DNA template
U Pu+
Q
FW primer
Q
PrPr+
RV primers

+

+

T

PCR

Pr U Pu Q

DNA

TTranslation
Purification

M

Addition
Quantification of MS-QBiC peptide
Light version ofQKquantification-tag
Pu
QT
MS-QBiC peptide M K*
K*
K*
Digestion
Trypsin
Q
K
Pu
T
Peptide
Q
M K*
K*
K*

+

LC-MS

Q
Q

Pu

K*

T

K*

Biological sample

Protein+

MS-QBiC peptide
Digestion
T
K
Peptide
T
K*

LC-MS

K*

K*

Addition
Quantification of target peptide

K

Retention timeQ

Signal intensityof MS

MS-QBiC peptide

Signal intensityof MS

because there is no need for culturing, harvesting, and disrupting
cells. Notably, cellular metabolism causes isotope scrambling anddilution, which is a problem where the homogeneity of isotopelabeled peptides is reduced due to conversion of labeled amino acids
into others, or vice versa (9). This problem can be overcome in a cellfree system by artificial adjustment of the system components (10).
Here we describe a workflow for multiplexed absolute quantification of proteins using SRM-based targeted proteomics. The
workflow, termed MS-based Quantification by isotope-labeled
Cell-free products (MS-QBiC), has several features that expand
advantages of the internal standard synthesis using a cell-free system. It is based on the use of the PURE system, a reconstituted
cell-free protein synthesis system (11). Because the PURE system
consists of purified factors and enzymes for E. coli translation
machinery, synthesized peptides are rarely challenged by proteasedegradation that usually occurs in cell-extract systems. Additionally, isotope scrambling or dilution is avoided without the need toadjust system components (10). The developed workflow was applied to the absolute quantification of core circadian clock proteinsin mouse livers across the circadian day. To obtain optimal peptides for the detection and quantification by the SRM-based targeted proteomics analysis, we synthesized 120 peptides for 20
circadian clock proteins. All of the peptides were successfully
synthesized from PCR-amplified genes by the PURE system. Dynamic changes of copy numbers for 16 proteins during the circadian
day were successfully quantified, demonstrating the potential of thismethod for the multiplexed targeted proteomics approaches.

Q
T
K*
K*
K*
Trypsin
R
K
K
R
R
R
R
K
R

M

Pu

T
T

K
K*

Retention time

Calculation of the absolute amount of target protein

Fig. 1. Development of the MS-QBiC workflow. Schematic description of
the MS-QBiC workflow. A purification tag, a quantification tag, and a tryptic
peptide of the target protein (target peptide) are sequentially arrayed as asingle peptide sequence (MS-QBiC peptide). The target peptide sequence is
attached by one- or two-step PCR. The MS-QBiC peptide is synthesized in the
PURE system in the presence of stable isotope-labeled Arg and Lys for isotopic
labeling both the quantification tag and the target peptide. Trypsin digestionof purified MS-QBiC peptide produces equal amounts of isotopically labeled
quantification tag and target peptide. The quantification tag is used tomeasure purified MS-QBiC peptide, and the target peptide is used as an internal standard for the target protein quantification.

The quantified MS-QBiC peptide was mixed with cellular protein
extracts from a mouse liver and subjected to tryptic digestion and
LC-SRM analysis. By comparing the ion peak intensities of thefragment peptides, the quantification of the target peptide could
be performed (Fig. S3D), indicating that the MS-QBiC workflow
can be applied to the SRM analysis.
Quantification of Core Circadian Clock Proteins. The workflow wasapplied to the quantification of core circadian clock proteins. The
mammalian circadian clock is driven through complex feedback
and feed-forward loops regulated by a variety of circadian clock
proteins and three clock-controlled elements (12–15) (Fig. 2A).
Although quantitative analyses of mRNA-level gene-expression of
circadian clock proteins have been performed previously (13, 16),
direct quantification of these proteins across the circadian day isessential for the accurate description and simulation of circadianclock networks. According to previous mRNA-level gene-expression
profiles (13, 16), 20 circadian clock proteins were examined and 120
tryptic peptides were selected as target peptides for quantification ofthese proteins (Fig. 2 A and B, Datasets S1 and S2, and SI Results).
Note that some peptides overlapped a common region of similar
genes for DEC1 (BHLHE40) and DEC2 (BHLHE41), CKIδand CKIe, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2),
RORα and RORβ, and DBP and TEF.
An experimental workflow used for quantification of circadianclock proteins is shown (Fig. 2C). Mice were entrained in a 12-h
light/12-h dark (LD) cycle for 2 wk. After transferring to constantdarkness, mice livers were collected at six time points with 4-hintervals at circadian times (CT0, CT4, CT8, CT12, CT16, andCT20). Livers from two mice were collected at each time point.
The 120 synthesized and purified MS-QBiC peptides were mixed
with protein extracts from the mice liver. The mixtures were
subjected to tryptic digestion and then prefractionated with super
Narumi et al.

DBP

BMAL1
CLOCK
CRY2

D-Box

CRY1
REV-ERBα

DEC1
REV-ERBβ

DEC2
RORα
PER1
RORβ

CKIδ

PER2

CKIε

PER3

RRE

D

RORγ

E
40

b3(+1) 16
y3(+1)y4(+1) 12
y5(+1)
y6(+1) 8

3
2

Light
Intensity (×1,000)

Amount of MS-QBiC peptide (picomoles)

C

Target protein

HLF

E/E'BoxTarget peptide(Detected peptide)
3 (2)BHLHE40 (DEC1)8 (0)BHLHE41 (DEC2)3 (1)BHLHE40/41 (DEC1/2)*
4 (3)BMAL1 (ARNTL)
3 (3)
CLOCK
8 (4)
CRY1
5 (2)
CRY2
3 (3)
CKIδ
4 (3)
CKIε
3 (3)
CKIδ/ε*
3 (2)
DBP
3 (2)
TEF
1 (1)
DBP / TEF*
3 (0)
HLF
6 (2)
NFIL3 (E4BP4)
5 (3)NR1D1 (REV-ERBα)
5 (3)NR1D2 (REV-ERBβ)1 (0)NR1D1/2 (REV-ERBα/β)*12 (3)PER1
10 (3)PER2
14 (3)
PER3
3 (0)
RORα
8 (0)
RORβ
1 (0)
RORα/β*2 (1)RORγ120 (47)
Total

PNAS PLUS

B
TEF

30

20
Heavy

10 20 30 40 50 60 70 80 90 100 110 120
SRM-ID

3
2
1

0

0
160
b3(+1)y3(+1) 120
y4(+1)y5(+1) 80
y6(+1)40

030
y4(+2)y7(+2)y3(+1) 20
y4(+1)y5(+1)
10

0

0

16
12
8

SRM-ID :Protein :Sample :Fraction # :

32.2
15
BMAL1
CT20
Fr-6

32.4

30.2 30.4
Retention time (min)
23
CRY1
CT4
Fr-14

y8(+1)y11(+1)y12(+1)y13(+1)

4

4

0
32.0
0

5

1

4

10

y4(+2)y7(+2)y3(+1)y4(+1)y5(+1)y8(+1)y11(+1)y12(+1)y13(+1)

Primers corresponding to
120 target peptides

Mice livers(sampled at 6 timepoints every 4 hwith two biological replicates)

PCR
DNA

AdditionProteins

Pure system

Digestion
120 of MS-QBiC peptides

PeptidesSCX Chromatography

Digestionwith LVTDLTK
120 of MS samples for
product quantification

18 fractions from each sample forquantification of clock proteins
Quantification of MS-QBiC peptides
and development of the SRM assays Quantification of clock proteins
by LC-SRM/MS
by LC-SRM/MS

F
1,000

r = 0.96
100

10

1
47.2

47.4

47
DBP
CT12
Fr-5

1

10

100

1,000

Concentration of replicate-1(attomoles per μg lysate)Fig. 2. Quantification of 20 circadian clock proteins in mice livers. (A) The transcriptional circuit of the mouse circadian clock. Clock-controlled elements (CCE;
rectangles), transcriptional factors (green or magenta ovals) with their effects (lines), and kinases (yellow ovals) are shown in the circuit. Green ovals represent
activators, and magenta ovals represent repressors. Gray lines indicate that the CCE is located on the promoter (or enhancer) regions of the correspondingtranscription factor genes. Dashed lines indicate that the putative CCE is encoded. (B) A list of target proteins to be quantified. The numbers indicate thenumber of prepared target peptides and, in parentheses, the number of detected target peptides in the LC-SRM analyses. An asterisk indicates that target
peptides are shared between two target proteins. (C) An experimental workflow for the absolute quantification of 20 circadian clock proteins. (D) Yields of
120 MS-QBiC peptides synthesized in the PURE system. The colors of each bar are same as those used in B. (E) Examples of SRM chromatograms corresponding
to target peptides derived from clock proteins (Upper) and their internal standards (Lower). (F) Comparison of the quantified values between two biologicalreplicates. Correlation coefficient (r) is indicated in the plot.

cation exchange (SCX) chromatography. The resultant 18 fractions
were individually subjected to LC-SRM analysis (Fig. S4 andDataset S2), and quantification of each peptide was performed.
Prefractionation by SCX was adopted in the workflow because it
was essential for the detection of proteins with low copy numbers(17, 18).
Quantification of the quantification tag showed that all of 120
MS-QBiC peptides were successfully synthesized from PCRamplified genes by the PURE system. The amounts of purified
peptide from the same volume of reaction mixtures (25 μL)
ranged between 1.4 and 33.3 pmol (Fig. 2D). Although the yields
were variable, implying dependency on the sequence of the target
peptide, they were sufficient for our SRM-based quantification.
These data demonstrate the versatility of our workflow for many
peptides regardless of their sequence.
Quantification of the target peptide (Fig. 2E) showed that 47
peptides (42 peptides were derived from a single protein and 5
peptides were in the common region of two proteins) could be
used for detection of circadian clock proteins in mice liver
samples (Fig. 2B and Dataset S2). The remaining 73 peptides
were classified into two classes: in one class, internal standards
were not detected probably because of unsuccessful separation
with SCX chromatography (black rows in Fig. S4C) and in theother class signal/noise ratios were not high enough for detection
Narumi et al.

of endogenous targets. The quantified values of 47 peptides at six
time points were compared between two biological replicates by
a double-logarithmic plot (Fig. 2F). The quantified values werehighly correlated between replicates, indicating the biologicalreproducibility of circadian clock protein dynamics as well as thetechnical reproducibility of the developed workflow. Fifteen proteins were detected with two or more internal standards whereas
one protein, RORγ, was detected with a single internal standard(Fig. 2A and Fig. S5A). Peptides for the common region of DEC1
(BHLHE40) and DEC2 (BHLHE41), CKIδ and CKIe, and DBP
and TEF were also successfully quantified, but four proteins[DEC2 (BHLHE41), HLF, RORα, and RORβ] were not detected
clearly in our analysis (Fig. S5 D and E and SI Results).
Time courses for the concentrations of 16 proteins (Fig. 3A)
were shown as averaged peptide concentrations derived from same
protein with errors between peptides. The differences in concentration between peptides (Fig. S5A and Dataset S3) were relatively
higher than those between biological replicates (Fig. S5B and
Dataset S3). Various factors, such as posttranslational modification, the presence of unknown protein isoforms, and inefficient or
missed cleavages by the proteolysis, in addition to measurement
error itself, may cause these differences, which are common to MSbased absolute quantification (20). Despite these problems, thedata showed similar values from peptides derived from same
PNAS | Published online May 31, 2016 | E3463

SYSTEMS BIOLOGY

E4BP4

Concentration of replicate-2(attomoles per μg lysate)

A15

BHLHE40 (DEC1)

10
5
0
CRY2

40

40

3

30

2

20

1

10

0

0

BMAL1 (ARNTL)

1.5
1
0.5
0

CKIδ

1.5

200

6

150

4

100

2

50

0

0

3

15

20

2

10

10

1

5

CLOCK

40
30

6
4

1

20

0
TEF

30

0.5
0
NFIL3 (E4BP4)

5
4

2

10
0

0
CKIε

10

0.6
0.4

2
10

2.5

0

0
DBP

50

0

30

0
PER1

15
10

PER2

1.5 20
1

15

0.5

4

0
8 12 16 20

0

DBP
TEF

7.5

15

5

10

2.5

5

0

0

0

NR1D2
(REV-ERBβ)

4

30

15

PER1

20
10

25

0

0

5

5

4

4

3

3

PER3

3

RORγ

NFIL3
(E4BP4)
NR1D1(REV-ERBα)

12
20

9

PER2

2
2

2

1

1

0
8 12 16 20

0
0

5
0
0

0

CRY2

75

10
5

5

CRY1

NR1D2 (REV-ERBβ)
10
20

2

0

20

CLOCK

NR1D1 (REV-ERBα)
100
30

50

0

20
15

10

0

BMAL1
(ARNTL)1

10
0.2

Delay between mRNAand protein peaks (h)-4 -2 0 2 4 6 8 10
BHLHE40(DEC1)

3

40

7.5

3

1

B

5
4

30

5

10

0
0

CRY1

40

20

20

10 250830

Copies per cell (×1,000)

4Relative expression of mRNA (AU)

A

4

1

4

0
8 12 16 20

6

10

PER3

3
0
0

4

0
8 12 16 20

RORγ

Circadian time (h)Fig. 3. A summary of the expression levels of circadian clock proteins and their corresponding mRNA. (A) Time course of the quantified proteins with theircorresponding mRNA. Expression levels of the protein are shown in the same color as used in Fig. 2B. Concentrations are expressed as copies per cell (left axis).
Copy numbers were calculated by assuming that the total amount of protein per cell is 0.5 ng (19). Bars on the graph indicate the SD of quantified values
using three or more internal standard peptides. Open circles indicate maximum and minimum quantified values using two or more peptides. Relative expressions of mRNA, which are the replots of Fig. S7, are shown in gray (right axis). (B) Phase delay of the variations between circadian clock proteins and theircorresponding mRNA. The phases of each variation were calculated by 24-h cosine curve fitting to time-course data of each internal standard peptide andmRNA expression (Dataset S3). The bars indicate the SD of calculated phases of proteins using three or more internal standard peptides. Open circles indicate
the maximum and minimum phase delay calculated from the phases of proteins using two or more internal standard peptides.

protein, which suggests that the observed values reflect absolute copy numbers in cells. Six proteins [DEC1 (BHLHE40),
CRY2, DBP, CKIe, TEF, and NFIL3 (E4BP4)] were quantified
with only two target peptides, and two proteins (CKIδ and RORγ)
were quantified with only a single peptide. Although target peptides
were selected from as many as possible according to our criteria(Dataset S2 and SI Results), the number of detected peptides depends on both a trypsin cleavage site in the amino acid sequence ofthe protein and the signal/noise ratio of each target peptide in the
SRM analysis. The use of alternative proteases may increase thenumber of usable peptides for more reliable quantification. However, absolute quantification depends on the efficiency and specificity of the selected proteases, which is highest with the use of atandem LysC (Lysyl Endopeptidase)/trypsin cleavage (Fig. S6).
Thus, this method is currently the best way for MS-based protein
quantification (21) and our studies (SI Results). To show errors
between peptides for the six proteins, maximum and minimum
quantified values from each measurement were additionally plottedin the graph (Fig. 3A). It should be noted that peptide-1 and peptide3 from CKIδ and peptide-1 from CKIe (Fig. S5A) were excluded
from protein quantification. The concentrations of these peptides
were consistently lower compared with the maximum peptide conE3464 | www.pnas.org/cgi/doi/10.1073/pnas.1603799113

centration for each protein, i.e., the coefficient of variation between
peptides was more than 50% at all time points (Dataset S3). Additional experiments showed that these peptides were phosphorylated (Fig. S5C), which indicated that the peptides were not suitablefor quantification. Phosphorylation of peptide-1 from CKIe was also
reported previously in phosphoproteomics analyses including that ofmice liver (22, 23).
The relative abundance of the mRNA corresponding to eachprotein, obtained by quantitative PCR (qPCR) using livers fromsame samples (Fig. S7), is additionally shown in the time course
in Fig. 3A, which shows that copy number of the 14 proteins, aswell as the abundance of mRNA, fluctuated across the circadianday except for CKIδ and CKIe (Dataset S3). The phase differences
between the proteins and mRNA, calculated by a 24-h cosine curve
fitting to the time-course data, varied with each protein (Fig. 3B).
There was relatively little phase delay for some proteins [DEC1(BHLHE40), DBP, TEF, E4BP4 (NFIL3), REV-ERBα (NR1D1),
REV-ERBβ (NR1D2), PER2, and RORγ], indicating that these
protein levels are governed mainly by mRNA abundance. Phase
differences were relatively large for BMAL1, CLOCK, CRY1,
CRY2, PER1, and PER3, suggesting that these expression
levels are regulated through complicated processes, including
Narumi et al.

PNAS PLUS

A
Normalized
expression level0

BT Detection in Wild-Type and Clock Mutant Mice. To validate themolecular timetable based on the protein-level expression profiles, mice livers were further collected at CT4 and CT16 withtwo biological replicates. The quantified values of 36 peptides
from livers were applied to BT estimation using the molecular
timetable method (Fig. S8A). The livers at CT4 were estimatedto be 3.7 and 3.3 h and those at CT16 to be 17 and 16.5 h, respectively, which were close to the actual circadian phase.
The circadian clock proteins in clock mutant mice were additionally quantified using the MS-QBiC method. Livers fromthree knockout (KO) or double knockout (DKO) mice (Bmal1
KO, Per1/2 DKO, and Cry1/2 DKO), which were previously shownto have deficiency in the circadian rhythmicity (34–36), were colNarumi et al.

24

0

0 4

B

2.0

8 12 16 20 0 4 8 12 16 20 0 4
Circadian time (h)8 12 16 20

CT0

CT4

CT8

CT12

CT16

CT20

12

24 0
12
24 0
Molecular peak time (h)

12

0
-2.0
2.0
0
-2.0
0

4

8

12

16

20

24

24

Fig. 4. Molecular timetable based on the protein expression of core clock
genes. (A) Time course of normalized expression levels of peptides. The
colors—in ascending order from green to orange (CT2-10, Left), from orange
to purple (CT10-16, Center), and from purple to green (CT16-2, Right)—were
assigned to represent the molecular peak time, which corresponds to themolecular timetable of each peptide. The color code is represented above
the diagrams. (B) Expression profiles of peptides at each circadian time
point. Normalized expression levels of peptides at each circadian time point
are plotted against their molecular peak time. Best-fitted cosine curves(black) and those based on the actual circadian time point (gray) are shown.
The peak time of the best-fitted curves indicates the estimated BT.

lected at CT4 and CT16, respectively, with three biological replicates. Quantified values of 16 proteins based on the quantificationof 47 peptides showed a markedly different behavior comparedwith those of the wild-type mice (Fig. S9 and SI Results). The large
differences of the expression level of DEC1 (BHLHE40), DBP,
TEF, E4BP4 (NFIL3), PER1, PER2, and PER3 between CT4 andCT16 in wild-type mice were diminished in the mutant mice, consistent with the deficiency in circadian rhythmicity.
The quantified values of 36 peptides for 14 circadian clock proteins were further applied to BT estimation of clock mutant miceusing the molecular timetable of the wild-type mice as a reference.
The plots of the normalized quantity of each peptide against themolecular timetable were similar between CT4 and CT16 in eachsample and indicated the same estimated time, which reflects a
deficiency in circadian rhythmicity (Figs. S8 B–D and S10). Considering that the variation of the expression of circadian clock
proteins was diminished in the mutant mice (Fig. S9), similar profiles between CT4 and CT16 may suggest that the expression levelof these proteins is constant across the circadian day (Fig. S8E).
Discussion
As the technologies for MS-based proteomics advance, quantification of entire cellular protein networks has become feasible(37–39). However, it is still a difficult task to quantitatively investigate dynamics of the networks using global proteomics approaches by comparatively analyzing multiple cellular extracts
under a variety of environments or perturbations. The quantification becomes far more difficult for low abundant proteins, suchas transcription factors and kinases. Two recent global proteomics
studies have revealed various proteins with circadian rhythmicity
PNAS | Published online May 31, 2016 | E3465

SYSTEMS BIOLOGY

peptides for 14 circadian clock proteins except for CKIδ and CKIe
showed circadian rhythmicity (Dataset S3). The time points for
maximum or minimum quantity varied among the proteins. Our
previous studies demonstrated that such data sets can provide amolecular timetable that acts as a reference for body-time (BT)detection from only one or two time points (31–33). BT detection
has been performed for mRNA-level gene-expression profiles ofmouse cells and the mouse and human blood metabolite profiles.
In the present study, the method was further applied for proteinlevel expression profiles using the quantified values of each peptide to show whether they indicate the endogenous state of thecircadian clock.
To construct a molecular timetable, the quantified values of 36
peptides for 14 circadian clock proteins at each time point werenormalized according to the previous study. Peak time of each
peptide was calculated by the time-course data of the normalized
values (Fig. 4A and Dataset S3), which indicated the molecular
timetable of each peptide. The calculated peak time corresponded
well if the peptide originated from a same protein, suggesting thatthe molecular timetable of each peptide represented that of thecorresponding protein. The normalized quantity of each peptide
at each time point was plotted against the calculated molecular
timetable, and then 24-h period cosine curves were fitted to theseplots, where the peak time of the best-fitted curve indicated theestimated BT (Fig. 4B). The estimated BT matched well with theactual circadian phase with a mean error of 0.67 ± 0.29 h, demonstrating that a molecular timetable can be constructed for theprotein-level expression profiles.

Molecular peak time (h)
8
12
16
20

2.0

0

Construction of a Molecular Timetable. The quantified values of

4

-2.0

Normalized expression level

posttranscriptional regulation, translation control, posttranslational
modification, and degradation (24–28). These observations demonstrated that the direct quantification of proteins is essential forthe description of protein-level dynamics of biological systems.
Circadian variation of PER1, PER2, CRY1, and CRY2 wasinvestigated previously by Western blot analyses (24). The time
points for the maximum and minimum quantity of these proteinsin that report corresponded well with this study. Both studies
showed that the amplitudes of PER1 and PER2 were larger
compared with CRY1 and CRY2. In contrast, significantly highvariations of BMAL1 and CLOCK expression level observed in
the Western blot analysis were not observed in our results. Although we have no explanation for this inconsistency, our observations suggested that the amplitude of the variation of these
proteins was relatively low. It is noteworthy that the copy numbers and variations of these proteins matched each other, whichreflects that they function through the formation of a heterodimer(29). It is interesting that these copy numbers also matched CRY1
at CT0, the peak time for CRY1. A recent report suggested thatthe three proteins form a ternary complex to inhibit the E/E′-Boxpromoters at this time point (30). Finally, the mRNA-level expression of all of the proteins, except for CKIδ and CKIe, is known
to fluctuate across the circadian day (13), which is consistent with
our analysis.

by analyzing time-series samples from large numbers of mice livers(40, 41). However, core circadian clock proteins, which consist
mainly of transcription factors and kinases, were not detected.
Targeted proteomics has grown in recent years as a strategy tocompensate for the weaknesses of global proteomics approaches.
It monitors only signal intensities with good signal/noise ratios
derived from target peptides under optimal conditions. It cananalyze low-abundance proteins more effectively, but the number of proteins that can be analyzed is reduced compared withthe global proteomics approaches. Therefore, targeted proteomics
approaches are suitable for analyzing the dynamics of specified
biological systems comprising low-abundance proteins.
In this study, we performed systems-level analysis of dynamics
of core circadian clock proteins by using SRM-based targeted
proteomics. Sixteen proteins from 20 circadian clock proteins
were successfully quantified using time-series samples from miceliver, and 14 proteins had circadian variation (Fig. 3 and Dataset
S3). The developed method can be applied to other organs or
tissues such as the brain that function as a central circadian clock.
It will be interesting to quantify the core circadian clock proteinsin the brain to compare the abundance between the central andperipheral organs using the developed method for future studies.
Four proteins [DEC2 (BHLHE41), HLF, RORα, and RORβ]
were not clearly quantified, although estimation of two proteins(HLF and RORα) was examined from peak intensities with low
signal/noise ratios (Fig. S5E). The quantification result from thecommon region of DEC1 (BHLHE40) and DEC2 (BHLHE41)
suggests that the expression level of DEC2 is extremely low (Fig.
S5D), and this may have resulted in unsuccessful detection of thesignal peaks for DEC2. The detection limit of DEC2 (BHLHE41)
and RORβ, estimated as threefold of the SD of noise peaks, isabout 1,000 and 100 copies per cell, respectively, when the mostsensitive standard peptides are used (Dataset S3). Thus, further
development of the SRM-based detection as well as the sensitivityof MS itself is necessary for the detection of proteins with extremely low expression levels (<1,000 copies per cell).
The MS-QBiC workflow allows for multiplexed preparation of
isotopically labeled peptides for use as internal standards. Multiple peptides can be prepared in parallel without any cDNA
cloning or gene synthesis step by using the MS-QBiC workflow.
The use of the reconstituted cell-free protein synthesis system
avoids isotope scrambling and dilution. Moreover, multiplexed
peptide preparation can increase the sensitivity of SRM analysis
by developing an optimal method that depends on the physicalproperties of individual peptides. Although the yield of synthesizedpeptides differs from one peptide to another, sufficient amounts ofthe peptide were successfully prepared regardless of their sequences, suggesting versatility in the developed workflow. Thisversatility also suggests that the developed method does not rely on
the characteristics of proteins and can be applied to proteins thatare difficult to prepare recombinantly, such as membrane-boundproteins and other DNA-binding proteins. Currently, SRM analyses highly depends on the performance of the MS, and thus detection of several hundreds of proteins is estimated to be the upper
limit according to previous reports (39, 42). However, this limit canbe increased by improvement of MS performance. Therefore, highthroughput synthesis of internal peptides using the PURE system
can be adapted to such improvements in the future. Presently,
the developed method is not able to be applied to the preparationof modified peptides that mimic posttranslational modification.
However, integration of the developed method with technologies
based on genetic code engineering (43), including site-specific incorporation of phosphoserine residues into the proteins or peptides(44), has the potential to overcome this difficulty, and we are now
exploring this possibility.
The core circadian clock proteins consist mainly of transcription
factors, which bind to DNA and function as activators or repressors for transcription. Interactions between DNA and BMAL1,
E3466 | www.pnas.org/cgi/doi/10.1073/pnas.1603799113

CLOCK, CRY1, CRY2, PER1, and PER2, which play crucial
roles in the formation of circadian rhythmicity, have been comprehensively analyzed using ChIP-seq analyses (45) demonstratingthat binding of these proteins to their binding sites shows circadianrhythmicity. The peak time of binding matches well with that ofthe protein concentrations quantified in this study, except for
CRY2, suggesting that the binding is principally regulated by theexpression levels of each protein. However, the binding may beregulated not only by the quantity of protein, but also by otherqualities (e.g., posttranslational modification status): The amplitude of BMAL1, CLOCK, CRY1, and CRY2 is quite low, and aconsiderable amount of the proteins may remain in cells at a
circadian phase when their binding to DNA is low and the peaktime of CRY2 concentration is different from that of its DNA
binding. These observations suggest that qualitative changes ofthese proteins are also important for the DNA-binding activities.
Calculating from our results and the ChIP-seq data, the numbersof proteins at each binding site are 5.6 BMAL1, 14.6 CLOCK, 7.2
PER1, 3.2 PER2, 3.4 CRY1, and 4.8 CRY2 at a single binding
site, respectively. The values suggest that 3–15 proteins are necessary at a binding site to regulate gene expression for the formation of the circadian clock.
The application of the molecular timetable methods to thequantified peptides from 14 circadian clock proteins demonstrates
that single-time-point data can predict body time, indicating that
expression levels of each circadian protein are promising markers
for future application of body time detection in human samples.
The accuracy of detection is comparable to the mRNA-level
expression-based timetable method rather than the metabolitebased method, which requires two-time-point data for detection(31–33). Furthermore, the constructed timetable, which is basedon the absolute quantification of cellular proteins, can be universal
compared with the previous two methods based on the relative
quantification, which may enable the single-time-point assay indifferent places. Resemblance of peptide expression profiles between CT4 and CT16 in three mutant mice suggests that the expression level of the proteins is fixed at the level of the designatedtime point. In Bmal1 KO mice, the absence of CLOCK/BMAL1
heterodimer represses the E/E′-Box promoters and then activates
the RRE promoters. In Per1/2 DKO and Cry1/2 DKO mice, theabsence of PER1 and PER2 or CRY1 and CRY2 activates the
E/E′-Box promoters and in turn activates the D-Box promoterswhereas the RRE promoters are suppressed to some extent(Figs. S8E and S9). Such fixation of the activities of each promoter may result in the stalled circadian gene-expression dynamics at the specific circadian phase.
Materials and Methods
Animals. All mice were carefully kept and handled according to the RIKEN
Regulations for Animal Experiments. See SI Materials and Methods for details.
Sample Preparation. Chemically synthesized peptides, MS-QBiC peptides, cell/
nuclear lysates from HEK293T cells, and mouse liver samples were prepared
for this study. They were subjected to enzymatic digestion and prefractionation before MS analyses. See SI Materials and Methods for details.
MS Analyses. Two types of mass spectrometers—an Orbitrap mass spectrometer and a triple quadruple mass spectrometer—were used in this study. See SI
Materials and Methods for details.
RNA Expression Analysis of Circadian Clock Genes by qPCR. The mRNA-level
expression profiles of circadian clock genes were measured by qPCR. See SI
Materials and Methods for details.
BT Detection by Molecular Timetable Methods. Estimation of body time byusing expression profiles of circadian clock proteins was performed according
to the molecular timetable methods as reported previously (31). See SI
Materials and Methods for details.

Narumi et al.

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Narumi et al.

PNAS | Published online May 31, 2016 | E3467

PNAS PLUS(A) 26710014 (to Y. Shimizu); Grant-in-Aid for Young Scientists (B) 25830146(to K.L.O.); Grant-in-Aid for Challenging Exploratory Research 26640134(to Y. Shimizu); Grant-in-Aid for Exploratory Research 15K13750 (to K.M.);
Grants-in-Aid for Japan Society for the Promotion of Science (JSPS) Fellows 25-1565 (to G.N.K.) and 25-5989 (to Y. Shinohara) from MEXT/JSPS;
the strategic programs for R&D (President’s Discretionary Fund) of RIKEN(H.R.U. and Y. Shimizu); an intramural Grant-in-Aid from the RIKEN Quantitative Biology Center (to H.R.U. and Y. Shimizu); the RIKEN Special Postdoctoral Research Program (G.N.K.); and a grant from Core Research
for Evolutional Science and Technology of Japan Science and Technology
Agency (to H.R.U.).

SYSTEMS BIOLOGYACKNOWLEDGMENTS. We thank the laboratory members at RIKEN QBiC,
in particular, J. Hara and G. A. Sunagawa for their kind help in preparing
knockout mice; A. Millius for his critical reading and editing of themanuscript; E. A. Susaki for his kind support in setting up the MS room;
and T. Kawashima for his early efforts on the molecular timetable. This
work was supported by the Program for Innovative Cell Biology by Innovative Technology and the Brain Mapping by Integrated Neurotechnologies
for Disease Studies from the Ministry of Education, Culture, Sports, Scienceand Technology (MEXT) of Japan (H.R.U.); Grant-in-Aid for Scientific
Research (S) 25221004 (to H.R.U.); Grant-in-Aid for Scientific Research on
Innovative Areas 23115006 (to H.R.U.); Grant-in-Aid for Young Scientists

Supporting Information
Narumi et al. 10.1073/pnas.1603799113
SI Results

Selection of the Target Peptides Used for the MS-QBiC Workflow.

Examination and Validation of the MS-QBiC Workflow. The MS-

Target peptides were selected from the tryptic digests of cellular protein extracts or nuclear protein extracts from HEK293T
cells overexpressing each protein and detected by LC-MS/MS
analyses (Dataset S1). When not enough peptides were identified by these analyses, peptides suitable for the use in the MSQBiC workflow were selected from the sequence information of
the original proteins (Dataset S2). The selection was according to
the following rules. The length of the target peptides was selected
to not exceed 17 residues to prepare the DNA template by PCR
within two steps. Each peptide contained only a lysine or arginine
residue at its C terminus. Peptides containing methionine in thesequence or those containing glutamine at their N terminus were
avoided because they produce unexpected products containing oxidized methionine or pyroglutamic acid. Peptides containing cysteine were permitted because we developed a protocol to convert thecysteine to its alkylated form almost completely by reduction andalkylation (SI Materials and Methods).

QBiC workflow was examined by synthesizing several test peptides using the PURE system, a reconstituted cell-free protein
synthesis system (11). The first trial was to synthesize a peptide(cell-free product-1) (Fig. S1A) including a purification tag and atarget peptide for purification and detection by LC-MS. A FLAGtag was used for purification, and a peptide from the circadianprotein CLOCK with a sequence of DQFNVLIK was selected as
the target peptide. LC-MS analysis of the purified products
showed that all of the products contained an intact purification tag
and the target peptide (Fig. S1B). Peptides lacking methionine
and containing oxidized methionine were also observed, which
does not prevent the construction of the MS-QBiC workflow.
However, the tryptic digestion efficiency of cell-free product-1
was extremely low presumably because of multiple aspartic acidresidues in the FLAG-tag (Fig. S1C). Therefore, a spacer peptide
sequence was introduced downstream of the FLAG tag in cellfree product-2 (Fig. S1A). Measurement of digestion efficiency
using LC-MS showed that the introduction of a spacer containing
more than four leucines with a lysine residue was sufficient forcomplete digestion (Fig. S1C). From there on, we used a spacer
with the sequence LLLLK.
We then introduced a quantification-tag sequence between thespacer and the target peptide. A peptide with a sequence of
LVTDLYK was selected as the quantification tag from tryptic
digests of BSA. This peptide was selected because the signal
intensity was relatively high and undigested fragments were not
identified in LC-MS analysis. In addition, the peptide did notcontain Gln at its N terminus nor Met or Cys in the sequence,
which can be nonenzymatically converted to pyroglutamic acid,and oxidized Met and Cys, respectively.
The resultant peptide (cell-free product-3) (Fig. S1A) was synthesized in the PURE system containing stable isotope-labeled
lysine, and then the products were subjected to tryptic digestion.
The quantification of the quantification tag was performed byusing both the chemically synthesized LVTDLYK peptide andtryptic digests of commercially available BSA. The measured
values were almost identical between experiments, indicating thatboth materials can be used for quantification of cell-free products(Fig. S1D). Afterward, both the chemically synthesized LVTDLYK
peptide and the target peptide (DQFNVLIK) were mixed with thedigested cell-free product-3, and resultant mixtures were subjectedto the LC-MS analysis. Quantification of both quantification tag
and target peptide by using chemically synthesized peptides asreferences showed that the amounts of these peptides were stoichiometrically equivalent, indicating that the quantified value ofthe quantification tag can be applied to the quantification of thetarget protein by using the target peptide as an internal standard(Fig. S1E). Other target peptides were also examined to demonstrate versatility in the developed workflow. The sequences of thetarget peptides were ILYISNQVASIFHCK (cell-free product-4)(Fig. S1A), containing cysteine that requires the reduction and alkylation for the MS analysis, and EAGVEVVTENSHTLYDLDR(cell-free product-5) (Fig. S1A), a relatively long peptide. These
peptides are from the circadian clock proteins PER2 and CRY1,
respectively. The results of LC-MS analyses demonstrated that themolar ratios between the quantification tag and the target peptide
were nearly 1:1, demonstrating versatility in the developed workflow (Fig. S1E).
Narumi et al. www.pnas.org/cgi/content/short/1603799113

Quantification of Peptides for Common Region of Two Proteins andPeptides That Were Not Clearly Detected. Peptides for the common

region of DEC1 (BHLHE40) and DEC2 (BHLHE41), CKIδ andCKIe, and DBP and TEF were successfully quantified (Fig. S5D).
The time course of the peptides shared by DEC1 (BHLHE40) andDEC2 (BHLHE41) resembled closely that of DEC1 (BHLHE40).
Quantification of DEC2 (BHLHE41) with peptides unique to
DEC2 was unsuccessful. These observations suggest that the expression level of DEC2 (BHLHE41) was much lower comparedwith DEC1 (BHLHE40). The amounts of peptides common for
CKIδ and CKIe were closer to CKIδ, consistent with the expression level of CKIδ that was 20-fold higher than that of CKIe. The
time course for the peptide common to DBP and TEF was a sumof protein quantities for DBP and TEF, which is reasonable giventhat we detected unique peptides from both DBP and TEF.
HLF, RORα, and RORβ could not be quantified similarly to
DEC2 (BHLHE41), suggesting a low expression level. However,signals that seemed to originate from HLF and RORα wereobserved, although signal/noise ratios were too close for quantification. Given this noisy signal, we estimated the copy numbersfor HLF and RORα as 3,400 and 1,280 copies/cell, respectively,
without circadian variation (Fig. S5E). In contrast, we could notdetect any peak corresponding to RORβ, suggesting a low expression level.
Protein Expression Phenotype in Clock Mutant Mice. The expressionsof CLOCK, DBP, TEF, REV-ERBα (NR1D1), REV-ERBβ(NR1D2), PER1, PER2, and PER3 were suppressed in Bmal1
KO mice (Fig. S9). CLOCK may have been destabilized by theabsence of BMAL1, both of which form a heterodimer to play arole in activation of E/E′-Box promoters (Fig. 2A). Suppressed
expression of DBP and TEF (16), the transcription of which isregulated by the E/E′-box, may have been caused by the absenceof the CLOCK/BMAL1 heterodimer. Suppressed expression of
REV-ERBα (NR1D1), REV-ERBβ (NR1D2), PER1, PER2,
and PER3, the transcription of which is regulated by either the
E/E′-box or D-box (16), may have been caused by the absence of
the CLOCK/BMAL1 or decreased expression of DBP/TEF/HLF.
Suppression of the E/E′-Box promoters in BMAL1 KO mice may
have also resulted in the continuous activation of the RRE promoters by the suppression of their repressors, REV-ERBα (NR1D1)
and REV-ERBβ (NR1D2). By this activation, the expression of
E4BP4 (NFIL3), CRY1, DEC1 (BHLHE40), and RORγ, which are
1 of 14

regulated by the RRE promoters (16, 46), may have been activatedcompared with the wild-type mice, which may have resulted in thedisappearance of the circadian rhythmicity.
The expression levels of each protein resembled each other in
Per1/2 KO and Cry1/2 KO mice, although there were subtle
differences between strains (Fig. S9). The expression of DEC1
(BHLHE40), DBP, TEF, REV-ERBα (NR1D1), REV-ERBβ(NR1D2), and RORγ was elevated in both KO mice, suggestingthat the E/E′-Box promoters were activated by the absence of
their repressors, PER1 and PER2 or CRY1 and CRY2. The
increased level of DBP and TEF may have activated in turn the
D-Box promoters, which may have resulted in the increased
expression level of PER3. In contrast, the effect on the RRE
promoters was complicated because levels of both repressors,
REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), and an activator, RORγ, were increased. Considering that mild repressionof BMAL1, CLOCK, and E4BP4 (NFIL3) was observed, theRRE promoters may have been repressed to some extent.
Thus, the quantified values of these proteins in diverse individuals can be beneficial for further study of those networks bysystems biological approaches using modeling and simulation. We
note that some peptides corresponding to PER1 and PER2 were
quantified to some extent even in Per1/2 KO mice. They may haveresulted from the method of gene deletion, in which a large part ofthe promoter and exon regions still remain in this strain (35, 47).

M2 Magnetic Beads (Sigma Aldrich) according to the manufacturer’s instruction. The peptides were eluted with 20 μL 0.2% trifluoroacetic acid (TFA) solution, and then they were diluted six
times with phase-transfer surfactant (PTS) buffer (12 mM sodium
deoxycholate, 12 mM sodium N-lauroylsarcosinate, and 50 mM
NH4HCO3) and stored at −80 °C until used. Peptides that were notsubjected to enzymatic digestion were diluted twice with water andstored. For quantification of the peptide using the quantification
tag, 5 μL of the diluted solution was mixed with 1 pmol of
LVTDLYK peptide with three technical replicates.

Preparation of Chemically Synthesized Peptides. Four peptides (DQFNVLIK, LVTDLYK, ILYISNQVASIFHCK, andEAGVEVVTENSHTLYDLDR) and 10 phosphopeptides shownin Fig. S5C were synthesized with a peptide synthesizer Syro
Wave (Biotage) using Fmoc solid-phase chemistry. Concentrations of the synthesized peptide were measured by an aminogroup determination method (48).

Preparation of Cell/Nuclear Lysates from HEK293T Cells. All of thecircadian clock genes used in this study [Dec1 (Bhlhe40), Dec2
(Bhlhe41), Bmal1 (Arntl), Clock, Cry1, Cry2, CKIδ, CKI«, Dbp,
Tef, Hlf, E4bp4 (Nfil3), Rev-Erbα (Nr1d1), Rev-Erbβ (Nr1d2),
Per1, Per2, Per3, Rorα, and Rorγ] except for Rorβ (isoform 1)
were subcloned into the pMU2 vector (50) and expressed under
the CMV promoter as a FLAG-tag fusion form in their N terminus.
Rorβ was subcloned into the pCMVTnT vector (Promega) and
expressed under the CMV promoter. The genes were derived froma mammalian gene collection library [Dec1 (Bhlhe40), CKIδ, andRorα] and an NIH 3T3 cDNA library [CKI«, Dbp, Tef, Hlf, E4bp4(Nfil3), Rev-Erbα (Nr1d1), Rev-Erbβ (Nr1d2), Rorγ, and Rorβ]. A
cDNA collection for Bmal1 (Arntl), Clock, Cry1, Cry2, Per1, Per2,
and Per3 is a kind gift from Hajime Tei (Kanazawa University,
Kanazawa, Japan) and cDNA of Dec2 (Bhlhe41) is a kind gift from
Ken-ichi Honma and Sato Honma (Hokkaido University, Sapporo,
Japan). For the expression of the genes, HEK293T cells weregrown in DMEM (Invitrogen) supplemented with 10% (vol/vol)
FBS (JRH Biosciences) and antibiotics (25 U/mL penicillin and 25
mg/mL streptomycin) (Invitrogen). At 24 h before transfection,
cells were plated at 106 cells per well in 10-cm dishes. Transfection
was performed with FuGene6 (Promega) according to the manufacturer’s instruction. A total of 2 μg of each expression vector or a
mixture of 1 μg BMAL1 vector and 1 μg CLOCK vector wastransfected to the HEK293T cells on 10-cm dishes. After 48 h, cells
were collected by 0.25% trypsin–EDTA (Life Technologies)
treatment. The cell pellets were lysed in 1 mL PTS buffer followedby extensive sonication. The cell lysates were then flash-frozen inliquid nitrogen and stored at −80 °C. For the detection of TEF,
BMAL1, CLOCK, REV-ERBα (NR1D1), REV-ERBβ (NR1D2),
and RORγ, we also performed nuclear isolation from the cellpellet. The cell pellet was suspended with 1 mL of the following fivebuffers (and at each time collected by centrifugation at 1,500 × g for
5 min): Buffer #1—DPBS (Life Technologies); #2 and #3—NB(10 mM Tris·HCl, pH 7.0, 10 mM NaCl, 3 mM MgCl2, and 30 mM
sucrose) supplemented with 0.5% (vol/vol) Nonidet P-40; #4 and#5—NB + 10 mM CaCl2. The resultant pellet, mainly consisting of
nuclei, was lysed in 1 mL PTS buffer followed by extensive sonication. The cell lysates were then flash-frozen in liquid nitrogen andstored at −80 °C.

Preparation of MS-QBiC Peptides. A DNA sequence (5′-ATGGACTACAAGGACGACGACGACAAGCTGCTGCTGCTGAAGCTGGTTACTGACCTGACTAAG-3′) that encodes a FLAG tag, spacer peptide, and a quantification tag(MDYKDDDDKLLLLKLVTDLTK) was cloned into a pURE1
vector (BioComber) containing a T7 promoter sequence and theribosome-binding site (Shine-Dalgarno sequence). Using theresultant plasmid as a template, templates for MS-QBiC peptide
synthesis were amplified by PCR with a T7 promoter primer(5′-GGGCCTAATACGACTCACTATAG-3′) as a forward primer
and appropriate reverse primers listed in Dataset S2. PCR mixtures(2.5 μL) were directly added to yield 25 μL PURE system reaction
mixtures (49) containing 13C6 15N4 L-Arginine and 13C6 L-Lysine(Thermo Scientific) as a substitute for nonlabeled L-Arginine and
L-Lysine, respectively. The mixtures were incubated at 37 °C for
30 min, and then synthesized peptides were purified using anti-FLAG

Enzymatic Digestion. Enzymatic digestion of MS-QBiC peptides,
mouse liver samples, and cell/nuclear lysates from HEK293T cells
was performed basically according to a PTS protocol as reported
earlier (51). Before the digestion, blocking treatment of test tubes
was performed for MS-QBiC peptides, in comparatively lower
peptide concentration, by reducing nonspecific adsorption of
peptides. Enolase from Baker’s yeast (Sigma Aldrich) was subjected to the enzymatic digestion using Lysyl Endopeptidase(LysC, Wako Pure Chemical Industries) as described below.
Digests (0.2 μg/μL) in a PTS buffer were added to new tubes andwere incubated at 4 °C overnight, followed by discarding of thecontents. A sample in a PTS buffer was reduced with 10 mM TCEP
at 37 °C for 1 h and then alkylated with 15 mM iodoacetamide at
37 °C for 30 min in case it contained cysteine residues. The resultant
sample was diluted five times with 50 mM NH4HCO3 solution.
Predigestion was performed by the addition of LysC at 2 ng/μL

SI Materials and Methods
Animals. All mice were carefully kept and handled according to the
RIKEN Regulations for Animal Experiments. Eight- to 10-wk-old
male mice of wild-type (C57BL/6N, Japan SLC) and mutant wereentrained under a 12 h–12 h light–dark condition (400 lx) for
2 wk. After transferring to constant darkness, wild-type mice
were killed every 4 h over 1 d (i.e., CT0, -4, -8, -12, -16, and -20)
for proteomic analyses or every 4 h over 2 d (i.e., CT0, -4, -8, -12,
-16, -20, -24, -28, -32, -36, -40, and -44) for qPCR analyses. All
mutant mice were killed in 4 and 16 h (i.e., CT4 and -16). Livers
were excised and then quickly frozen in liquid nitrogen followed
by storage at −80 °C. The mutant mice, all of which displayed acircadian arrhythmic behavior in constant darkness, were Bmal1
knockout mice (Bmal1 KO), Per1/Per2 double knockout mice
(Per1/2 DKO), and Cry1/Cry2 double knockout mice (Cry1/2
DKO), which were originally generated by Shimba et al. (34),
Bae et al. (35), and van der Horst et al. (36), respectively.

Narumi et al. www.pnas.org/cgi/content/short/1603799113

2 of 14(MS-QBiC peptides), 1:50 (wt/wt) (mouse liver samples), or
1:100 (wt/wt) (cell/nuclear lysates from HEK293T), which were incubated at 37 °C for 8 h. Subsequently, digestion was performed by
the addition of same amounts of trypsin (Roche) as of LysC, whichwere incubated at 37 °C for 16 h. After the digestion, an equalvolume of ethyl acetate was added to the sample, which was acidified with 0.5% TFA to transfer the detergents into an organic
phase. After the sample was centrifuged at 10,000 × g for 10 min atroom temperature, an aqueous phase containing peptides was collected and dried with SpeedVac (Thermo Scientific). The sample
was dissolved in a solution containing 2% acetonitrile and 0.1%
TFA, and then it was desalted by using self-prepared stage tips (52)(MS-QBiC peptides and cell/nuclear lysates from HEK293T cells)or 1 cc Oasis HLB cartridge (10 mg sorbent; Waters) (mouse
liver samples), which was dried again with SpeedVac and storedat −80 °C until use.
Prefractionation of Mouse Liver Samples with SCX Chromatography.

Digested mouse liver samples were prefractionated into 18
fractions by SCX chromatography as follows: The dried peptide
mixtures were dissolved in 500 μL of a SCX-A (25% acetonitrile
and 10 mM H3PO4, pH 2.7). A total of 200 μL of the solution
was applied to a PolySULFOETHYL A column (100 × 2.1 mm,
5-μm particles with a 300-Å pore diameter; PolyLC) using theElite LaChrom HPLC system (Hitachi). Using two mobile phases, SCX-A and SCX-B (1 M KCl, 25% acetonitrile, and 10 mM
H3PO4, pH 2.7), peptides were eluted with a gradient (0% B for
3 min, 0–7% B in 25 min, 7–22% B in 12 min, 22–65% B in
7 min, 40–100% B in 4 min, and 100% B for 5 min, 200 μL/min).
The elution was collected every 1 min up to 60 min, and theresulting 60 fractions were dried with SpeedVac. Then they weredissolved in a solution containing 2% acetonitrile and 0.1% TFA
and combined into 18 fractions as shown in Fig. S4A. Resultant
fractions were desalted by using self-prepared stage tips (52),dried again with SpeedVac, and stored at −80 °C until use.
Mass Spectrometers. Two types of mass spectrometers were used inthis study. One was an Orbitrap mass spectrometer (positivemode, scan range of 350–1,500 m/z, 30,000 FWHM resolution at
400 m/z, LTQ Orbitrap Velos Pro mass spectrometer, Thermo
Scientific), which was used for a high-resolution full-scan analysis.
The other was a triple quadruple mass spectrometer (positive mode,
scan width of 0.002 m/z, Q1 and Q3 resolutions of 0.7 FWHM, acycle time of 1 s, a gas pressure of 1.8 mTorr, TSQ Vantage EMR
mass spectrometer, Thermo Scientific), which was used for SRM
analyses. Both of them were equipped with a nanoLC interface(AMR), a nano-Advance UHPLC system (Bruker Daltonics), andan HTC-PAL autosampler (CTC Analytics) with a trap column(0.3 × 5 mm, L-column, C18; Chemicals Evaluation and ResearchInstitute). Analytical samples were separated by reversed-phase
chromatography in which we used a home-made capillary column(length of 200 mm and inner diameter of 100 μm) packed with 2 μm
C18 resin (L-column 2, Chemicals Evaluation and Research Institute) using two mobile phases of RP-A (0.5% acetic acid) and
RP-B (0.5% acetic acid and 80% acetonitrile). Elution was directly
electrosprayed (2.4 kV) into the MS.
Mass Spectrometric Analyses of MS-QBiC Peptides and Cell/Nuclear
Lysates from HEK293T Cells. To develop and examine the MS-QBiC

workflow, in vitro-synthesized MS-QBiC peptides or their tryptic
digests were analyzed with both the Orbitrap mass spectrometerand the triple quadrupole mass spectrometer. Analytical samples
were separated with a linear gradient from 15 to 40 min at a flowrate of 300 nL/min using RP-A and RP-B. Tryptic digests of cell/
nuclear lysates from HEK293T cells were analyzed with the
Orbitrap mass spectrometer to identify expressed circadian clock
proteins. Analytical samples were separated with a linear gradientfor 210 min at a flow rate of 300 nL/min using RP-A and RP-B.
Narumi et al. www.pnas.org/cgi/content/short/1603799113

The MS scans to analyze precursor ions were performed in the
Orbitrap mass analyzer, and then the MS/MS scans to analyze
fragment ions were performed in the LTQ mass analyzer where
each of the most intense precursor ions (up to top 10) was isolated
and fragmented by collision-induced dissociation (CID) with thenormalized collision energy (35%). The CID raw spectra wereextracted using Proteome Discoverer 1.3 (Thermo Scientific) andsearched against the mouse International Protein Index (IPI)
database (version 3.87) using Mascot (v2.3, Matrix Science) withfollowing settings: The parameter of the cleavage was set to trypsin
and the missed cleavage was allowed up to 1. The mass toleranceswere set to 5 ppm for precursor ion and 0.6 Da for fragment ion,
respectively. On the conditions of the modification, we set carbamidomethylation at cysteine as fixed modification and oxidation atmethionine as variable (nonfixed) modification. The significancethreshold of P < 0.05 was set. The list of the identified peptides incircadian clock proteins is indicated in Dataset S1.
Development of SRM Methods. Optimal SRM methods containing
SRM transitions (from three to five per peptide), which wererepresented as specific pairs of m/z values associated with precursor and fragment ions, and collision energies (CE) associatedwith each SRM transition were developed by LC-SRM/MS
analyses of tryptic digests containing MS-QBiC peptides and1 pmol of LVTDLYK using primary SRM transitions developed
as follows: For m/z values of precursor ions, those of both the
doubly and triply charged ones were used. For m/z values offragment ions, those of singly charged y3 to yn−1 were used if the
precursor ion was doubly charged (n represents the number ofresidues of the precursor ion). If the precursor ion was triply
charged, values of both singly and doubly charged y3 to yn−1 wereused. In the case in which the precursor ion was short enough(n ≤ 10), b ions were also used according to the same rule as yions. Primary CE for each SRM transition was determined according to the following formulas, as previously reported (17):CE = 0.034 × m/zprecursor − 0.848 for doubly charged precursor
ions and CE = 0.022 × m/zprecursor + 5.953 for triply charged
precursor ions. Up to 10 candidate transitions with strong intensities were selected from SRM chromatograms obtained byprimary SRM transitions using Pinpoint software (version 2.3.0;
Thermo Scientific). Optimization of CE was further performed foreach candidate transition by the LC-SRM/MS analyses accompanied with seven CE values (primary CE, primary CE ± 2 eV,primary CE ± 4 eV, and primary CE ± 6 eV). Obtained chromatograms were analyzed and the best CE for each transition wasdetermined. Finally, up to five transitions per peptide were developed as optimal SRM methods. A total of 574 SRM transitions(1,149 transitions for light and heavy peptides) was obtained for
120 target peptides.
Quantification of Circadian Clock Proteins with SRM. Prefractionatedmouse liver samples by the SCX chromatography were analyzed
for quantification of circadian clock proteins. If no target peptide
was detected in the first 3 fractions from 18 fractions, theremaining 15 fractions were analyzed. The dried peptide mixtures
were dissolved in 10 μL of a solution containing 2% acetonitrile
and 0.1% TFA. A total of 2 μL of each sample was applied to thetriple quadruple mass spectrometer and analyzed with usingdeveloped SRM methods. Samples were separated with a gradientusing RP-A and RP-B (5–45% B in 50 min, 45–95% B in 1 min,
95% for 2 min, 95–5% B in 1 min, and 5% B for 6 min) at a flowrate of 300 nL/min. After each run, 30 min washing of the LC line
and equilibration of the analytical column for the next run were
performed. SRM chromatograms for each SRM transition wereextracted using Qual Browser of Xcaliber 2.2 (Thermo Scientific).
Quantification of the target peptides was performed by using thepeak with the highest ratio of signal to noise in the peaks, which
3 of 14was associated with one target peptide and not overlapped bynoise peaks.
Analysis of Digestion Efficiency and Specificity of Several Proteases.

Enzymatic digestion of BSA was performed according to a PTS
protocol as reported earlier (51). BSA (20 μg) in a PTS buffer was
reduced with 10 mM TCEP at 37 °C for 1 h and then alkylatedwith 15 mM iodoacetamide at 37 °C for 30 min in the dark. The
resultant sample was diluted five times with a 50-mM NH4HCO3
solution. Enzymatic digestion was performed using several conditions in addition to the same condition with liver proteins to compare several proteases where 1:50 (wt/wt) of Trypsin, 1:50 (wt/wt)of LysC, 1:50 (wt/wt) of chymotrypsin (Princeton Separations), or
1:20 (wt/wt) endopeptidase Glu-C (New England BioLabs) with
0.5 mM Glu-Glu, respectively, was added and followed by a 24-h
incubation at 37 °C. After the digestion, peptide mixtures wereextracted using ethyl acetate and TFA as described above and desalted by using self-prepared stage tips (52). BSA digests (100 fmol)
were subjected to LC-MS/MS analysis. The data were analyzed byProteome Discoverer 1.4 (Thermo Scientific) and Mascot (v2.3,
Matrix Science) using the bovine IPI database (v3.73): The parameter of the cleavage was set to each protease or none, whichmeans searching for nonspecific cleavage, and missed cleavages
were allowed up to 7. The mass tolerances were set to 5 ppm forprecursor ion and 0.6 Da for fragment ion, respectively. On theconditions of the modification, we set carbamidomethylation at
cysteine as a fixed modification and oxidation at methionine modification and pyroglutamylation at N-terminal glutamine as variable(nonfixed). The hit with an ion score >20 was adopted by a peptide
spectrum match (PSM).
Detection of Phosphorylated Peptides. Analyses of phosphorylationof peptide-1 and peptide-3 from CKIδ and peptide-1 from CKIe
(Fig. S5A) were performed by a targeted proteomics approach
using isotope labeling with chemical dimethylation according to
the previous report (53). Chemically synthesized 10 phosphopeptides (Fig. S5C) were dimethyl-labeled with isotope-labeledformaldehyde (13CD2O, ISOTEC) to introduce heavy isotopes.
They were dissolved in 2% acetonitrile and 0.1% TFA andstored at −80 °C until use. Enzymatically digested mouse liver
samples including 2 mg of proteins were dimethyl-labeled withformaldehyde (Sigma-Aldrich) on solid-phase extraction cartridges[solid phase extraction (SPE) cartridges, Oasis HLB 30 mg, Waters]. The samples eluted from the SPE cartridges were combinedwith 10 isotope-labeled phosphopeptides that were diluted with thesolution containing 50 ng/μL Enolase digest, 2% acetonitrile, and
0.1% TFA. The resultant mixtures were applied to Fe-IMAC(Immobilized Metal Ion Affinity Chromatography) according to aprevious report (54) to enrich the phophopeptides and then subjected to the SRM analysis. The developed SRM methods for
those phosphopeptides are shown in Dataset S2.
RNA Expression Analysis of Circadian Clock Genes by qPCR. The
mRNA-level expression profiles of circadian clock genes were
measured by qPCR. Mouse livers were sampled every 4 h over 2 d(n = 2). Total RNA was prepared from the livers at each time
point using TRIzol reagents (Life Technologies) according to the
manufacturer’s instructions. The cDNA was synthesized from0.25 μg of total RNA with random hexamer (Promega) and Su-

Narumi et al. www.pnas.org/cgi/content/short/1603799113

perScript II Reverse Transcriptase (Life Technologies) according
to the manufacturer’s instructions. The qPCR was performed
using QuantStudio7 Real-Time PCR System (Life Technologies)
and SYBR Premix Ex Taq GC (TaKaRa). Samples contained 1 ×
SYBR Premix Ex Taq, 0.8 μM primers, and 1/20 synthesized
cDNA in a 10-μL volume. PCR conditions were as follows: 1 min
at 95 °C, then 45 cycles of 15 s at 95 °C, and 1 min at 59 °C.
Absolute cDNA abundance was calculated using the standard
curve obtained from mouse genomic DNAs. Tbp expression levels
were quantified and used as the internal control (55). Oligonucleotide sequences of qPCR primers for the circadian clock genes
are described in Dataset S2.
Phase Determination of Peptide and mRNA Levels by Cosine Curve
Fitting. To assess the circadian rhythmicity and determine thephases for peptides and mRNA, 24-h cosine curve fitting to thetime-course data of each peptide and mRNA was performed.
Pearson’s correlations between the time-series data of each
peptide and 24-h period cosines with different peak times at
10-min intervals were calculated, and then the best-fit cosine
curve that gave a maximum Pearson’s correlation was selected.
Circadian rhythmicity of each peptide and mRNA was evaluatedby a permutation test.
BT Detection by Molecular Timetable Methods. Estimation of bodytime by using expression profiles of circadian clock proteins was
performed according to the molecular timetable methods asreported previously (31). The timetable was created by using the
calculated phases ti (i = 1 . . . 36) for 36 peptides from 14 circadian clock proteins, which showed circadian rhythmicity by
24-h cosine curve fitting. Using the timetable, we estimated the
body time as follows: We normalized an absolute amount Xi (i =1 . . . 36) of the peptide i (i = 1 . . . 36) by using its average μi andSD σ i. The normalized expression level Yi is described as follows:
Yi = (Xi − μi)/σ i. We created an expression profile {ti, Yi} (i =1 . . . 36) composed of the peak time ti of peptide i and its
normalized expression level Yi. To estimate the BT of an expression profile, we calculated the Pearson’s correlation over the
peptides (i = 1 . . . 36) between an expression profile {ti, Yi} and a
24-h cosine curve {ti, √2 Cos(2π(ti − b)/24)} with a peak timeb (0 ≤ b < 24). We prepared 24-h cosine curves with a peak timeb from 0 to 24 h in increments of 10 min. We then selected thebest-fit cosine curve that gave a maximum Pearson’s correlation(c) and body time (bc). Then we evaluated the statistical significance of the maximum Pearson’s correlation as follows: We first
generated random expression data by obtaining the sum of a
24-h period cosine at random time [√2 Cos(2π(tr − bi)/24)]
and a measurement noise (Ni), where tr represents random
time (0 ≤ tr < 24). Ni was a randomly selected value in the noisedata set of the each peptide, which were obtained by subtracting the
calculated values [√2 Cos(2π(ti − bc)/24)] from the experimental
values (Yi) over the indicated time points. For the random expression data, we evaluated a maximum Pearson’s correlation. We repeated this procedure 10,000 times to create the distribution of
maximum Pearson’s correlations. The statistical significance(P value) was determined by dividing the number of greater correlations obtained from random expression data than the correlation obtained from the experimental expression data by 10,000.

4 of 14

FLAG-tag

Atarget peptide

cell-free product-1: M-DYKDDDDK-DQFNVLIK
FLAG-tagspacertarget peptide

cell-free product-2: M-DYKDDDDK-[L]nK-DQFNVLIK
FLAG-tagspacerquantification-tag

target peptide

cell-free product-3: M-DYKDDDDK-LLLLK-LVTDLYK-DQFNVLIK
FLAG-tagspacerquantification-tag

target peptide

cell-free product-4: M-DYKDDDDK-LLLLK-LVTDLYK-ILYISNQVASIFHCK
FLAG-tagspacerquantification-tag

target peptide

cell-free product-5: M-DYKDDDDK-LLLLK-LVTDLYK-EAGVEVVTENSHTLYDLDR

B

Relative Abundance

100
Relative Abundance

C

25.88

80
60

24.02

100

710.662 710.996
z=3
z=3

Efficiency of digestion

711.329z=350

Sequence

Tar. / (Tar. + Undi.)

711.663z=3711.997z=3cell-free product-1

0.019

712
m/z

cell-free product-2 (n=1)

0.420cell-free product-2 (n=2)

0.991

cell-free product-2 (n=3)

0.997cell-free product-2 (n=4)

1.000cell-free product-2 (n=5)

1.000

0
710

40

711

713

20
22.93

0
0
RT
(min)

10

20

30
40
Time (min)50
Observed Calculatedm/z (Z=3) m/z (Z=3)

Peptide Sequence

22.93 DYKDDDDKDQFNVLIK

657.650657.650

24.02 [Formyl:N-term]M[oxidation]DYKDDDDKDQFNVLIK

715.994715.994

25.88 [Formyl:N-term]MDYKDDDDKDQFNVLIK

710.662710.662

Tar. means intensity of the target peptide
Undi. means intensity of the undigested productChemically synthesized peptide: LVTDLYK(BSA peptide)
KLVTDLYKVBovine serum albumin (BSA):

Estimated amount ofcell-free product-3 (attomole)

D

E

15
10
5
0
BSA
peptide

BSA

Ratio of the amount of target peptid
to that of quantification-tag

RT: Retention Time [Formyl:N-term]: Formylation of the N-terminal amino acid[oxidation]: Oxidation of the amino acid

1.5
1
0.5
0
DQF

ILY

EAG

Fig. S1. Examination and validation of the MS-QBiC workflow. (A) Amino acid sequences of test peptides used for the examination and validation of
the MS-QBiC workflow. (B) LC-MS analysis of the synthesized cell-free product-1. A base peak chromatogram of the purified peptide and a mass
spectrum of the ion peak with highest intensity (Inset) are shown. The table indicates three identified peptides that correspond to the three ion peaksin the chromatogram. (C ) Effect of spacer sequence on the tryptic digestion efficiency. Digestion efficiency was calculated as the ion peak intensities ofthe digested peptide divided by the sum of those of the digested and undigested peptide. (D) Measuring the abundance of the quantification tag.
Stable isotope-labeled cell-free product-3 was mixed with chemically synthesized LVTDLYK peptide (BSA peptide) or the tryptic digests of commerciallyavailable BSA. Concentration of the quantification tag was determined by comparing ion peak intensities of the stable isotope-labeled quantification
Legend continued on following page

Narumi et al. www.pnas.org/cgi/content/short/1603799113

5 of 14

tag with nonlabeled peptides. (E ) Validation of the quantification of a target peptide. Stable isotope-labeled cell-free product-3, -4, and -5 were mixed
with the chemically synthesized target peptides DQFNVLIK (DQF), ILYISNQVASIFHCK (ILY), and EAGVEVVTENSHTLYDLDR (EAG), respectively. Concentration of the target peptide was determined by comparing ion peak intensities of the stable isotope-labeled target peptide with nonlabeled
peptides.

FLAG-tagspacer quantification-tag target peptide

MDYKDDDDK*LLLLK*LVTDLTK*DQFNVLIK*+ LVTDLTK (Known concentration)+ DQFNVLIK (Target)
Tryptic digestion
LC-MS

Quantification of MS-QBiC peptide
LVTDLTKLVTDLTK*

Intensity (×107)

Intensity (×106)5398.24912395.7418

DQFNVLIK DQFNVLIK*
488.777

LVTDLTK*LVTDLTK
395.240
16

Quantification of target peptide
DQFNVLIKDQFNVLIK*

398.7514
396.2424
489.278491.7863
2

492.2881

489.779

0

0
392

394

396

398
m/z

400

402

486

488

490

492

494

496

m/z

Fig. S2. Peptide quantification by the MS-QBiC method. Quantification of the quantification tag and the target peptide using LC-MS. Purified MS-QBiC
peptide (Upper) was digested by trypsin. The quantification tag was quantified by comparing ion peak intensities of the stable isotope-labeled quantification
tag with those of the chemically synthesized quantification tag (Lower Left). The target peptide was quantified by comparing the stable isotope-labeled target
peptide with the nonlabeled target peptide (Lower Right). The target peptide was derived from CLOCK, a circadian protein.

Narumi et al. www.pnas.org/cgi/content/short/1603799113

6 of 14

b3 b4 b5 b6 b7
y6 y5

m/z
Sequence
Precursor Product577.320LVTDLTK
395.240676.388583.340LVTDLTK*
398.250682.408
190.155
239.791296.710370.245434.274
196.085
253.106
302.64359.182415.725
328.194379.302478.370592.413739.481867.540DQFNVLIK*
391.162505.205604.273717.357
830.441379.302478.370592.413739.481867.540491.787
391.162505.205604.273717.357
830.441C

B 100

DQ F I V L I K
y7 y6 y5 y4 y3
Ion
ChargeCE Type Precursor Product
y5
13
1
2
y6
y5
13
1
2
y6
y3
y4
y5
y6
y7
2
b3
b4
b5
b6
b7
13
3
y3
y4
y5
y6
1
y7
b3
b4
b5
b6
b7
y3
y4
y5
y6
y7
1
16
2
b3
b4
b5
b6
b7

Product ions from DQFNVLIK*(+2)
y6 (+1)y3 (+1)y4 (+1)y5 (+1)b6 (+1)b5 (+1)b7 (+1)b3 (+1)
1.0

0.5

50

LVTDLTK(chemically synthesized)

0
6
4
2
0
16

LVTDLTK*(from MS-QBiC peptide)

DQFNVLIK*(+2)(from MS-QBiC peptide)

8
0

14

15

16
17
Retention time (min)

0.8
0.6
0.4
0.2
0
y3 y4 y5 y6 y7 b3 b4 b5 b6 b7
Product ions from DQFNVLIK*(+2)

D

SRM transitions for DQFNVLIK and DQFNVLIK*
TargetPeptidePrecursor ProductIon types
CE
mass
mass
(charge)373.281 20 y3(+1)
472.35 20 y4(+1)
DQFNVLIK 488.777 586.393 20 y5(+1)
733.461 18 y6(+1)
830.441 16 b7(+1)
379.302 20 y3(+1)
478.37 20 y4(+1)
DQFNVLIK* 491.787 592.413 20 y5(+1)
739.481 18 y6(+1)
830.441 16 b7(+1)
DQFNVLIKy3(+1)y4(+1)y5(+1)
y6(+1)
b7(+1)

DQFNVLIK*y3(+1)y4(+1)y5(+1)
y6(+1)
b7(+1)

5.0

0.4

0.2

2.5
0
40
30
20
10

0
10

12 14 16 18 20
Collision energy (eV)

22

18

1.0

Intensity (×1,000)

Relative abundance

0.8

intensity (×100,000)

L VTDL TK

Relative abundance

A

0
32.8
33.0
Retention time (min)33.2

Fig. S3. Application of the MS-QBiC workflow to the SRM analysis. (A) Development of the primary SRM method for screening of optimal SRM transitions. The
table shows an example for cell-free product-1. Optimal SRM transitions for LVTDLTK and LVTDLTK* (the asterisk indicates that the amino acid is isotopelabeled) to quantify the MS-QBiC peptide and primary SRM transitions for DQFNVLIK* to quantify the target peptide are shown in the table. The primarytransitions were developed as follows: Both doubly and triply charged monoisotopic precursors for the target peptide were considered. For the doubly charged
precursors, singly charged monoisotopic products y3 to yn−1 were monitored (n represents the number of residues of the peptide). For the triply charged
precursors, both singly and doubly charged monoisotopic products y3 to yn−1 were monitored. In the case that the peptide was short enough (n ≤ 10), b ionswere used according to the same rule as y ions. (B) SRM chromatograms of LVTDLTK (Upper Top), LVTDLTK* (Upper Middle), and DQFNVLIK* (Upper Bottom).
(C) Optimization of collision energy for each SRM transition. (D) Quantification of the target peptide in a mouse liver using LC-SRM. The SRM transitions(Upper) were developed by using purified MS-QBiC peptide. The target peptide was quantified by comparing ion peak intensities of the fragment peptides
from the stable isotope-labeled target peptide (Lower ) with the nonlabeled target peptide in mice livers (Lower). CE, collision energies.

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A

1

2

C

3 4 5 6 7 8 9 1011121314151617 18

Number of SCX fractions
3

Absorbance at 220 nm

250

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18

200
10
150
20
100
30
50
40
0
0

10

20

30

40

50

60

70

Retention time (min)Target peptide: FSQGQQLVTK* (SRM-ID 21)
4

Fr4

Fr5

Fr6

Fr7

Fr8

Fr9

Fr10

Intensity (×1,000)

3

SRM-ID

B

50

60

70

2
1

80

0
21.4

22.2 21.4

22.2 21.4

22.2 21.4

22.2 21.4

22.2 21.4

22.2 21.4

22.2

Target peptide: YFGSIDSSENNHK* (SRM-ID 91)
3

Fr11

Fr12

Fr13

Fr14

Fr15

Fr16

90
Fr17

2

100

1
110

0
23.0 23.4

23.0 23.4

23.0 23.4

23.0 23.4

23.0 23.4

Retention time (min)23.0 23.4

23.0 23.4
120

Fig. S4. Prefractionation of tryptic digests composed of cellular protein extracts from mice livers and MS-QBiC peptides using SCX chromatography. (A) A
chromatogram of the tryptic digests separated with the SCX chromatography. Numbers above the chromatogram indicate numbers of the SCX fractions.
(B) Examples of the SRM chromatogram. SRM chromatograms of FSQGQQLVTK* (SRM-ID 21, Upper) contained in the SCX fractions from no. 4 to no. 10 and
those of YFGSIDSSENNHK* (SRM-ID 91, Lower) in the fractions from no. 11 to no. 17 are shown. (C) Distribution of the signal intensities corresponding to thetarget peptides originated from the MS-QBiC peptide in the SCX fractions from no. 3 to no. 18. A red color indicates that the corresponding signal was detectedin the fraction. Black rows indicate that the signal was not able to be detected in any fraction.

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80

10

4

40

0

Concentration (attomoles per μg lysate)

CRY2

0

0

40

10 200

0

0
TEF

100

160
80

NFIL3 (E4BP4)

300

20

40

10

20

30

8

20
10

0

0
PER1

50

0
100

PER2

30
20
10

0
0
0 4 8 12 16 20

10

0

0

0

NR1D2 (REV-ERBβ)
30

6

40

4

0

0
PER3

10

10

0
0
0 4 8 12 16 20

0
RORγ

6 100

12
8

0

0

GAPVNV[pS]SSDLTGRGAPVNVS[pS]SDLTGRGAPVNVSS[pS]DLTGRGAPVNVSSSDL[pT]GR

CKIδ3-1
CKIδ3-2
CKIδ3-3
CKIδ3-4

IQQ[pT]GNTSPRIQQTGN[pT]SPR
IQQTGNT[pS]PR

CKIε1-1
CKIε1-2
CKIε1-3

40

PER1

30
20

50

4

30
20

2
0
15

8
4

0

4

0
8 12 16 20

CKIδ

4

4

0

10

0
0
0 4 8 12 16 20

2

0

0
RORγ

6 100

16
20

10

30

80
4

12
8

20

60
40

2

10

4

20

0
0
0 4 8 12 16 20

00
0 4 8 12 16 20

60
40
20
0
120
80
40

0
50

52

54

44

46

0
66

DBP/TEF

240 400160

40

0
8 12 16 20

Peptide-2

68

62

64

70

72

56

58

Retention time (min)CKIδ1-1 CKIδ1-2 CKIδ1-3 CKIδ3-1 CKIδ3-2 CKIδ3-3 CKIδ3-4

120
100

300

80
60
DBP

100
0
0

40
20

TEF

4

0
8 12 16 20

Circadian time (h)Peptide-1

6

0

30 20

8

40
20

PER3

30

60 20

7

80

200

0

80

200

14

5

120 200

400

00

NR1D2 (REV-ERBβ)

21

200

CKIε

0

0

28

20
10

10

0

0
35

10

CKIδ/ε

600

40

10

Copies per cell (×103)

BHLHE40

0

20

4

3
2
1
0

40

E
30

10

Peptide-40

50 BHLHE40/41(DEC1/2) 800

80

Circadian time (h)

D

12

100

0
0
0 4 8 12 16 20

*

PER2

40
30

8

4 100
0

0

0
DBP

120

300

12 80
60
8
40
4 20

0
0
0 4 8 12 16 20

ID

Concentration(attomoles per μg lysate)

0

0

20
10

0

NR1D1 (REV-ERBα)

8

30
80

12

30

12

10

40

10

NFIL3 (E4BP4)

20

20

50

0

30

10

0
CKIε

20
80

40

120

10 40

240 40
160

40
20

60

0
0
0 4 8 12 16 20

48

40

30 50

20

0

0
CKIδ

0

80

10

Signal intensity (×103)

CKIδ1-1
CKIδ1-2
CKIδ1-3

TEF

80

10 40

20 40020

Detection

VA[pS]SGLQSVVHR
VAS[pS]GLQSVVHR
VASSGLQ[pS]VVHR

0

10 200

6
ID

0

4

C
Monitored phosphopeptides

40

40

100

20

40

2

4

CRY1
40
30

20

30 600

80

30

60

80

40 800120

80
4Circadian time (h)Peptide-2
Peptide-3

Peptide-1

2

0

16
20

8

60 20
20

30 20

12 80
60
8
40
4 20

40

40

4 100
0

20

4

80

200

0

40

80

NR1D1 (REV-ERBα)
12

0

30

8

0

10

CRY2

120

8

CLOCK
120

30

20

10

DBP

12

30

10

0

40

60

0

20

0

30 50

80

0
CKIε

240 4020 40020

BMAL1 (ARNTL)
40

12 120

40
30

80

10 40

0

0
CKIδ

BHLHE40 (DEC1)
50

30

20

10 40

30 6008080

2040 800

120

30

30

20

40

120

Copies per cell (×1,000)

8

B

CRY1
40

RORα

10

10

CKIε1-1

HLF

3 24

6

8
2 16

6

4

4

1

8

0
8 12 16 20

0

2

2
0
0

4

0

4

0
8 12 16 20

Circadian time (h)12Copies per cell (×103)

30

CLOCK
120

Concentration(attomoles per μg lysate)

BMAL1 (ARNTL)
40

12 12040

Concentration (attomoles per μg lysate)

BHLHE40 (DEC1)
50

Copies per cell (×1,000)

A

Peptide-3

Fig. S5. A summary of the expression levels of each peptide. (A, B, D, and E) Concentrations are expressed as attomol per microgram of lysate (Left axis) or
copies per cell (Right axis). Copy numbers were calculated by estimating a total amount of proteins per cell is 0.5 ng (19). (A) Quantification of each of thepeptides from a protein. Time courses of the quantified peptides arranged according to their parental proteins are shown. The correspondence of detected
peptides to plots is shown in Dataset S2. (B) Time course of the quantified proteins calculated from two biological replicates. Closed circles indicate average
peptide concentrations derived from same protein in each biological replicate. (C) SRM detection of phosphorylated peptide-1 and peptide-3 from CKIδ andpeptide-3 from CKIe. Monitored phosphopeptides are summarized in the table (Left). SRM chromatograms (Right) corresponding to peptides derived frommouse liver (Upper) and isotope-labeled internal standards (Lower) are shown. An asterisk (*) indicates that the peptides were not separated by chromatography and therefore could not be differentiated from each other in the chromatogram. (D) Quantification of peptides for common region of two circadian
Legend continued on following page

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shown. The correspondence of detected peptides to plots is shown in Dataset S2. Gray lines indicate the time course of quantified parental proteins.
(E) Quantification of peptides with low signal intensities. Time courses of the peptides for HLF and RORα are shown.

Number of PSMs

A

LysC and Trypsin

Trypsin

LysC

GluC

Chymotrypsin

1,200

1,200

1,200

300

300

800

800

800

200

200

400

400

400

100

100

0

0

0
0 1 2 3 4 5

0 1 2 3 4 50
0 1 2 3 4 50
0 1 2 3 4 5

0 1 2 3 4 5

Number of incomplete digestions

B

Number of PSMs

1,200

LysC and Trypsin

1,200

Trypsin

1,200

800

800

800

400

400

400

0

0
A C D E F G H I K L M N P Q R S T VW Y

500

GluC

0
A C D E F G H I K L M N P Q R S T VW Y

250

400

200

300

150

200

100

100

50

LysC

A C D E F G H I K L M N P Q R S T VW Y

Chymotrypsin00
A C D E F G H I K L M N P Q R S T VW Y

A C D E F G H I K L M N P Q R S T VW Y

Amino acid residue
Fig. S6. Digestion efficiency and specificity of alternative proteases. BSA digests, which were prepared by both trypsin and LysC (Lysyl Endopeptidase), trypsin,
LysC, GluC (Glutamyl endopeptidase), or chymotrypsin, were analyzed by LC-MS/MS. The numbers of PSMs were classified by the number of incomplete digestions (A) to show efficiency and by the C-terminal amino acid residues of the identified peptides (B) to show specificity of selected proteases. The amino acidresidues specific to each protease are highlighted in red.

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3.5

Bhlhe40 (Dec1)

Bhlhe41 (Dec2)

Bmal1 (Arntl)

1.6

4

3

6

1.4

2.5

5

1.2

3

2

4

1

2.5

1.5

3

1

2

0.4

1

0.5

1

0.2

0.5

0
4

0

Cry1

6

3

5

1

2

0.5

1

14

Dbp

12

1.5

0

CKIδ

1

0.5

CKIε

0.4
0.3

0.6
0.2

0.4

0.1

0.2
0

0

0

2

0.6

0.8

3

1.5

0.8

1.2

4

2

Clock

3.5

0

Cry2

7

3.5
2.5

Relative expression of mRNA (AU)

7

Tef

2.5

0

Hlf

5

5

2

4

4

1.5

3

3

1

2

2

0.5

1

1

Nfil3 (E4bp4)

10
8
6
4
2
0
20

0

Nr1d1 (Rev-Erbα)

160

Nr1d2 (Rev-Erbβ)

14
15

1.2

6
5

2

0.6

8

1.5

0.4

1

4
0.2

2

Per3

3

Rorα

5

0.25

4

2

3
2

0

Rorβ

0.3

2.5

1.5

0.5

0

0

0

Per2

2.5

0.8

10

3.5
3

1

12

10

3.5

0

Per1

10

Rorγ

8

0.2

6

0.15

0.5
0
0 4 8 12 16 20 24 28 32 36 40 44

4

0.1

1
1

0.05

0

0
0 4 8 12 16 20 24 28 32 36 40 44

20
0 4 8 12 16 20 24 28 32 36 40 44

0 4 8 12 16 20 24 28 32 36 40 44

Circadian time (h)Fig. S7. Time course of the relative expression levels of mRNA for 20 circadian clock genes. Expression levels of each mRNA were quantified by qPCR with twobiological replicates.

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B

0
-3
3

CT16

0
-3
0

0

24 0
Molecular peak time (h)

4

8

Mice

12

Normalized expression level

CT43

Normalized expression level

A

24

16

20

Environment

Estimated time (h)(Statistical significance)

CT4

3.7
3.3
(P < 0.0001) (P < 0.0001)

CT4

3
0
-3
3

CT16

0
-3

0

24 0
240
Molecular peak time (h)

0

24

4
Mice

Wild-type

Normalized expression level

Normalized expression level

D

CT4

0
-3
3

CT16

0
24 0
240
Molecular peak time (h)

0

4

8

Mice

12

24

16

20

Normalized expression level

CT4

19.3
20.5
20.2
(P = 0.1136) (P = 0.1889) (P = 0.1424)

CT16

20.7
(P < 0.05)

-3
3

CT16

0
-3

0

24 0
240
Molecular peak time (h)

0

4

8

Mice

16.5
(P < 0.001)

CT16

16.2
16.5
15.7
(P < 0.0005) (P < 0.0001) (P < 0.0005)

12

16

24
20

24

Environment

Estimated time (h)(Statistical significance)

CT4

16
16
15.8
(P < 0.0001) (P < 0.0005) (P < 0.0001)

CT16

16
15.3
15.7
(P < 0.0005) (P < 0.0001) (P < 0.0005)

16.3
16(P < 0.001) (P < 0.0005)

CT4

19.8
20.3
(P = 0.1537) (P = 0.055)

024Cry1/2 DKO

Bmal1 KO

Per1/2 DKO

Cry1/2 DKO(Estimated time: 19.7 h, P = 0.0974)(Estimated time: 15.8 h, P < 0.0001)(Estimated time: 15.8 h, P < 0.0005)

Upregulated

Upregulated

Upregulated

PER3
NR1D2 BHLHE40
NR1D1

RORγ
NR1D1 BHLHE40

NFIL3

NR1D2
CLOCK

TEF

DBP

DBP TEF

PER2
PER3

NR1D2
RORγ NFIL3

NFIL3

PER1 NFIL3

0

CRY1
CRY2

BMAL1

Downregulated
-8

24

CT4

Per1/2 DKO

820

Environment

3

Estimated time (h)(Statistical significance)

Environment

E

16

Estimated time (h)(Statistical significance)

17
16.5(P < 0.005) (P < 0.0001)

C3

0

12

Bmal1 KO
CT16

-3

8

24

NR1D1

24 0
0

CRY1

Molecular peak time (h)
4

8

12

NFIL3

DBP TEF
BMAL1

Downregulated

0

16

PER1
PER3

NFIL3

CRY2

CLOCK

RORγ

BHLHE40

PER2

CLOCK

Downregulated
24 0
20

24
24

Fig. S8. BT detection of wild-type and clock mutant mice by molecular timetable methods using a single-time-point data set. BT of (A) wild-type, (B) Bmal1
KO, (C) Per1/2 DKO, and (D) Cry1/2 DKO mice at CT4 and CT16 was estimated using datasets of expression profiles of peptides. Data sets were obtained withtwo (wild-type) and three (mutant) biological replicates. Best-fit cosine curves (black) and those based on the actual circadian time point (gray) are shown. The
peak time of the best-fit curves indicates the estimated BT, which is also shown with its statistical significance. Statistical significance was obtained by estimating the goodness of the Pearson correlation in the cosine curve fitting of the experimental profile by comparing with those of 10,000 random profiles(details are described in SI Materials and Methods). The colors, in ascending order from green to red to blue, represent the molecular peak time, whichcorresponds to the molecular timetable of each peptide. The color code is represented below the diagrams. CT was defined as “extrapolated entrained time ofmice in a LD cycle before sampling.” (E) Averaged expression profiles of peptides in Bmal1 KO, Per1/2 DKO, and Cry1/2 DKO mice at both CT4 and CT16
[normalized expression levels at CT4 (n = 3) and CT16 (n = 3) are averaged]. Error bars indicate SDs. Parental proteins of each peptide are designated.

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Bmal1 KO

Wild-typeProteins CT
BHLHE40(DEC1)BMAL1(ARNTL)

0

1

2

3

4 0

1

2

3

Ratio
4 0

Per1/2 DKO
1

2

3

Cry1/2 DKO
4 0

1

2

3

4

4
16
4
16
4

CLOCK
16
4
CRY1
16
4
CRY2
16
4
CKIδ
16
4
CKIε
16
4
DBP
16
4
TEF
16
NFIL3
(E4BP4)

4
16

4
NR1D1
(REV-ERBα) 16
4
NR1D2(REV-ERBβ) 16
4
PER1
16
4
PER2
16
4
PER3
16
4
RORγ
16

Fig. S9. Expression levels of circadian clock proteins in clock mutant mice. Livers from three knockout mice (Bmal1 KO, Per1/2 DKO, and Cry1/2 DKO) werecollected at CT4 and CT16 with three biological replicates. Relative concentrations of each peptide, in which the concentration was divided by the median valueof that across the circadian day in the wild type, are summarized according to parental proteins. Red and blue lines indicate the maximum and minimum
concentration of the peptides across the circadian day in wild type, respectively. CT was defined as “extrapolated entrained time of mice in a LD cycle before
sampling.”

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Wild-type
CT4

8
0
-8

CT16

8
0
-8

0
4

24 0
Molecular peak time (h)
8

16

24
20

0
-8
CT16

8
0
-8

0

24 0
24 0
Molecular peak time (h)
0

4

8

12

-8
CT16

8
0
-8

0

24 0
24 0
Molecular peak time (h)
0

4

8

D

Per1/2 DKO
CT4

8

0

24

Normalized expression level

Normalized expression levelC

12

Bmal1 KOCT4

B 8
Normalized expression level

Normalized expression level

A

16

24
20

24

12

16

24
20

24

Cry1/2 DKO
CT4

8
0
-8

CT16

8
0
-8

0

24 0
24 0
Molecular peak time (h)
0

4

8

12

16

24
20

24

Fig. S10. Expression profiles of peptides used for the BT detection of wild-type and mutant mice. Profiles of (A) wild-type, (B) Bmal1 KO, (C) Per1/2 DKO, and(D) Cry1/2 DKO mice in Fig. S8 are shown with a wider y-axis range. Best-fit cosine curves (black) and those based on the actual circadian time point (gray) are
shown. The colors, in ascending order from green to red to blue, represent the molecular peak time, which corresponds to the molecular timetable of each
peptide. CT was defined as “extrapolated entrained time of mice in a LD cycle before sampling.”

Other Supporting Information Files
Dataset S1 (XLSX)
Dataset S2 (XLSX)
Dataset S3 (XLSX)

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