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RESEARCH ARTICLE
Strategy for comprehensive identification of human
N-myristoylated proteins using an insect cell-free protein
synthesis system
Takashi Suzuki1�, Koko Moriya2�, Kei Nagatoshi2, Yoshinobu Ota2, Toru Ezure1, Eiji Ando1,Susumu Tsunasawa3 and Toshihiko Utsumi2,4
1 Clinical and Biotechnology Business Unit, Shimadzu Corporation, Kyoto, Japan2 Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi, Japan3 Institute for Protein Research, Osaka University, Osaka, Japan4 Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan
Received: November 26, 2009
Revised: January 26, 2010
Accepted: February 1, 2010
To establish a strategy for the comprehensive identification of human N-myristoylated
proteins, the susceptibility of human cDNA clones to protein N-myristoylation was evaluated
by metabolic labeling and MS analyses of proteins expressed in an insect cell-free protein
synthesis system. One-hundred-and-forty-one cDNA clones with N-terminal Met-Gly motifs
were selected as potential candidates from �2000 Kazusa ORFeome project human cDNA
clones, and their susceptibility to protein N-myristoylation was evaluated using fusion
proteins, in which the N-terminal ten amino acid residues were fused to an epitope-tagged
model protein. As a result, the products of 29 out of 141 cDNA clones were found to be
effectively N-myristoylated. The metabolic labeling experiments both in an insect cell-free
protein synthesis system and in the transfected COS-1 cells using full-length cDNA revealed
that 27 out of 29 proteins were in fact N-myristoylated. Database searches with these 27 cDNA
clones revealed that 18 out of 27 proteins are novel N-myristoylated proteins that have not
been reported previously to be N-myristoylated, indicating that this strategy is useful for the
comprehensive identification of human N-myristoylated proteins from human cDNA
resources.
Keywords:
Animal proteomics / Comprehensive analysis / Insect cell-free protein synthesis
system / Metabolic labeling / MS analysis / Protein N-myristoylation
1 Introduction
Protein N-myristoylation is a well-recognized form of
lipid modification that occurs in eukaryotic and viral
proteins [1–5]. In general, N-myristoylation is an irreversi-
ble cotranslational protein modification. In this process,
myristic acid, a 14-carbon saturated fatty acid, is attached
to the N-terminal Gly residue of the protein at the extreme
N-terminus after removal of the initiating Met. A stable
amide bond links myristic acid irreversibly to the protein.
The N-myristoyltransferase (NMT) that catalyzes the
transfer of myristic acid from myristoyl-CoA to the
N-terminal Gly is a member of the GNAT superfamily
of proteins [6]. The precise substrate specificity of this
enzyme has been characterized using purified enzyme
and synthetic peptide substrates [7–9]. The requirement for
Gly at the N-terminus is absolute and no other amino acid
can take its place.
Abbreviations: KOP, Kazusa ORFeome project; NMT, N-myris-
toyltransferase; QIT, quadrupole ion trap �These authors have contributed equally to this work.
Correspondence: Professor Toshihiko Utsumi, Department of
Biological Chemistry, Faculty of Agriculture, Yamaguchi
University, Yamaguchi 753-8515, Japan
E-mail: utsumi@yamaguchi-u.ac.jp
Fax: 181-83-933-5820
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
1780 Proteomics 2010, 10, 1780–1793DOI 10.1002/pmic.200900783
Many N-myristoylated proteins play key roles in regulat-
ing cellular structure and function. They include proteins
involved in a wide variety of cellular signal transduction
pathways, such as protein kinases, phosphatases, guanine
nucleotide-binding proteins, and Ca21-binding proteins. In
many cases, the functions of these N-myristoylated proteins
are regulated by reversible protein�membrane and
protein�protein interactions mediated by protein N-myris-
toylation.
Recently, a sophisticated program for the automated
prediction of protein N-myristoylation from the substrate
protein sequence has been developed and this prediction
program is available on a public WWW-server [10]. Large-
scale application of this predictor using GenBank (from
the National Center for Biotechnology Information (NCBI)
data produces lists of thousands of potential NMT substrates
[11]. However, because of the lack of a simple and easy
strategy to detect protein N-myristoylation, the number of
experimentally verified N-myristoylated proteins is far lower
than the predicted number. In the previous study, it was
shown that metabolic labeling in a newly developed cell-
free protein synthesis system (Transdirect insect cell)derived from insect cells [12] is a simple and sensitive
method to detect protein N-myristoylation [13]. In addition,
it has recently been demonstrated that MALDI-TOF MS
and MALDI-quadrupole ion trap (QIT)-TOF MS analysis
of proteins synthesized with the insect cell-free protein
synthesis system provided detailed structural information
about N-myristoylated proteins, such as the exact location of
the modification and the structure of the attached functional
group [14]. In this study, to establish a strategy for
comprehensive identification of human N-myristoylated
proteins, the susceptibility of human cDNA clones in
human cDNA resources to protein N-myristoylation was
evaluated by metabolic labeling and MS analyses of
proteins expressed using an insect cell-free protein synthesis
system. For this analysis, 141 cDNA clones with an
N-terminal Met-Gly motif were selected as potential candi-
dates from �2000 Kazusa ORFeome project (KOP) human
cDNA clones. The susceptibility of these cDNA clones to
protein N-myristoylation was first evaluated using fusion
proteins, in which the N-terminal ten amino acid residues
were fused to an epitope-tagged model protein. Then,
protein N-myristoylation on the gene product of the full-
length cDNA was evaluated by metabolic labeling experi-
ments both in an insect cell-free protein synthesis system
and in transfected COS-1 cells. As a result, the products of
27 out of �2000 cDNA clones were found to be N-myris-
toylated. This corresponds to 1.4% of the total number of
cDNA clones tested. Database searches using these 27
cDNA clones revealed that 18 out of 27 proteins were novel
N-myristoylated proteins that have not been reported to be
N-myristoylated previously. These results indicate that the
strategy proposed in this study is useful for the compre-
hensive identification of human N-myristoylated proteins
from human cDNA resources.
2 Materials and methods
2.1 Materials
Transdirect insect cell, which is based on a Sf21 extract, is a
commercial product of Shimadzu (Kyoto, Japan). The TNT
T7 Insect Cell Extract Protein Expression System is a
coupled transcription and translation system using the same
insect cell extract as the Transdirect insect cell kit and was
purchased from Promega (Madison, WI, USA). Restriction
endonucleases and DNA modifying enzymes were obtained
from Toyobo (Osaka, Japan), New England Biolabs (Ipswich,
MA, USA), Nippon Gene (Toyama, Japan), and Promega.
Factor Xa was purchased from Novagen (Madison WI,
USA). [3H]leucine, [3H]myristic acid, and Amplify were
from GE Healthcare (Piscataway, NJ, USA). Myristoyl-CoA,
CHCA, DHB, ANTI-FLAG M2-Agarose, and FLAG peptide
were obtained from Sigma (St. Louis, MO, USA). Strep-
Tactin Superflow was purchased from Qiagen (Valencia,
CA, USA).
2.2 Human cDNA resources
Human cDNAs collected in the KOP, KOP clones [15–17],
were purchased from Promega and used as sources for
obtaining the cDNA clones. Human N-myristoylated
proteins were screened for among 1929 KOP clones regis-
tered in March, 2008.
2.3 Prediction of N-myristoylated proteins using
prediction programs
Two public WWW-server based prediction programs for
N-myristoylation, the MYR Predictor (http://mendel.imp.
ac.at/myristate/SUPLpredictor.htm) [10] and Myristoylator
(http://www.expasy.org/tools/myristoylator/) [18], were
used for the prediction of N-myristoylated proteins. The
entire amino acid sequences deduced from the nucleotide
sequences of the ORFs were used as the query.
2.4 Construction of the expression vectors
The expression vector pTD1-tGelX for screening N-myris-
toylated proteins was constructed as follows. To introduce
the Factor Xa recognition sequence between Ser-10 and His-
11 of the truncated human gelsolin (tGelsolin), PCR was
carried out using primers tGelX1-F and tGelX1-R and pTD1-
tGelsolin [14] as the template. The amplified DNA fragment
was then treated with T4 polynucleotide kinase. After
treatment, the DNA fragment was self-ligated and used to
transform Escherichia coli DH5a. The obtained plasmid was
designated as pTD1-tGelX-tGelsolin. The KpnI site in
the protein-coding region of pTD1-tGelX-tGelsolin was
Proteomics 2010, 10, 1780–1793 1781
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
disrupted using mutagenic primers. The plasmid was then
digested with KpnI and HindIII, and the 1.5 kbp DNA
fragment was isolated, which was then subcloned into the
KpnI-HindIII sites of a pTD1 vector [19].
The expression vector pTD1-FL used for the cell-free
protein synthesis of the ORFs of human cDNA clones were
constructed as follows. The pENTR1A vector (Invitrogen) was
digested with EcoRV and KpnI. The resulting 0.5 kbp DNA
fragment was inserted into the EcoRV-KpnI site of the pTD1
vector. The resulting plasmid was designated as pTD1-ccdB.
The dsDNA containing the EcoICRI recognition sequence,
FLAG tag sequence, and stop codon was synthesized by
annealing the two oligonucleotides, EF-F and EF-R. The
annealed dsDNA was inserted into the KpnI-XbaI sites of the
pTD1-ccdB vector. The resulting plasmid was designated as
pTD1-ccdB-FLAG. To introduce a SgfI site immediately
downstream at the polyhedrin 50 untranslated region, a
translational enhancer sequence, PCR was performed using
the primers Flexi-Fw and Flexi-Rv and pTD1-ccdB-FLAG as
the template. The amplified DNA fragment was then treated
with T4 polynucleotide kinase. After the treatment, the DNA
fragment was self-ligated and used to transform E. coli DB3.1.
For in vivo mammalian expression, a pcDNA3 vector was
modified as follows. To introduce a SgfI site into the
multiple cloning site of the pcDNA3 vector, synthetic oligo-
nucleotides SgfI-F and SgfI-R were annealed and the
resulting dsDNA was inserted into the KpnI-EcoRI sites of
the pcDNA3 vector. The resulting plasmid was named as
pcDNA3-SgfI. The pTD1-FL vector was digested with SgfI
and EcoRI, and the resulting 0.5 kbp DNA fragment was
inserted into the SgfI-EcoRI sites of the pcDNA3-SgfI vector.
The generated plasmid was designated as the pcDNA3-ccdB.
The dsDNA containing an EcoRV recognition sequence,
FLAG tag sequence, and stop codon was synthesized using
oligonucleotides EV-F and EV-R. The annealed dsDNA was
inserted into the EcoRI-XhoI sites of the pcDNA3-ccdB
vector. The resulting plasmid was designated as pcDNA3-
FL, and used as the mammalian expression vector.
The DNA sequences of these constructs were confirmed
by the dideoxynucleotide chain termination method. The
primers used for the vector construction are listed in
Supporting Information Table S1.
2.5 Subcloning of target nucleotide sequences into
the expression vectors
pTD1 plasmids containing the cDNAs coding for the tGel-
solin fusion proteins with N-terminal ten amino acid
sequence of the ORF of the KOP cDNA clones at the
N-terminus were constructed as follows. The two oligonu-
cleotides, 50-ATGGGNNNNNNNNNNNNNNNNNNNNNN-
NNNGGTAC-30 (sense strand) and 50-CNNNNNNNNNNN-
NNNNNNNNNNNNNNCCCAT-30 (antisense strand), coding
for the N-terminal ten amino acid sequence of the ORF of the
KOP cDNA clones were annealed. The annealed dsDNAs
were individually ligated into EcoRV-KpnI sites of the pTD1-
tGelX vector. The resulting plasmids were designated as
pTD1-tGelX-NNNN, where NNNN indicates the number of
the product ID of the KOP cDNA clones.
For the construction of the pTD1 plasmids including full-
length KOP cDNA clones, the pTD1-FL vector was digested
with SgfI and EcoICRI and the KOP cDNA clones digested
with SgfI and PmeI were subcloned into the vector. For the
construction of pcDNA3 plasmids containing the full-length
KOP cDNA clones, pcDNA3-FL was treated with SgfI and
EcoRV and the KOP cDNA clones digested with SgfI and PmeI
were subcloned into the vector. The sequences of the oligo-
nucleotides used are listed in Supporting Information Table S1.
2.6 In vitro coupled transcription and translation
reaction
The pTD1 plasmids were extracted using PureYield Plasmid
Miniprep System (Promega). The plasmids were concen-
trated by ethanol precipitation and used as the template for
in vitro coupled transcription and translation. In vitrocoupled transcription and translation reactions were
performed using a TNT T7 Insect Cell Extract Protein
Expression System in the presence of [3H]leucine or
[3H]myristic acid. A mixture composed of 10.0 mL of TNTR
T7 ICE Master Mix, 1.0 mL of plasmid DNA template (1 mg),
and 1.5 mL of [3H]-labeled compound (2.0 mCi of [3H]-leucine
or 20.0 mCi of [3H]-myristic acid) were incubated at 291C for
4 h. After incubation, the samples were analyzed by SDS-
PAGE and fluorography.
2.7 In vitro transcription and translation reaction
PCR was performed using two primers pTD1-161–179 and
pTD1-845–827 (Supporting Information Table S1) and the
expression clones for cell-free protein synthesis as the
template. The amplified DNA fragments were used as the
template for in vitro transcription. The mRNAs were
synthesized at a 40 mL scale using a T7 RiboMAX Express
Large Scale RNA Production System (Promega) in accor-
dance with manufacturer’s instructions. After completion of
the reaction, 60mL of 25 mM EDTA was added to the in vitrotranscription mixture. The mixture was then used as the
template for in vitro translation. In vitro translation was
performed at a 1 mL scale using a Transdirect insect cell in
the presence of 50mM myristoyl-CoA in accordance with the
manufacturer’s instructions.
2.8 Affinity purification of the proteins synthesized
using the cell-free protein synthesis system
Affinity purification of the strep- and FLAG-tagged proteins
were performed as described previously [14, 20]. The affinity
1782 T. Suzuki et al. Proteomics 2010, 10, 1780–1793
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
column eluate was concentrated to �20 mL by ultrafiltration
(molecular weight cut off 5 10 kDa). The purity and the yield
of the expressed proteins were estimated by SDS-PAGE
using purified tGelsolin [14] as the standard. The concen-
trated samples were stored at �201C until use.
2.9 Transfection of COS-1 cells and determination of
N-myristoylated proteins
Simian-virus-40-transformed African green monkey kidney
cell line COS-1 was maintained in DMEM (Gibco BRL)
supplemented with 10% FCS (Gibco BRL). Cells (2� 105)
were plated onto 35-mm diameter dishes 1 day before
transfection. pcDNA3 constructs (2 mg) containing cDNAs
coding for FLAG-tagged proteins was used to transfect each
plate of COS-1 cells along with 4mL of Lipofectamine (2 mg/
mL, Gibco BRL) in 1 mL of serum-free medium. After
incubation for 5 h at 371C, the cells were re-fed with
serum-containing medium and incubated again at 371C
for 24 h. The cells were then washed twice with 1 mL of
serum-free DMEM and incubated for 6 h at 371C in 1 mL
of DMEM with 2% FCS containing [3H]myristic acid
(100 mCi/mL). Subsequently, the cells were washed three
times with Dulbecco’s PBS, collected with a cell scraper, and
then lyzed with 200mL of RIPA buffer (50 mM Tris-HCl
(pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, protease inhibitors) on ice for
20 min. After that the samples were analyzed by SDS-PAGE
and fluorography.
2.10 SDS-PAGE and fluorography
The samples were denatured by boiling for 3 min in SDS-
sample buffer followed by analysis by SDS-PAGE using a
12.5% gel. Thereafter, the gel was fixed and soaked in
Amplify for 30 min. The gel was dried under vacuum and
exposed to an X-ray film (Kodak) for an appropriate period.
2.11 MS
For the analyses of tGelsolin-fusion proteins, sample
preparation for MS was carried out as follows. Three
microliters of affinity-purified proteins (typically about
1–2 mg) were incubated at 251C for 3 h in 10 mL of a solution
of 50 mM Tris-HCl, pH 7.5, containing 20 mU Factor Xa.
After the incubation, 40mL of 70% v/v ACN containing
0.1% v/v TFA was added to the mixture and these were used
as the samples. In the case of in vitro synthesized full-length
cDNA products, the affinity-purified proteins (about
0.5–1.0 mg) were separated by SDS-PAGE and then stained
with Coomassie brilliant blue R-250. Protein bands were
reduced and alkylated with iodoacetamide. The gel piece was
washed with 100 mM ammonium bicarbonate solution,
shrunk by dehydration in ACN, and dried in a vacuum
centrifuge. The gel piece was then rehydrated with 2 mL of
50 mM ammonium bicarbonate solution containing
0.2% w/v n-octyl-b-D-glucopyranoside (Wako Pure Chemical
Industries) and 30 ng of trypsin (Promega). After incubation
for 5 min on ice, 10 mL of 50 mM ammonium bicarbonate
solution was added to the gel piece and incubated overnight
at 371C. The tryptic digests were extracted twice using
60% v/v ACN containing 0.1% v/v TFA. The extracted solu-
tion was dried, then dissolved in 10 mL of 50% v/v ACN
containing 0.1% v/v TFA. The sample (0.5–1.0 mL) was
mixed with 0.3 mL of CHCA or DHB solution (5 mg/mL in
50% v/v ACN containing 0.1% v/v TFA). The MS spectra
and MS/MS spectra were acquired in reflectron positive ion
mode with an AXIMA-CFR-plus MALDI-TOF MS instru-
ment and an AXIMA-QIT MALDI-QIT-TOF hybrid mass
spectrometer (Shimadzu/Kratos, Manchester, UK), respec-
tively.
3 Results
3.1 Establishment of a strategy for screening cDNAs
coding for N-myristoylated proteins from cDNA
resources
To establish a strategy for the comprehensive identification of
human N-myristoylated proteins, the susceptibility of human
cDNA clones to protein N-myristoylation was evaluated using
fusion proteins in which the N-terminal ten amino acid
residues were fused to an epitope-tagged model protein,
tGelsolin. For this strategy, an expression vector, pTD1-tGelX,
was constructed to express tGelsolin-fusion proteins using an
insect cell-free protein synthesis system. In this vector, the
factor Xa recognition sequence was inserted between the site
for insertion of the coding sequence for the N-terminal ten
amino acid residues of the target cDNA and the tGelsolin
sequence starting at His-11 (Fig. 1A). Therefore, the
N-terminal ten amino acid residues can be obtained by clea-
vage of the in vitro-translated products with factor Xa.
To evaluate the performance of the vector in the identifi-
cation of N-myristoylated gene products, pTD1-tGelX-tGel-
solin and pTD1-tGelX-1301, in which the N-terminal ten
amino acid residues of known N-myristoylated proteins
tGelsolin and G protein a subunit (Gai1), respectively, were
subcloned into the pTD1-tGelX vector were constructed
(Fig. 1B), and metabolic labeling experiments were
performed. These proteins were synthesized using an in vitrocoupled transcription and translation system in the presence
of [3H] leucine or [3H] myristic acid, and then analyzed by
SDS-PAGE and fluorography. Luciferase, a non-myristoylated
control protein, and two tGelsolin-fusion proteins were effi-
ciently synthesized in the cell-free protein synthesis system,
as shown by the incorporation of [3H] leucine (Fig. 1C). On
the other hand, incorporation of [3H] myristic acid was only
observed in tGelsolin-fusion proteins. To determine whether
Proteomics 2010, 10, 1780–1793 1783
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
these tGelsolin-fusion proteins were myristoylated at the
N-terminus, factor Xa digests of the in vitro synthesized and
affinity-purified proteins were analyzed by MALDI-TOF MS.
Each peak with the highest intensity in the MS spectra
produced from these samples was consistent with the
theoretical m/z value of N-myristoylated factor Xa fragment
(Figs. 2A and C). These ions were respectively identified as
N-myristoylated factor Xa fragment by MS/MS analysis
(Figs. 2B and D). These results suggested that both tGelsolin-
fusion proteins synthesized in vitro were efficiently N-myris-
toylated, and these strategies to identify the N-myristoylated
gene products were effective.
3.2 Identification of N-myristoylated proteins from
141 human cDNA clones with N-terminal Met-
Gly motifs
One-hundred-and-forty-one cDNA clones with N-terminal
Met-Gly motifs were selected as potential candidates from
1929 KOP human cDNA clones, and their susceptibility to
protein N-myristoylation was evaluated by the strategy
described above. The samples analyzed are listed in
Supporting Information Table S2. As shown in the upper
panels of Fig. 3, all of the cDNA clones were expressed as
determined by the incorporation of [3H] leucine. In contrast,
the incorporation of [3H] myristic acid was observed for 34
out of 141 clones, as shown in the middle panels of Fig. 3.
The results of the prediction for protein N-myristoylation
using two prediction programs, the MYR Predictor and
Myristoylator are shown in the lower panels of Fig. 3. Many
of the cDNA clones predicted to be ‘‘reliable’’ or ‘‘high
confidence’’ were found to be N-myristoylated. However, the
reliability of both programs was not complete and some
cDNA clones predicted as non-preferable substrate for
NMTs were effectively N-myristoylated (e.g. Lanes 55 and
101 in Fig. 3). To confirm the N-myristoylation of the gene
products of 34 cDNA clones found to be N-myristoylated by
the [3H] myristic acid incorporation, in vitro-synthesized
tGelsolin-fusion proteins were affinity-purified and digested
using factor Xa, then the liberated N-terminal peptide
fragments were analyzed by MALDI-TOF MS. Because an
N-terminal Met-Gly sequence can serve as a substrate for
methionineaminopeptidases and NMTs/N-acetyltransferase
A, this study searched for ions corresponding to the free
amino terminus (10), N-acetylated (142.0), or N-myristoy-
lated (1210.2) factor Xa peptide fragment in the MS spectra.
Theoretical and observed m/z values are summarized in
Supporting Information Table S3. A peak corresponding to
the N-myristoylated factor Xa digest fragment was detected
in 28 out of 34 tGelsolin-fusion proteins (Supporting
Information Table S3 and Fig. 4). Interestingly, both ions
corresponding to the N-myristoylated and N-acetylated
factor Xa fragment were clearly detected in the MS spectra
produced from gene products of tGelX-1517 and tGelX-1828
(Supporting Information Table S3 and Fig. 4). These results
support the previous observation that the N-terminal
sequence requirements for the two N-terminal protein
modifications partially overlap [21]. N-myristoylated peptide
ions were not observed in the MS spectra produced from
A B
C
tGelX-tGelsolin
tGelX-1301
-COOHstrep
NH2-
-COOHstrep
NH2-
[3H]-leucine [3H]-myristic acid
-Pla
smid
Lu
cife
rase
tGel
X-t
Gel
solin
tGel
X-1
301
-Pla
smid
Lu
cife
rase
tGel
X-t
Gel
solin
tGel
X-1
301
60
45
kDa
MGLGLSYLSS
-COOH
MGXXXXXXXX
FXC NNNN (cDNA clones)-
(Factor Xa site)
IEGR
tGelX-NNNN
+
+
tGelsolinNH2
NH2
NH2
- -COOH
strep11
MGXXXXXXXX
-COOHstrep
IEGR
-
MGCTLSAEDK
tGelsolin11
Figure 1. Detection of protein
N-myristoylation of the
tGelX-tGelsolin and tGelX-
1301 by metabolic labeling in
an insect cell-free protein
synthesis system. (A) Sche-
matic representation of the
generation of tGelX-NNNN
with N-terminal 10 amino
acid sequences of the ORFs
of KOP cDNA clones at its N-
terminus. (B) Schematic
structure of tGelX-tGelsolin
and tGelX-1301. (C) tGelX-
tGelsolin and tGelX-1301
were synthesized using an in
vitro coupled transcription
and translation using a TNT
T7 Insect Cell Extract Protein
Expression System in the
presence of [3H]leucine or
[3H]myristic acid. The labeled
translation products were
analyzed by SDS-PAGE and
fluorography.
1784 T. Suzuki et al. Proteomics 2010, 10, 1780–1793
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
factor Xa digests of six tGelsolin-fusion proteins (Supporting
Information Table S3 and Fig. 4). In five tGelsolin-fusion
proteins among these six proteins, the efficiency of [3H]
myristic acid incorporation was low as compared with that
of [3H] leucine (Fig. 3). However, only N-acetylated peptide
ions were detected in the MS spectrum produced from
tGelX-0782 protein despite the result that a significant
incorporation of [3H] myristic acid was observed in this gene
product (Fig. 3, lane 55). The reason why a difference was
generated between these two assay systems was not clear.
3.3 Analyses of protein N-myristoylation occurs on
in vitro synthesized full-length cDNA products
To determine whether the experimental results obtained
with the tGelsolin fusion proteins were consistent with
those with the full-length cDNA products, metabolic label-
ing experiments were performed using 29 full-length
cDNAs in which efficient incorporation of [3H] myristic acid
was observed with the tGelsolin fusion proteins. The
samples analyzed are listed in Supporting Information
Table S4. As shown in the lower panels of Fig. 5, all the
cDNA clones were expressed as determined by the incor-
poration of [3H] leucine. In addition to the protein band with
an expected molecular mass, some protein bands with lower
molecular mass were observed with several cDNA clones,
such as FXC00244 (lane 2), 00557 (lane 5), 00905 (lane 12),
00950 (lane 13), and 01173 (lane 16). The results of [3H]
myristic acid incorporation revealed that all the products of
the 29 cDNA clones were N-myristoylated, as shown in the
upper panels of Fig. 5.
MS analyses were performed on in vitro synthesized gene
products of FXC00876 and FXC01873. The in vitro synthe-
sized and affinity-purified gene product of FXC00876 was
detected as a main band with an apparent molecular mass of
48 kDa by SDS-PAGE analysis (Fig. 6A). The protein band
was reduced and S-alkylated and then digested with trypsin.
The tryptic digests were analyzed by MALDI-TOF MS. MS
spectrum of this sample revealed that the protein band was
the gene product of FXC00876 (Fig. 6B). A peak equivalent
to the N-myristoylated tryptic peptide was observed at m/z643.26 (Fig. 6B). This ion was identified as the N-myris-
toylated tryptic fragment of the FXC00876 product,
N-myristoyl-Gly-Asn-Ser-Arg, by MS/MS analysis (Fig. 6C).
Using a similar strategy, it was demonstrated that the in
y10y11y13y14MS MS/MS
y13MS
X5
Myristoyl-
Precursor ion80
100%Int.
1719
.94
%Int. X5%Int.
1719
.94
50
100
b4
y14y13
y10
y11
m/z 1719.94
20
40
601.
93
3.90
b4
b5-H2O
b6-H2Oy14
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m/z
1.93
3.90
0500 1000 1500
b7
01000 1200 1400 1600 1800 2000
169
1883
tGelX-tGelsolin
b7
169
1883
Myristoyl
y6y7y8y10
%Int
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%Int
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MS
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-
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%Int. y7
G S E D K GAC T L -
%Int.
1746
.90
y6 y8
y10
Precursor ion
m/z1746.90
20
40
60
1075
.47
1718
.91 50
y6 y8
y10
Precursor ion
m/z1746.90
1075
.47
1718
.91
500 1000 1500m/z m/z
m/z m/z
1000 1200 1400 1600 1800
tGelX-1301
00
G L G L S Y L S S G T I E G R
T I E G R
b3 b4 b5 b6 b7
2000
b3
A B
C DFigure 2. Detection of protein
N-myristoylation of the tGelX-
tGelsolin and tGelX-1301 by
MS analysis. MALDI-mass
(A, C) and MALDI-MS/MS
(B, D) spectra of the N-terminal
peptides from the tGelX-tGel-
solin (A, B) and tGelX-1301
(C, D). MS/MS analyses were
performed for the peaks at m/z
1719.94 observed for the
tGelX-tGelsolin (A) and at m/z
1746.90 for the tGelX-1301 (B).
The observed fragment ions
were indicated in the sequen-
ces shown.
Proteomics 2010, 10, 1780–1793 1785
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
vitro synthesized gene product of FXC01873 was N-myris-
toylated (data not shown).
3.4 Analyses of protein N-myristoylation occurring
on full-length cDNA products expressed in
transfected mammalian cells
To determine whether the results obtained by the in vitrometabolic labeling in insect cell-free protein synthesis
system reflect the in vivo behavior of the cDNA products,
metabolic labeling in transfected COS-1 cells was performed
using the 29 full-length cDNAs analyzed in the insect
cell-free protein synthesis system. The samples analyzed
are listed in Supporting Information Table S4. In contrast
to the insect cell-free protein synthesis system, not all the
cDNA clones were expressed in transfected COS-1 cells.
Protein synthesis was not observed for seven cDNA
clones, as determined by the Western blotting analysis
(indicated by arrows in the lower panels of Fig. 7). To
determine the effect of the molecular size of the synthesized
protein on the protein expression in COS-1 cells, samples of
the protein expression assays in two expression systems
were aligned in accordance with the theoretical molecular
mass of the gene products. The samples analyzed are
listed in Supporting Information Table S5. As shown in
the lower panels of Fig. 8, a relatively constant level of
protein expression was observed in the insect cell-free
protein synthesis system. In contrast, the level of protein
expression in transfected cells was strongly affected by the
molecular size of the gene products, and six cDNA clones
with products with molecular weights higher than 110 kDa
were not expressed (indicated by arrows in the upper panels
of Fig. 8). As for protein N-myristoylation, obvious incor-
poration of [3H] myristic acid was observed on 20 cDNA
clones out of 22 expressed clones, as indicated by asterisks
in the upper panels of Fig. 7. Thus, the results obtained by
in vitro metabolic labeling with [3H] myristic acid in an
Sample No.
Sample No.
Sample No.
[3H]-leucine
[3H]-myristicacid
[3H]-leucine
[3H]-myristicacid
[3H]-leucine
[3H]-myristicacid
MYRMyristoylator
Myristoylation
MYRMyristoylator
Myristoylation
MYRMyristoylator
Myristoylation
Sample No.
Sample No.
Sample No.
[3H]-leucine
[3H]-myristicacid
[3H]-leucine
[3H]-myristicacid
[3H]-leucine
[3H]-myristicacid
Figure 3. Identification of N-myristoylated proteins from 141 human cDNA clones with N-terminal Met-Gly motifs. One-hundred-and-forty-
one tGelsolin fusion proteins with N-terminal Met-Gly motifs derived from KOP human cDNA clones were synthesized using an in vitro
coupled transcription and translation with a TNT T7 Insect Cell Extract Protein Expression System in the presence of [3H]leucine
or [3H]myristic acid. The labeled translation products were analyzed by SDS-PAGE and fluorography (upper and middle panels).
The results of the protein N-myristoylation assay are summarized in the lower panels, 11: efficiently N-miristoylated, 1: weakly
N-myristoylated. The results of the prediction for protein N-myristoylation using two prediction programs, the MYR Predictor and
Myristoylator, are shown in the lower panels. R, T, and blank represents ‘‘Reliable’’, ‘‘Twilight zone’’, and ‘‘No’’ prediction in the MYR
Predictor, respectively. H, M, L, and blank represents ‘‘High confidence’’, ‘‘Medium confidence’’, ‘‘Low confidence’’, and ‘‘No’’ predictions
in the Myristoylator program, respectively.
1786 T. Suzuki et al. Proteomics 2010, 10, 1780–1793
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
insect cell-free protein synthesis system were highly
consistent with the susceptibility of the cDNA product to
protein N-myristoylation in intact mammalian cells.
3.5 Eighteen out of 27 cDNA products found to
be N-myristoylated are novel N-myristoylated
proteins that have not been reported to be
N-myristoylated previously
The characteristics of the gene products of 27 cDNA clones
found to be N-myristoylated in this study are summarized in
Table 1 and Supporting Information Table S6 [22–35].
Database searches with these cDNA ;clones suggested that
nine proteins are previously reported N-myristoylated
proteins and 18 proteins are novel. Thirteen proteins out of
these 18 novel N-myristoylated proteins were functionally
unknown proteins. The rest of the proteins were key players
in various cellular signal transduction pathways, such as
phosphatase, ubiquitin E3-ligase, cytoskeletal regulatory
protein, apoptosis-related protein, and amino acid trans-
porter. Therefore, it is quite probable that the N-myristoy-
lation of these proteins plays critical roles in the structure
and function of these proteins.
Free Ac0 42
Myr210.2
Free Ac0 42
Myr210.2
Sample No. Sample No.
14
9
80
81
83
3
32
16
× 50
84
× 1097
2922
50
44
105
101
10655
56
59
106
113
55
114
126
127
133
60
62
61
64
65
75
134
137
13875
Free Ac0 42
Myr210.2
Free Ac0 42
Myr210.2
0
100
1500 1550 1600 1650 1700
0
100
1550 1600 1650 1700 1750
0
100
1700 1750 1800 1850
0
100
1600 1650 1700 1750 1800
0
100
1650 1700 1750 1800 1850
0
100
1500 1550 1600 1650
0
100
1450 1500 1550 1600 1650
1650 1700
0
100
1500 1550 1600 1650 1700
1600 1650 1700 1750 1800
100 100
0
100
1600 1650 1700 17500
100
1550 1600 1650 1700 1750
0
100
1500 1550 1600 1650 17000
100
1650 1700 1750 1800 1850
0
100
1400 1450 1500 1550
01600 1650 1700 1750 1800
01550 1600 1650 1700 1750
0
100
1500 1550 1600 1650 1700
100100
0
100
1600 1650 1700 1750 1800
0
100
1500 1550 1600 1650
01500 1550 1600 1650
0
100
1550 1600 1650 1700
01500 1550 1600 1650 1700
0
100
1650 1700 1750 1800 1850
0
100
1650 1700 1750 1800
0
100
1500 1550 1600 1650 1700
0
100
1450 1500 1550 1600 16500
100
1550 1600 1650 1700 1750
0
100
1550 1600 1650 1700
0
100
1700 1750 1800 1850 1900
0
100
1500 1550 1600 1650 1700
1650 1700 1750 1800
100
0
100
1550 1600 1650 1700 1750
0
100
1600 1650 1700 1750 1800
1550 1600 1650 1700
0
100
1650 1700 1750 1800100
01500 1550 1600 1650 1700
01500 1550 1600 1650
m/zm/z
in fig 3in fig 3
Figure 4. MALDI-MS of the
factor Xa digests of 34 tGelso-
lin-fusion proteins. Thirty-
four purified tGelsolin-fusion
proteins found to be N-myris-
toylated by [3H]myristic acid
incorporation were digested
with factor Xa, and then the
liberated N-terminal peptide
fragments were analyzed by
MALDI-TOF MS. The ions
corresponding to the free
amino terminus (10), N-acety-
lated (142.0), or N-myristoy-
lated (1210.2) fragments are
indicated by arrows.
Proteomics 2010, 10, 1780–1793 1787
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
F XC NNNN
(M)G F L AGpTD1-FLAG-FXCNNNN
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
10585
S ample No.
50
37
29
[3H] Myr
[3H] Leu
20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
10585
S ample No.
50
37
29
20
kDa
Figure 5. Detection of the
protein N-myristoylation of the
gene products of 29 full-length
cDNAs by the metabolic label-
ing in in vitro coupled trans-
cription and translation. The
gene products of 29 full-length
cDNAs in which efficient incor-
poration of [3H] myristic acid
was observed with the tGelso-
lin fusion proteins were
synthesized by in vitro coupled
transcription and translation
using TNT T7 Insect Cell Extract
Protein Expression System in
the presence of [3H]leucine or
[3H]myristic acid. The labeled
translation products were
analyzed by SDS-PAGE and
fluorography.
%Int.
F XC 00876-F L AGF XC 00876-F L AG70
80
90
100 MS
105
250
160
30
40
50
60
m/z 643.26
75
5050
0
10
20
500 1000 1500 2000 2500 3000
/
m/z
35
30
100
%Int.
Prec urs or ion
y2y3 y2 y1
MS/MS
(kDa)
60
70
80
90 G N S Rmyris toyl
b1 b2 b3
P rec urs orm/ z 643.26y1 b1
/
20
30
40
50y3
b2
b3
0
10
100 150 200 250 300 350 400 450 500 550 600 650m/z
00876- F LAG
A
B
C
Figure 6. Detection of protein
N-myristoylation of the in vitro
synthesized gene product of
FXC00876 by MS analysis. The
purified gene product of
FXC00876 (1 mg) was separated by
electrophoresis in an SDS-poly-
acrylamide gel (10%) (A). MALDI-
MS of the tryptic peptides from
the gene product of FXC00876 (B).
The peaks of the tryptic peptides
derived from the gene product
of FXC00876 are indicated by
asterisks. MS/MS analysis was
performed for the peak at m/z
643.26 (C). The observed fragment
ions are indicated in the sequence
shown.
1788 T. Suzuki et al. Proteomics 2010, 10, 1780–1793
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
4 Discussion
The identification of N-myristoylated proteins expressed in
cells or tissues was usually performed by MS analysis. The
protein samples were extracted from cells or tissues and
purified by 2-D PAGE or LC, digested with proteases, and
then applied for MS analysis. Protein N-myristoylation was
identified from the difference between the detected mole-
cular mass and the theoretical molecular mass of the
peptide. In this case, only the major N-myristoylated
proteins included in the cells or tissues could be detected
because only the limited sets of genes were expressed in the
particular cells or tissues.
Another strategy to detect protein N-myristoylation is
metabolic labeling utilizing the transfected mammalian
cells [36, 37]. For this analysis, the cDNA coding for the
query protein was transfected to the mammalian cells. The
transfected cells were incubated in the presence of radio-
isotope ([3H] or [14C])-labeled myristic acid, and protein
N-myristoylation was detected by the specific incorporation
of RI-labeled myristic acid into the expressed protein. This
method was useful in determining the susceptibility of the
particular gene product to protein N-myristoylation.
However, it usually takes several weeks to obtain these
results because this strategy utilizes the cultured mamma-
lian cells to express the protein and the efficiency of the
incorporation of RI-labeled myristic acid into the target
protein is not high. In addition, not all the cDNAs could be
expressed in the intact cells. For example, it is known that
the expression levels of cytotoxic proteins, multi-spanning
membrane proteins, or proteins with large molecular size
are very low in transfected cells as compared with those of
non-cytotoxic soluble proteins with small molecular size.
Therefore, this strategy is not applicable to the high-
throughput proteomic analysis of the N-myristoylated
proteins.
In addition to the transfected mammalian cells,
metabolic labeling of N-myristoylated proteins could be
performed in a cell-free protein synthesis system [21, 38].
The cell-free protein synthesis system is a cell or
tissue extract having an ability to synthesize proteins. In
this system, any desired protein can be produced by
the addition of the mRNA coding for the target protein.
It was found that these cell-free protein systems
have activities for various co- and post-translational
modifications of proteins. Protein N-myristoylation in
intact cells is not a single enzymatic reaction catalyzed by
NMT. This modification appears to be a highly regulated
reaction involving the coordinated participation of the
protein synthesis machinery (ribosomes) and several
different enzymes/proteins, such as N-methionyl-amino-
peptidase, fatty acid synthase, long chain acyl-CoA
synthase, acyl-CoA-binding proteins, etc. In addition, other
cotranslational protein modification such as protein
N-acetylation also affected the protein N-myristoylation
reaction. Because the cell-free protein synthesis system
using rabbit reticulocyte lysate contains all the com-
ponents involved in cotranslational protein N-myristoyla-
W.B .
p
F XC NNNN
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
pcDNA3-F L AG -F XC NNNN(M)G F L AG
16 17 18 19 20 21 22 23 24 25 26 27 28 29 S ample No.
10585
50
37
[3H] Myr
37
29
20
16 17 18 19 20 21 22 23 24 25 26 27 28 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15S ample No.
10585
50
37
29
20
kDa
Figure 7. Detection of protein
N-myristoylation of the gene
products of 29 full-length
cDNAs expressed in COS-1
cells by metabolic labeling. The
29 full-length cDNAs analyzed
in Fig. 5 were transfected into
COS-1 cells, and metabolic
labeling with [3H]myristic acid
was performed. The labeled
translation products were
separated by SDS-PAGE and
then analyzed by Western
blotting using anti-FLAG anti-
body or fluorography. The
samples in which protein
N-myristoylation was observed
are indicated by asterisks in the
upper panels. The samples that
showed no protein expression
are indicated by arrows in the
lower panels.
Proteomics 2010, 10, 1780–1793 1789
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
tion and N-acetylation, the use of this system to study
N-terminal cotranslational protein modifications seemed
to be appropriate [21]. In fact, using this assay system,
we have previously demonstrated that the amino acid
residue at position 3 in an N-myristoylation consensus
motif, Met-Gly-X-X-X-Ser-X-X-X, strongly affected the
susceptibility of protein to two cotranslational protein
modifications, N-myristoylation and N-acetylation [38].
Thus, metabolic labeling in the cell-free protein synthesis
system using rabbit reticulocyte lysate is a useful strategy to
detect protein N-myristoylation.
In addition to the rabbit reticulocyte lysate system,
we have recently demonstrated that a newly developed cell-
free protein synthesis system (Transdirect insect cell)
derived from insect cells [12] can be used for in vitro meta-
bolic labeling assays, and that the metabolic labeling in
this insect cell-free system is a simple and sensitive method
to detect protein N-myristoylation [13]. Analysis of the
N-myristoylation of a series of model proteins with mutated
N-myristoylation motifs revealed that the amino acid
sequence requirements for the N-myristoylation reaction in
this system are quite similar to those observed in the rabbit
reticulocyte lysate system [13]. The advantage of the use of
this insect cell-free system over the use of the rabbit reti-
culocyte lysate system is that the MS analysis can be
performed on the proteins expressed with this cell-free
protein synthesis system [14, 20]. In fact, we have recently
demonstrated that the MS analyses of the in vitro synthe-
sized proteins provide with detailed structural informations
about the N-myristoylated protein, such as the exact location
of the modification and the structure of the attached func-
tional group [14].
To perform a genome-wide comprehensive analysis of
human N-myristoylated proteins using the cell-free protein
synthesis system, the presence of well-characterized cDNA
resources that cover all human cDNAs is indispensable.
During the last decade, several large-scale government and
academic programs have collected and characterized human
cDNA clones [39]. The largest collections comprise cDNAs
with full-length protein coding sequences. The major
programs include the NEDO(FLJ) Project, the KOP, the
Mammalian Gene Collection (MGC), the German Human
cDNA Project, the Harvard Institute of Proteomics (HIP),
and the ORFeome program of the Center for Cancer Systems
Biology (CCSB) at the Dana-Farber Cancer Institute [39]. In
this study, KOP human cDNA clones were chosen as a model
cDNA resource to establish a strategy for the comprehensive
identification of human N-myristoylated proteins [15]. The
KOP human cDNA clones are a set of fully sequenced and
well-characterized collection of relatively long full-length
protein coding sequences and is composed of 1929 human
cDNAs (This collection has recently been expanded to �6000
clones). The susceptibility of KOP human cDNA clones to
protein N-myristoylation was evaluated by metabolic labeling
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
S ample No.
10585
10585
(W.B .)C OS -1 cells
50
37
29(W.B .)C OS -1 cells
50
37
29
2020
10585
C ell-free s ys tem
50
37
29([3H] L eu)
20
kDa
20
S ample No.
Figure 8. Effect of the molecular size of synthesized protein on protein expression in COS-1 cells. To determine the effect of the molecular
size of the synthesized protein on their protein expression in COS-1 cells, the samples of two protein expression assays performed in
Figs. 5 and 7 were aligned in accordance with the theoretical molecular masses of the gene products. The gene products of the cDNA
clones expressed in the transfected COS-1 cells were analyzed by Western blotting using anti-FLAG antibody. The samples that showed
no protein expression are indicated by arrows (upper panels). The gene products synthesized by in vitro coupled transcription and
translation using a TNT T7 Insect Cell Extract Protein Expression System were analyzed by SDS-PAGE and fluorography (lower panels).
1790 T. Suzuki et al. Proteomics 2010, 10, 1780–1793
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
and MS analyses of proteins expressed using a insect cell-free
protein synthesis system.
Because the N-terminal Met-Gly sequence is critical and
the N-terminal ten amino acids are sufficient to direct protein
N-myristoylation, cDNA clones with a Met-Gly sequence at
their N-termini were selected from the KOP human cDNA
clones, and the nucleotide sequences encoding the N-term-
inal ten amino acids were fused to the 50-end of a cDNA
coding for the model protein, tGelsolin. In this fusion
construct, the factor Xa recognition sequence was inserted
between the N-terminal ten amino acid residues of the
query protein and the tGelsolin sequence. Then the
N-myristoylation of the fusion protein was evaluated by
metabolic labeling and MS analysis of the factor Xa digests of
the fusion proteins expressed with the insect cell-free protein
synthesis system. Using this strategy, we could detect protein
N-myristoylation of any protein without obtaining full-length
cDNA clones. As a result of testing 141 tGelsolin fusion
proteins with N-terminal Met-Gly motifs selected from 1929
KOP human cDNA clones, 29 fusion proteins were found to
be effectively N-myristoylated. The results obtained by meta-
bolic labeling experiments were highly consistent with those
obtained using MS analysis. The metabolic labeling experi-
ments in an insect cell-free protein synthesis system using
full-length cDNAs revealed that all the 29 cDNA clones were
expressed and all the products were N-myristoylated. These
results clearly indicated that the use of the tGelsolin fusion
proteins with the N-terminal ten amino acid residues of the
target proteins was quite useful in searching for N-myris-
toylated proteins present in cDNA resources. When these 29
cDNAs were transfected into COS-1 cells, seven cDNA clones
were not expressed. The gene products of six out of these
seven cDNA clones had theoretical molecular masses larger
than 110 kDa. The product of one cDNA (FXC00782) other
than these six cDNAs has been reported to be a multi-span-
ning transmembrane protein with eleven transmembrane
domains [25]. These results suggested that the insect cell-free
protein synthesis system has an ability to produce proteins
that are difficult to express in transfected mammalian cells.
The [3H] myristic acid labeling of 22 cDNA clones expressed
in COS-1 cells revealed that the products of 20 cDNA clones
were N-myristoylated. From these observations, it was
concluded that the gene products of 27 cDNAs out of 1929
KOP cDNA clones are N-myristoylated.
Database searches with these 27 cDNA clones revealed
that 9 out of 27 proteins are known N-myristoylated proteins
Table 1. The characteristics of the gene products of the 27 cDNA clones found to be N-myristoylated in this study
FXC No. MW (Da) Myristoylation Predictions Symbol Protein function Reference
1 FXC00128 38 767 Novel R/H FAM131B Unknown2 FXC00244 108 379 Novel N/H KIAA1522 Unknown3 FXC00316 191 600 Known R/H AKAP12 Akinase anchoring protein [22]4 FXC00401 63 142 Known N/H RFTN1 Raft-linking protein [23]5 FXC00557 60 750 Novel R/H MGRN1 E3 ubiquitin ligase [24]6 FXC00721 45 189 Novel R/H KIAA1045 Unknown7 FXC00782 50 492 Novel N/N SERINC1 Serine incorporator [25]8 FXC00792 96 776 Known R/H KIAA1274 Negative regulator of
insulin signaling[26]
9 FXC00876 51 044 Novel T/H KIAA1609 Unknown10 FXC00892 62 426 Novel T/L EEPD1 Unknown11 FXC00896 47 737 Novel T/H KIAA1715 Unknown12 FXC00905 53 940 Novel T/N DIXDC1 F-actin-binding protein [27]13 FXC00950 73 575 Novel R/H RNF157 Unknown14 FXC01083 330 999 Novel R/H ZZEF1 Unknown15 FXC01163 122 013 Novel R/H ANKIB1 Unknown16 FXC01173 126 361 Novel R/M BTBD7 Unknown17 FXC01184 124 213 Novel T/H FMNL2 Regulator of actin cytoskeleton [28]18 FXC01201 153 095 Known T/H PIK3R4 Adapter protein for
phosphatidylinositol 3-kinase[29]
19 FXC01301 40 359 Known R/H GNAI1 Trimeric G-protein a subunit [30]20 FXC01376 105 711 Known N/N PDE2A Phosphodiesterase [31]21 FXC01406 124 948 Known T/N ABL1 Non-receptor tyrosine kinase [32]22 FXC01433 40 525 Novel R/H AIFM2 Apoptosis-inducing protein [33]23 FXC01513 17 443 Novel N/M RNF11 Unknown24 FXC01517 20 696 Known T/H ARF1 Arf family GTPase [34]25 FXC01701 116 864 Novel N/M FMNL3 Unknown26 FXC01828 42 446 Novel N/H PPM1A Unknown27 FXC01873 34 058 Known N/H CDK5R1 Cyclin-dependent kinase
regulatory subunit[35]
For prediction of protein N-myristoylation, the same abbreviations as those in Fig. 3 were used.
Proteomics 2010, 10, 1780–1793 1791
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
that play critical roles in cellular signal transduction path-
ways, such as Gai1, ADP-ribosylation factor (Arf1),
phospho-diesterase (PDE2A), and non-receptor tyrosine
kinase (Abl1). The role of protein N-myristoylation in the
function of these proteins has been well characterized [30,
31, 32, 34]. Thirteen proteins out of the 18 novel N-myris-
toylated proteins were functionally unknown proteins. The
rest of the novel N-myristoylated proteins were key players
in various cellular signal transduction pathways, such as
phosphatase, ubiquitin E3-ligase, cytoskeletal regulatory
protein, apoptosis-related protein, and amino acid trans-
porter. Therefore, it is quite probable that protein N-myris-
toylation plays critical roles in the function of these proteins.
A bioinformatic approach is a powerful strategy to perform
the comprehensive identification of N-myristoylated proteins
[10, 40–42]. Two prediction programs, the MYR Predictor and
Myristoylator are available as public WWW-servers [10, 43]. To
evaluate the reliability of these prediction programs, experi-
mental results obtained in this study were compared with the
results of the prediction obtained by the two prediction
programs. As listed in the lower panels of Fig. 3, different sets
of samples in 141 candidate proteins having N-terminal Met-
Gly sequences present among 1929 KOP human cDNA clones
were predicted to be N-myristoylated by the two prediction
programs. The MYR Predictor predicted 22 proteins, whereas
the Myristoylator predicted 32 proteins. In the case of the MYR
Predictor, 19 proteins out of 22 predicted samples were found
to be N-myristoylated, as summarized in Table 1. In contrast,
23 proteins out of 32 predicted samples were N-myristoylated
in the case of the Myristoylator. Thus, the reliability of the
MYR Predictor was high; however, there existed a considerable
number of false-negative predictions. The Myristoylator
predicted many more N-myristoylated proteins, but there
existed a lot of false-positive predictions. When the results
obtained by these two prediction programs were combined, the
number of the correct prediction increased to 25. Even in this
case, two N-myristoylated proteins (FXC00782, FXC01376)
were not predicted to be N-myristoylated. These results clearly
indicated that the experimental approach as proposed in this
study is indispensable to detect the complete set of
N-myristoylated proteins present in cDNA resources. In this
case, however, it should be noted that this method is useful to
screen human N-myristoylated proteins, but the actual modi-
fication status should be confirmed using human-originated
materials such as human cell-lines and tissues.
From the bioinformatic analysis, it was proposed that
0.5% of all proteins encoded in the human genome are
N-myristoylated [10]. However, in this study, 27 N-myris-
toylated proteins were identified among 1929 human cDNA
clones. This corresponds to 1.4% of the total number of
cDNA clones tested. The number of human proteins having
N-terminal Met-Gly sequence in all the human proteins
listed in Swiss-Prot protein knowledgebase (20 039) is 1569.
This corresponds to 7.8% of the total proteins. In the case of
KOP cDNA clones used in this study, the ratio is 7.3% (141/
1929). Therefore, it seems likely that there is no bias of the
protein content in KOP cDNA clones against the whole
protein-coding sequences.
Thus, it is expected that a lot of novel N-myristoylated
proteins that have not been predicted to be N-myristoylated
will be identified when the same approach is performed on
larger cDNA resources that cover many of the human
genome cDNAs.
Part of this work was supported by a Grant-in-Aid forScientific Research (No. 20580099) from the Ministry ofEducation, Science, and Culture of Japan.
The authors have declared no conflict of interest.
5 References
[1] Resh, M. D., Trafficking and signaling by fatty-acylated and
prenylated proteins. Nat. Chem. Biol. 2006, 2, 584–590.
[2] Spiegel, A. M., Backlund, P. S., Butrynski, J. E., Jones, T. L. Z.
et al., The G protein connection: molecular basis of membrane
association. Trends Biochem. Sci. 1991, 16, 338–341.
[3] Boutin, J. A., Myristoylation. Cell. Signal. 1997, 9, 15–35.
[4] Resh, M. D., Fatty acylation of proteins: new insights into
membrane targeting of myristoylated and palmitoylated
proteins. Biochim. Biophys. Acta 1999, 1451, 1–16.
[5] Farazi, T. A., Waksman, G., Gordon, J. I., The biology and
enzymology of protein N-myristoylation. J. Biol. Chem.
2001, 276, 39501–39504.
[6] Dyda, F., Klein, D. C., Hickman, A. B., GCN5-related N-acet-
yltranseferases: a structural overview. Annu. Rev. Biophys.
Biomol. Struct. 2000, 29, 81–103.
[7] Towler, D. A., Gordon, J. I., Adams, S. P., Glaser, L., The
biology and enzymology of eukaryotic protein acylation.
Annu. Rev. Biochem. 1988, 57, 69–99.
[8] Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S.
et al., Myristoyl CoA: protein N-myristoyltransferase activ-
ities from rat liver and yeast possess overlapping yet
distinct peptide substrate specificities. J. Biol. Chem. 1988,
263, 1784–1790.
[9] Rocque, W. J., McWherter, C. A., Wood, D. C., Gordon, J. I.,
A comparative analysis of the kinetic mechanism and
peptide substrate specificity of human and Saccharomyces
cerevisiae myristoyl-CoA: protein N-myristoyltransferase.
J. Biol. Chem. 1993, 268, 9964–9971.
[10] Stroh, S. M., Eisenhaber, B., Eisenhaber, F. J., N-terminal
N-myristoylation of proteins: prediction of substrate
proteins from amino acid sequence. J. Mol. Biol. 2002, 317,
541–557.
[11] Stroh, S. M., Gouda, M., Novatchkova, M., Schleiffer, A.
et al., MYRbase: analysis of genome-wide glycine myris-
toylation enlarges the functional spectrum of eukaryotic
myristoylated proteins. Genome Biol. 2004, 5, R21.
[12] Ezure,T., Suzuki, T., Higashide, S., Shintani, E. et al., Cell-
free protein synthesis system prepared from insect cells by
freeze-thawing. Biotechnol. Prog. 2006, 22, 1570–1577.
1792 T. Suzuki et al. Proteomics 2010, 10, 1780–1793
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
[13] Sakurai, N., Moriya, K., Suzuki, T., Sofuku, K. et al., Detec-
tion of co- and post-translational protein N-myristoylation
by metabolic labeling in an insect cell-free protein synthesis
system. Anal. Biochem. 2007, 362, 236–244.
[14] Suzuki, T., Ito, M., Ezure, T., Shikata, M. et al., N-Terminal
protein modifications in an insect cell-free protein synthesis
system and their identification by mass spectrometry.
Proteomics 2006, 6, 4486–4495.
[15] Nagase, T., Yamakawa,H., Tadokoro, S., Nakajima, D.
et al., Exploration of human ORFeome: high-throughput
preparation of ORF clones and efficient characterization of
their protein products. DNA Res. 2008, 15, 137–149.
[16] Ohara, O., Nagase, T., Ishikawa, K., Nakajima, D. et al.,
Construction and characterization of human brain cDNA
libraries suitable for analysis of cDNA clones encoding
relatively large proteins. DNA Res. 1997, 4, 53–59.
[17] Nagase, T., Koga, H., Ohara, O., Kazusa mammalian
cDNA resources: towards functional characterization of KIAA
gene products. Brief. Funct. Genomic. Proteomics 2006, 5,
4–7.
[18] Bologna1, G., Yvon, C., Duvaud, S., Veuthey, A., N-Terminal
myristoylation predictions by ensembles of neural
networks. Proteomics 2004, 4, 1626–1632.
[19] Suzuki, T., Ito, M., Ezure, T., Kobayashi, S. et al., Perfor-
mance of expression vector, pTD1, in insect cell-free
translation system. J. Biosci. Bioeng. 2006, 1, 69–71.
[20] Suzuki,T., Ito, M., Ezure, T., Shikata, M. et al., Protein
prenylation in an insect cell-free protein synthesis system
and identification of products by mass spectrometry.
Proteomics 2007, 7, 1942–1950.
[21] Utsumi, T., Sato, M., Nakano, K., Takemura, D. et al., Amino
acid residue penultimate to the amino-terminal Gly residue
strongly affects two cotranslational protein modifications,
N-myristoylation and N-acetylation. J. Biol. Chem. 2001,
276, 10505–10513.
[22] Wang, H. Y., Tao, J., Shumay, E., Malbon, C. C., G-protein-
coupled receptor-associated A-kinase anchoring proteins:
AKAP79 and AKAP250 (gravin). Eur. J. Cell Biol. 2006, 85,
643–650.
[23] Saeki, K., Miura, Y., Aki, D., Kurosaki, T. et al., The B
cell-specific major raft protein, Raftlin, is necessary for the
integrity of lipid raft and BCR signal transduction. EMBO J.
2003, 22, 3015–3026.
[24] Whatley, B. R., Li, L., Chin, L-S., The ubiquitin-proteasome
system in spongiform degenerative disorders. Biochim.
Biophys. Acta 2008, 1782, 700–712.
[25] Inuzuka, M., Hayakawa, M., Ingi, T., Serinc, an activity-regu-
lated protein family, incorporates serine into membrane lipid
synthesis. J. Biol. Chem. 2005, 280, 35776–35783.
[26] Huang, S.-M. A., Hancock, M. K., Pitman, J. L., Orth, A. P. et al.,
Negative regulators of insulin signaling revealed in a
genome-wide functional screen. PLoS ONE 2009, 4, e6871.
[27] Wang, X., Zheng, L., Zeng, Z., Zhou, G. et al., DIXDC1
isoform, l-DIXDC1, is a novel filamentous actin-
binding protein. Biochem. Biophys. Res. Commun. 2006,
347, 22–30.
[28] Zhu, X.-L., Liang, L., Ding, Y.-Q., Overexpression of FMNL2
is closely related to metastasis of colorectal cancer. Int. J.
Colorectal Dis. 2008, 23, 1041–1047.
[29] Panaretou, C., Domin, J., Cockcroft, S., Waterfield, M. D.,
Characterization of p150, an adaptor protein for the human
phosphatidylinositol (PtdIns) 3-kinase. J. Biol. Chem. 1997,
272, 2477–2485.
[30] Preininger, A. M., Parello, J., Meier, S. M., Liao, G. et al.,
Receptor-mediated changes at the myristoylated amino
terminus of Gail proteins. Biochemistry 2008, 47, 10281–10293.
[31] Russwurm, C., Zoidl, G., Koesling, D., Russwurm, M., Dual
acylation of PDE2A splice variant 3. J. Biol. Chem. 2009, 284,
25782–25790.
[32] Gu, J. J., Ryu, J. R., Pendergast, A. M., Abl tyrosine kinases
in T-cell signaling. Immunol. Rev. 2009, 228, 170–183.
[33] Marshall, K. R., Gong, M., Wodke, L., Lamb, J. H. et al., The
human apoptosis-inducing protein AMID is an oxido-
reductase with a modified flavin cofactor and DNA binding
activity. J. Biol. Chem. 2005, 280, 30735–30740.
[34] Donaldson, J. G., Honda, A., Localization and function of Arf
family GTPases. Biochem. Soc. Trans. 2005, 33, 639–642.
[35] Asada, A., Yamamoto, N., Gohda, M., Saito, T. et al.,
Myristoylation of p39 and p35 is a determinant of cyto-
plasmic or nuclear localization of active cyclin-dependent
kinase 5 complexes. J. Neurochem. 2008, 106, 1325–1336.
[36] Utsumi, T., Sakurai, N., Nakano, K., Ishisaka, R., C-terminal
15 kDa fragment of cytoskeletal actin is posttranslationally
N-myristoylated upon caspase-mediated cleavage and
targeted to mitochondria. FEBS Lett. 2003, 539, 37–44.
[37] Sakurai, N., Utsumi, T., Posttranslational N-myristoylation
is required for the anti-apoptotic activity of human tGelso-
lin, the C-terminal caspase cleavage product of human
gelsolin. J. Biol. Chem. 2006, 281, 14288–14295.
[38] Utsumi, T., Nakano, K., Funakoshi, T., Kayano, Y. et al.,
Vertical-scanning mutagenesis of amino acids in a model
N-myristoylation motif reveals the major amino-terminal
sequence requirements for protein N-myristoylation. Eur. J.
Biochem. 2004, 271, 863–874.
[39] Temple, G., Lamesch, P., Milstein, S., Hill, D. E. et al.,
From genome to proteome: developing expression clone
resources for the human genome. Hum. Mol. Genet. 2006, 15,
R31–R43.
[40] Stroh, S. M., Eisenhaber, B., Eisenhaber, F., N-terminal N-
myristoylation of proteins: refinement of the sequence
motif and its taxon-specific differences. J. Mol. Biol. 2002,
317, 523–540.
[41] Martinez, A., Traverso, J. A., Valot, B., Ferro, M. et al., Extent
of N-terminal modifications in cytosolic proteins from
eukaryotes. Proteomics 2008, 8, 2809–2831.
[42] Boisson, B., Giglione, C., Meinnel, T., Unexpected protein
families including cell defense components feature in the N-
myristoylome of a higher eukaryote. J. Biol. Chem. 2003,
278, 43418–43429.
[43] Bologna, G., Yvon, C., Duvaud, S., Veuthey, A.-L., N-Term-
inal myristoylation predictions by ensembles of neural
networks. Proteomics 2004, 4, 1626–1632.
Proteomics 2010, 10, 1780–1793 1793
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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