Strategy for comprehensive identification of human N-myristoylated proteins using an insect cell-free protein synthesis system

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: [email protected]

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


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


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


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.



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

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50

100

b4

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m/z 1719.94

20

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0500 1000 1500

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01000 1200 1400 1600 1800 2000

169

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tGelX-tGelsolin

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Myristoyl

y6y7y8y10

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%Int.

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Precursor ion

m/z1746.90

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y6 y8

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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


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.



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


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


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


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.



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


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


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


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).



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


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.

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Strategy for comprehensive identification of human N-myristoylated proteins using an insect cell-free protein synthesis system