Antibody-free peptide substrate screening of serine/threonine kinase (protein kinase A) with a biotinylated detection probe

Article abstract of DOI:10.1016/j.ab.2011.02.005

Being different from anti-phosphotyrosine antibodies, anti-phosphoserine- or anti-phosphothreonine-specific antibodies with high affinity for the detection of serine/threonine kinase substrates are not readily available. Therefore, chemical modification m

Full text of DOI:10.1016/j.ab.2011.02.005

Analytical Biochemistry 413 (2011) 30–35
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Antibody-free peptide substrate screening of serine/threonine kinase (protein
kinase A) with a biotinylated detection probe
Mira Kim , Yong-Sun Park , Dong-Sik Shin , Jaehi Kim , Byung-Gee Kim a,b, Yoon-Sik Lee a,
a
a
a
a
⇑
a
School of Chemical and Biological Engineering, Seoul National University, San 56-1, Shilim-dong, Kwnak-gu, Seoul 151-744, South Korea
Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, San 56-1, Shilim-dong, Kwnak-gu, Seoul 151-744, South Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Being different from anti-phosphotyrosine antibodies, anti-phosphoserine- or anti-phosphothreonine-
specific antibodies with high affinity for the detection of serine/threonine kinase substrates are not read-
ily available. Therefore, chemical modification methods were developed for the detection of phosphoser-
ine or threonine in the screening of protein kinase substrates based on b-elimination and Michael
addition. We have developed a biotin-based detection probe for identification of the phosphorylated
serine or threonine residue. A biotin derivative induced a color reaction using alkaline phosphate-
conjugated streptavidin that amplified the signal. It was effective for the detection and separation of
the target peptide on the resin. The detection probe was successfully used in identifying PKA substrates
from peptide libraries on resin beads. The peptide library was prepared as a ladder-type, such that the
active peptides on the colored resin beads were readily sequenced with the truncated peptide fragments
by MALDI-TOF/MS analysis after releasing the peptides from the resin bead through photolysis.
Ó 2011 Elsevier Inc. All rights reserved.
Received 25 October 2010
Received in revised form 7 January 2011
Accepted 2 February 2011
Available online 3 March 2011
Keywords:
Ser/Thr kinase
Combinatorial chemistry
Peptide library
Phospho-peptide
b-Elimination
Protein kinases catalyze the phosphorylation of Ser, Thr, and Tyr
ture the phosphorylated peptide and to detect the phosphorylation
event [13–15]. Despite versatility and ease of use, the chelating
reaction has a disadvantage because of competition reactions of a
metal ion complex on a phosphate group with other anionic com-
pounds including ATP and negatively charged amino acid
sequences.
Alternatively, an antibody-based detection method has been
developed to identify phosphorylated proteins [16,17]. Recently,
our group reported a screening method for Tyr kinase substrates
residues in peptides and proteins with adenosine-triphosphate
1
(
ATP) [1–3], which regulates cellular functions, such as cell-cycle
regulation, apoptosis, and metabolism [4]. Moreover, protein kinases
are involved in diseases including cancer and inflammation. Identify-
ing phosphorylation in specific peptide sequences of substrates plays
a crucial role for developing kinase inhibitors for drug discovery
[
5–7]. Various approaches for detecting phosphoryl residue in
peptides have been investigated with the goal of determining the
primary sequence of the protein kinase phosphorylation site [8–10].
using a ladder-type peptide library. The substrate specificities of
32
c-src
Radiolabeling of target peptides with [
c
-
P]ATP is a traditional
protein Tyr kinases, such as p60
, ZAP-70, and Brk, were identi-
method for the detection of phosphorylation [11,12]. However, it
requires long analysis time and special handling procedures for
radioactivity measurements with X-ray instrumentation. In addi-
tion, a fluorophore-conjugated metal ion complex was used to cap-
fied using an anti-phospho-Tyr antibody [18,19]. Although anti-
phospho-Tyr antibodies have shown good affinity, selectivity, and
availability, only a few antibodies that specifically recognize a cer-
tain substrate of a Ser/Thr kinase-mediated phosphorylation have
been discovered [20–22].
⇑
Corresponding author. Fax: +82 2 876 9625.
E-mail addresses: redcat00@snu.ac.kr1 (M. Kim), gastight@hotmail.com (Y.-S.
As an alternative detection heuristic, chemical modification
methods based on b-elimination and Michael addition were devel-
oped to detect phospho-Ser/Thr residues and to avoid nonspecific
interactions with immune-affinity and metal-affinity systems
Park), dongss2@snu.ac.kr (D.-S. Shin), susia45@yahoo.co.kr (J. Kim), byungkim@
snu.ac.kr (B.-G. Kim), yslee@snu.ac.kr (Y.-S. Lee).
1
Abbreviations used: Fmoc, N-(9-fluorenylmethoxycarbonyl; DMF, dimethyl-
[
23–27]. Here, we report a new chemical modification method
formamide; NMP, N-methylpyrrolidone; DIEA, N,N-diisopropylethylamine; NHS,
N-hydroxysuccinimide; DCC, dicyclohexylcarbodiimide; PS, polystyrene; BOP,
benzotriazol-1-yloxy-tris(dimethylamino) phosphonium hexafluorophosphate;
HOBt,1-hydroxy-benzotriazole; Tricine, N-(tri(hydroxymethyl) methyl)glycine;
MALDI-TOF/MS, matrix-assisted laser desorption ionization time-of-flight mass
spectrometry; BCIP, 5-bromo-4-chloro-3 -indolyl phosphate p-toluidine salt; NBT,
nitroblue tetrazolium chloride; TEG, tetraethyleneglycol; AP, alkaline phospha-
tase; SA, succinic acid.
for the detection of phospho-Ser/Thr, which is suitable for the
screening of protein kinase substrates, in lieu of an antibody-based
method.
A thiol group containing a biotin derivative was designed as a
detection probe for phospho-Ser/Thr. After phosphorylation of
the Ser/Thr-containing ladder-type peptides on resin beads, the
0
0
003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2011.02.005

Antibody-free phospho-Ser/Thr detection / M. Kim et al. / Anal. Biochem. 413 (2011) 30–35
31
phosphorylated peptides were converted by b-elimination to the
a
,
HiCore resin was prepared from chloromethyl (CM) PS resin using
a method previously reported in the literature [30]. For introduc-
tion of the linker and spacer to the HiCore resin, the photolabile
b-unsaturated peptide residues which could be captured by the
thiol group-containing biotin probe and recognized by alkaline
phosphatase (AP)-conjugated streptavidin-derived colorimetric
reaction. Consequently, the active peptide substrates on the col-
ored resin bead could be sequenced by MALDI-TOF/MS after re-
lease from the bead (Fig. 1).
linker [31,32] and spacer, bAla-eACA-bAla-eACA (bebe, where b is
b-alanine and is -aminocaproic acid), were coupled to the resin
e
e
with the Fmoc strategy of solid-phase peptide synthesis.
To synthesize Kemptide on the resin in a ladder-type configura-
tion, each of the corresponding Fmoc amino acids (4 eq) was cou-
pled in series to the NH
0.2 eq), BOP (4.2 eq), HOBt (4.2 eq), and DIEA (8.4 eq). The prod-
uct, Ac-Gly-L-R-R-A-S-L-G-b –PLL–resin, was confirmed by
sequencing with MALDI-TOF/MS analysis after UV photocleavage
360 nm).
For a ladder-type random peptide library to be used in a Ser/Thr
2
–bebe–PLL–resin in the presence of Ac-Gly
Materials and methods
(
ebe
Synthesis of biotin–TEG–Cys–NH
2
(
For biotin–TEG (tetraethyleneglycol)–Cys–NH
TEG, and biotin were coupled to a Rink-amide resin using the
solid-phase BOP/HOBt-coupling method and Fmoc chemistry
2
synthesis, Cys,
kinase assay, the spacer and linker-conjugated resin (1.6 g) was di-
vided into 16 equal parts, which corresponded to 16 amino acids
(Scheme 1).
(
the natural amino acids except for Ser, Thr, Tyr, and Cys). The
Fmoc–TEG–succinic acid (SA), used as a building block, was pre-
ladder-type peptide library was prepared via the split-and-mix
method. At every coupling steps, each resin portion was reacted with
Fmoc amino acid (4 eq), Ac-Gly (0.2 eq), BOP (4.2 eq), HOBt (4.2 eq),
and DIEA (8.4 eq) in NMP. After coupling, the resins were combined
in a reaction tube, washed, and dried under reduced pressure.
For quantification of amino groups, ca. 100 mg of resin was
treated with a 20% piperidine/DMF solution and filtered, and the
filtrate solution was titrated by UV (290 nm) absorbance to calcu-
late the amount of available amine groups after partial capping
with Ac-Gly. After removing the Fmoc group, the resin was divided
into 16 equal parts and reacted with Fmoc amino acid. These steps
were repeated, and the side-chain protecting groups were depro-
tected by cleavage cocktail (Reagent K) at the final step.
pared separately in solution-phase synthesis [28,29]. 4,7,10-Triox-
a-1,13-tridecanediamine (10 mmol) was reacted with succinic
anhydride (10 mmol) in acetonitrile to yield H
Fmoc–TEG–SA was prepared from the reaction of H
2
N–TEG–SA. Then,
N–TEG–SA
2
and Fmoc–OSu (11 mmol) with DIEA in acetonitrile. The purity of
the product was 93%, and the yield was 59%.
Biotin-NHS was synthesized from biotin (5 mmol), NHS
(
5 mmol), and DCC (5 mmol) in DMF.
Fmoc–Cys(Trt)-OH (2 eq) and Fmoc–TEG–succinic acid were
sequentially coupled to a Rink-amide MBHA resin (0.6 NH mmol/
2
g) in the presence of BOP (2 eq), HOBt (2 eq), and DIEA (4 eq). The
Fmoc group was removed from the resin in a 20% piperidine/DMF
solution. Biotin–NHS (2 eq) was coupled to the NH
amide resin in the presence of DIEA (2 eq). Biotin–TEG–Cys–NH
and the Trt protecting group on the Cys residue were cleaved from
the resin by Reagent K (TFA:phenol:H O:thioanisole:1,2-ethanedi-
thiol = 82.5:5:5:5:2.5). The purity of biotin-TEG–Cys–NH was con-
2
–TEG–Cys–Rink
2
Kinase assay and detection
2
2
Kemptide-coupled resin (5 mg) was reacted with 1000 U PKA
firmed by MADLI-TOF/MS and HPLC (66% purity and 71% yield
based on the amount of Cys).
(
protein kinase A) and 1 mM ATP in an assay buffer solution con-
taining Tricine (0.9% w/v), MgSO (0.12% w/v), DTT (0.04% w/v),
4
EDTA (0.0016% w/v), and BSA (0.1% w/v) at 37 °C for 30 min. As a
control reaction, Kemptide-coupled resin was reacted with the
same buffer solution without PKA.
Ladder-type peptide synthesis on the resin
Kemptide (LRRASLG) and the random peptide library were
synthesized on a HiCore resin (core–shell-type PEGylated PS resin).
b-Elimination of phosphorylated peptide resin was accom-
plished under alkaline conditions with 0.5 N NaOH:DMSO:i-PrOH
Fig.1. Outline of the detection method of phospho-Ser/Thr. (AP, alkaline phosphatase; NBT, nitroblue tetrazolium; BCIP, bromo-chloroindolyl phosphate).

3
2
Antibody-free phospho-Ser/Thr detection / M. Kim et al. / Anal. Biochem. 413 (2011) 30–35
Scheme 1. Preparation of biotin-TEG–Cys–NH
piperidine/DMF; (e) biotin–NHS, DIEA, DMF; and (f) Reagent K (TFA, phenol, H
2
: (a) Fmoc–Cys(Trt), BOP, HOBt, DIEA, NMP; (b) 20% piperidine/DMF; (c) Fmoc–TEG–COOH, BOP, HOBt, DIEA, NMP; (d) 20%
O, thioanisole, 1,2-ethandithiol).
2
(
3:5.5:1.5) at 50 °C for 4 h. Michael addition was performed with
tetrazolium chloride (NBT) in Tris buffer (pH 9.5). After the color
reaction, the reaction was quenched by adding 1 N HCl solution.
PKA substrate screening was performed on the peptide library-
conjugated resin (25 mg) using 2500 U PKA and 1 mM ATP in an
assay buffer solution. After b-elimination and Michael addition
biotin–TEG–Cys–NH
(
tase-conjugated streptavidin (AP-ST, 1 U/mL) for 1 h. The AP on
the resin promoted a color reaction (purple) with 5-bromo-4-
chloro-3 -indolyl phosphate p-toluidine salt (BCIP) and nitroblue
2
(2 mM) in 0.05 N NaOH:DMSO:i-PrOH
3:5.5:1.5). Then each sample was treated with alkaline phospha-
0
2
with biotin–TEG–Cys–NH , color reaction was performed through
Scheme 2. Preparation of the ladder-type peptide library: (a) Fmoc-photolabile linker (PLL), BOP, HOBt, DIEA, NMP; (b) 20% piperidine/DMF; (c) Fmoc-
Fmoc-b-alanine, BOP, HOBt, DIEA, NMP; (d) 20% piperidine/DMF; (e) Fmoc-Ala (4 eq), Ac-Gly (0.2 eq), BOP, HOBt, DIEA, NMP; (f) 20% piperidine/DMF; (g) Fmoc-amino acid,
AcGly (2 eq), BOP, HOBt, DIEA, NMP; and (h) Reagent K (TFA, phenol, H O, thioanisole, 1,2-ethandithiol).
e-aminocaproic acid or
2

Antibody-free phospho-Ser/Thr detection / M. Kim et al. / Anal. Biochem. 413 (2011) 30–35
33
AP-ST treatment and BCIP/NBT reaction. One hundred reddish-
brown colored beads were picked during fourfold assay
a
(
4 ꢀ 25 mg resin), and each peptide together with its ladder-type
fragments was released from the resin by UV irradiation. The pep-
tide sequences were identified by MALDI-TOF/MS analysis and 88
beads were sequenced successfully. Detailed conditions of each
experimental step are described in the Supplementary Material.
Results and discussion
Synthesis of biotin–TEG–Cys–NH
2
Biotin–TEG–Cys–NH was designed as a thiol-containing biotin
2
probe. Cys was added because the thiol group could act as a nucle-
ophile in the Michael addition step. Biotin at the end of the probe
served as a binding ligand of streptavidin for a colorimetric reac-
tion. TEG was added to the probe for water compatibility. The
detection probe was prepared using the Fmoc strategy for solid-
phase peptide synthesis. Fmoc–Cys, Fmoc–TEG–-SA, and biotin
were successively coupled to the Rink-amide resin. Then, biotin-
N-hydroxysuccinimide (biotin–NHS) was coupled to the TEG–Cys
residue on the resin. After cleavage from the resin, the biotin–
Fig.2. Fmoc titration of peptide resin at each coupling step: the gray solid bar
represents the free amine group at each coupling step, and the striped bar
represents truncated peptide.
and the NBT/BCIP colorimetric reaction. We could clearly distin-
guish the color difference between the phosphorylated sample
and the control sample.
2
TEG–Cys–NH was obtained as a sticky solid.
Ladder- type peptide library synthesis on the resin
PKA substrate screening with the random peptide library
A photolabile linker and spacer (b
ebe) were successfully intro-
The ladder-type peptide library on the resin was incubated with
PKA and ATP and treated with an alkaline solution for b-elimina-
tion followed by the addition of biotin–TEG–Cys–NH . After the
2
duced to the resin bead. The b was added to avoid the overlap
ebe
of peaks in mass analysis and to increase the accessibility of
biomolecules.
colorimetric reaction, dark brown resin beads were hand-picked
under a microscope (Fig. 3). The full-length peptide and its subpep-
tides containing truncated sequences were released from the resin
by UV photocleavage, and the sequence of the peptide was easily
identified by mass analysis.
The PKA substrate profiling results are summarized in Fig. 4,
and the priorities of amino acid sequences are listed in Table 1.
Arg specificity at Xꢁ3 (57%) and hydrophobic residue specificity
Then, the ladder-type peptide was constructed on the resin
using a partial capping method. To prepare the ladder-type peptide
library, Ac-Gly was chosen as a capping reagent instead of acetic
acid (as in our previous work) because capping was easier to con-
trol than the use of acetic acid. To produce a longer and more ran-
dom peptide library, precise control of capping is necessary to
perform the reliable bioassay with full sequence peptides and, at
the same time, to properly interpret sequencing information.
(
1
73%) (e.g., Val, Ala at the X position) were clearly observed in
First, Ac-Gly-L-R-R-A-S-L-G-bebe-PLL-resin was prepared for
the model reaction with PKA and we confirmed that the ladder-
type peptide was successfully synthesized through MALDI-TOF/
MS analysis after UV photocleavage.
Next, a random peptide library was synthesized on the spacer-
conjugated resin using the split-and-mix synthesis method in a
one-bead one-compound (OBOC) approach (Scheme 2). Quantita-
tive analysis of a truncated peptide library pool showed that the
amount of available free amine groups for coupling on the resin
gradually decreased over the course of the synthesis. The amount
of each ladder peptide fragment equals the difference of available
free amine groups before and after coupling (Fig. 2).
Model test with Kemptide-coupled resin and PKA
To verify the detection system, Kemptide, a well-known peptide
substrate of protein kinase A [33,34], was synthesized on a HiCore
resin (PEGylated PS resin) as a ladder-type peptide by partial cap-
ping with N-acetylglycine (Ac-Gly) at each coupling step. After
phosphorylation, the Kemptide-coupled resin was treated under
alkaline conditions for b-elimination [35,36]. Then, biotin–TEG–
Cys–NH
We determined that the optimal conditions for b-elimination were
0 °C for 2 h and for Michael addition were 25 °C for 1 h. Kemp-
2
was added to the resulting resin for Michael addition.
5
tide-coupled resin, when treated with ATP and PKA, turned to a
brown color, while a control sample, which was treated without
PKA, remained yellow after AP-conjugated strepatavidin addition
Fig.3. Optical microscope images of ladder-type peptide libraries on resin beads
after PKA assay and colorimetric reaction.

3
4
Antibody-free phospho-Ser/Thr detection / M. Kim et al. / Anal. Biochem. 413 (2011) 30–35
Fig.4. PKA substrate peptide profiling results.
Table 1
Sequence frequency at each position of the PKA substrates.
change that could be distinguished by the naked eye, and were eas-
ily separated from the resin bead mixture. We expect that our
screening tool can be applied to other Ser/Thr kinase substrate
screening systems without the use of an anti-phospho-antibody.
Occurrence
frequency
X
ꢁ3
X
ꢁ2
X
ꢁ1
X
1
X
2
Most
Second most
R (57%)
K/Q (23%)a
V (15%)
R (14%)
L/I (12%)
V (36%)
A (21%)
A (25%)
V (23%)
K/Q (14%)a
b
Acknowledgments
a
K(Lys) and Q(Gln) have similar molecular weights.
L(Leu) and I(Ile) have identical molecular weights.
b
This work was supported by the Korea Biotech R&D Group of
the Next-Generation Growth Engine Project (F104AB010005-
0
7A0201-00510) and by the Seoul R&BD program (10538).
Table 2
Proteins matched to the screened substrate sequence of PKA (NetworKIN) [40].
Appendix A. Supplementary data
Screened peptide
sequence
Real protein
sequence
Protein substrate of PKA
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ab.2011.02.005.
RKLSVA
RKVSVA
RKLSVA
RKVSLA
ATP-binding cassette subfamily A
member 1
Cystic fibrosis transmembrane
conductance regulator
References
[
[
1] K. Helmbrecht, E. Zeise, L. Rensing, Chaperones in cell cycle regulation and
mitogenic signal transduction: a review, Cell. Prolif. 33 (2000) 341–365.
2] T.B. Kuhn, P.J. Meberg, M.D. Brown, B.W. Bernstein, L.S. Minamide, J.R. Jensen,
K. Okada, E.A. Soda, J.R. Bamburg, Regulating actin dynamics in neuronal
growth cones by ADF/cofilin and rho family GTPases, J. Neurobiol. 44 (2000)
the PKA assay. Lys (or Gln; Lys and Gln have identical same molec-
ular weights) was frequently found at the Xꢁ3 (15%) and Xꢁ2 (23%)
positions. These results are in good agreement with the previous
PKA assay results, which revealed that PKA has two Glu residues
as anionic binding sites and a hydrophobic site [37–39]. The anio-
nic sites correspond to the Xꢁ3 and Xꢁ2 positions, and the hydro-
126–144.
[
3] J. Herrington, L.S. Smit, J. Schwartz, C. Carter-Su, The role of STAT proteins in
growth hormone signaling, Oncogene 19 (2000) 2585–2597.
[4] S.
superfamily: kinase (catalytic) domain structure classification, FASEB J. 9
1995) 576–596.
5] A.J. Bridges, Chemical inhibitors of protein kinases, Chem. Rev. 101 (2001)
541–2572.
K Hanks, T. Hunter, Protein kinases 6 the eukaryotic protein kinase
phobic site corresponds to the X
the phosphorylation site is Arg or Lys at the Xꢁ3 and Xꢁ2 position
and a hydrophobic residue at the X position. We found that some
1
position. Therefore, the motif of
(
[
1
2
peptide sequences are identical or similar to the substrate protein
sequences through a kinase substrate data base search (Networ-
KIN) (Table 2) [40].
[6] P. Cohen, Protein kinases – the major drug targets of the twenty-first century?,
Nat Rev. Drug. Discov. 1 (2002) 309–315.
[
7] M.E.M. Noble, J.A. Endicott, L.N. Johnson, Protein kinase inhibitors: insights
into drug design from structure, Science 303 (2004) 1800–1805.
The detailed results of the synthesis and PKA assay of
Kemptide-coupled resin and additional peptide sequences in
substrate screening are described in the Supplementary Material.
[8] G.W. Tessmer, J.R. Skuster, L.B. Tabatabai, D.J. Graves, Studies on the specificity
of phosphorylase kinase using peptide substrates, J. Biol. Chem. 252 (1977)
5666–5671.
[
9] Z. Songyang, K.L. Carraway, M.J. Eck, S.C. Harrison, R.A. Feldman, M.
Mohammadi, J. Schlessinger, S.R. Hubbard, D.P. Smith, E. Eng, M.J. Lorenzo,
B.A.J. Ponder, B.J. Mayer, L.C. Cantley, Catalytic specificity of protein–tyrosine
kinases is critical for selective signaling, Nature 373 (1995) 536–539.
Conclusion
[
10] M Kim, D.S. Shin, J. Kim, Y.S. Lee, Substrate screening of protein kinases:
detection methods and combinatorial peptide libraries, Biopolym. Pept. Sci. 94
We developed a thiol-containing biotin compound as a detec-
tion probe of phospho-Ser/Thr in a protein kinase assay. Our detec-
tion system played a key role in the screening of the peptide
substrate specificity of PKA. Resin beads containing phosphory-
lated peptides after PKA treatment were easily recognized by color
(
2010) 753–762.
[11] J.J. Witt, R. Roskoski Jr., Rapid protein kinase assay using phosphocellulose-
paper absorption, Anal. Biochem. 66 (1975) 253–258.
12] P. Geer, T. Hunter, Phosphopeptide mapping and phosphoamino acid analysis
by electrophoresis and chromatography on thin-layer cellulose plates,
Electrophoresis 15 (1994) 544–554.
[

Antibody-free phospho-Ser/Thr detection / M. Kim et al. / Anal. Biochem. 413 (2011) 30–35
35
[
[
[
13] E. Hough, L.K. Hansen, B. Birknes, High-resolution (1.5 A) crystal structure of
phospholipase C from Bacillus cereus, Nature 338 (1989) 357–360.
[26] S. Akita, N. Umezawa, N. Kato, T. Higuchi, Array-based fluorescence assay for
serine/threonine kinases using specific chemical reaction, Bioorgan. Med.
Chem. 16 (2008) 7788–7794.
[27] S. Akita, N. Umezawa, T. Higuchi, On-bead fluorescence assay for serine/
threonine kinases, Org. Lett. 7 (2005) 5565–5568.
[28] A. Song, X. Wang, J. Zhang, J. Marik, C.B. Lebrilla, K.S. Lam, Synthesis of
hydrophilic and flexible linkers for peptide derivatization in solid phase,
Bioorg. Med. Chem. Lett. 14 (2004) 161–165.
[29] Z.G. Zhao, J.S. Im, K.S. Lam, D.F. Lake, Site-specific modification of a single-
chain antibody using a novel glyoxylyl-based labeling reagent, Bioconjugate
Chem. 10 (1999) 424–430.
14] C.A. Chen, R.H. Yeh, D.S. Lawrence, Design and synthesis of a fluorescent
reporter of protein kinase activity, J. Am. Chem. Soc. 124 (2002) 3840–3841.
15] S. Aoki, M. Zulkefeli, M. Shiro, M. Kohsako, K. Takeda, A luminescence sensor of
inositol 14 5-triphosphate its model compound by ruthenium-templated
2
+
0
assembly of a bis (Zn – cyclen) complex having a 2 2 -bipyridyl linker
cyclen = 1,4,7,10-tetraazacyclododecane), J. Am. Chem. Soc. 127 (2005) 9129–
139.
(
9
[
16] A. Pandey, A.V. Podtelejnikov, B. Blagoev, X.R. Bustelo, M. Mann, H.F. Lodish,
Analysis of receptor signaling pathways by mass spectrometry: identification
of vav-2 as a substrate of the epidermal and platelet-derived growth factor
receptors, Proc. Natl. Acad. Sci. USA 97 (2000) 179–184.
17] D.L. Foletti, R. Lin, M.A.F. Finley, R.H. Scheller, Phosphorylated syntaxin 1 is
localized to discrete domains along a subset of axons, J. Neurosci. 20 (2000)
[30] H.Y. Kim, J.K. Cho, W.J. Chung, Y.-S. Lee, Core–shell-type resins for solid-phase
peptide synthesis: comparison with gel-type resins in solid-phase photolytic
cleavage reaction, Org. Lett. 6 (2004) 3273–3276.
[
[
[31] C.P. Holmes, D.G. Jones, Reagents for combinatorial organic synthesis:
4
535–4544.
development of a new O-nitrobenzyl photolabile linker for solid phase
synthesis, J. Org. Chem. 60 (1995) 2318–2319.
[32] R. Wieboldt, D. Ramesh, E. Jabri, P.A. Karplus, B.K. Carpenter, G.P. Hess,
Synthesis and characterization of photolabile O-nitrobenzyl derivatives of
urea, J. Org. Chem. 67 (2002) 8827–8831.
18] Y.G. Kim, D.S. Shin, E.M. Kim, H.Y. Park, C.S. Lee, J.H. Kim, B.S. Lee, Y.S. Lee, B.G.
Kim, High-throughput identification of substrate specificity for protein kinase
by using an improved one-bead-one-compound library approach, Angew.
Chem. Int. Ed. 46 (2007) 5408–5411.
[
[
[
19] D.S. Shin, Y.G. Kim, E.M. Kim, M. Kim, H.Y. Park, J.H. Kim, B.S. Lee, B.G. Kim, Y.S.
Lee, Solid-phase peptide library synthesis on HiCore resin for screening
substrate specificity of Brk protein tyrosine kinase, J. Comb. Chem. 10 (2008)
[33] B.E. Kemp, R.B. Pearson, Protein kinase recognition sequence motifs, Trends.
Biochem. Sci. 15 (1990) 342–346.
[34] R.B. Pearson, B.E. Kemp, Protein kinase phosphorylation site sequences and
consensus specificity motifs: tabulations, Methods. Enzymol. 200 (1991) 62–
81.
[35] W. Li, R.A. Boykins, P.S. Backlund, G. Wang, H.C. Chen, Identification of
phosphoserine and phosphothreonine as cysteic acid and b-methylcysteic acid
residues in peptides by tandem mass spectrometric sequencing, Anal. Chem.
74 (2002) 5701–5710.
[36] F. Thaler, B. Valsasina, R. Baldi, J. Xie, A. Stewart, A. Isacchi, H.M. Kalisz, L.
Rusconi, A new approach to phosphoserine phosphothreonine analysis in
peptides proteins: chemical modification enrichment via solid-phase
reversible binding and analysis by mass spectrometry, Anal. Bioanal. Chem.
376 (2003) 366–373.
2
0–23.
20] S. Kane, H. Sano, S.C. Liu, J.M. Asara, W.S. Lane, C.C. Garner, G.E. Lienhard, A
method to identify serine kinase substrates. Akt phosphorylates a novel
adipocyte protein with a Rab GTPase-activating protein (GAP) domain, J. Biol.
Chem. 277 (2002) 22115–22118.
21] M. Gronborg, T.Z. Kristianzen, A. Stensballe, J.S. Andersen, O. Ohara, M. Mann,
O.N. Jensen, A. Pandey, A mass spectrometry-based proteomic approach for
identification of serine/threonine-phosphorylated proteins by enrichment
with phospho-specific antibodies: identification of a novel protein Frigg as a
protein kinase A substrate, Mol. Cell. Proteomics 1 (2002) 517–527.
22] G. Redlich, U.M. Zanger, S. Riedmaier, N. Bache, A.B.M. Giessing, M. Eisenacher,
C. Stephan, H.E. Meyer, O.N. Jensen, K. Marcus, Distinction between human
cytochrome P450 (CYP) isoforms and identification of new phosphorylation
sites by mass spectrometry, J. Proteome. Res. 7 (2008) 4678–4688.
23] G. Moorhead, R.W. MacKintosh, N. Morrice, T. Gallagher, C. MacKintosh,
Purification of type 1 protein (serine/threonine) phosphatases by microcystin-
Sepharose affinity chromatography, FEBS Lett. 356 (1994) 46–50.
24] P. Fadden, T.A. Haystead, Quantitative and selective fluorophore labeling of
phosphoserine on peptides and proteins: characterization at the attomole
level by capillary electrophoresis and laser-induced fluorescence, Anal.
Biochem. 225 (1995) 81–88.
[
[37] Z. Songyang, S. Blechner, N. Hoagland, M.F. Hoekstra, H. Piwnica-Worms, L.C.
Cantley, Use of an oriented peptide library to determine the optimal substrates
of protein kinases, Curr. Biol. 4 (1994) 973–982.
[
[
[38] P.T. Tuazon, W.C. Spanos, E.L. Gump, C.A. Monnig, J.A. Traugh, Determinants for
substrate phosphorylation by p21-activated protein kinase
Biochemistry 36 (1997) 16059–16064.
(c-PAK),
[39] J.A. Ubersax, J.E. Ferrell Jr., Mechanisms of specificity in protein
phosphorylation, Nat. Rev. Mol. Cell. Biol. 8 (2007) 530–541.
[40] R. Linding, L.J. Jensen, G.J. Ostheimer, M.A. van Vugt, C. Jørgensen, I.M. Miron, F.
Diella, K. Colwill, L. Taylor, K. Elder, P. Metalnikov, V. Nguyen, A. Pasculescu, J.
Jin, J.G. Park, L.D. Samson, J.R. Woodgett, R.B. Russell, P. Bork, M.B. Yaffe, T.
[
25] H. Jaffe, Veeranna, H.C. Pant, Characterization of serine and threonine
phosphorylation sites in b-elimination/ethanethiol addition-modified proteins
by electrospray tandem mass spectrometry and database searching,
Biochemistry 37 (1998) 16211–16224.
Pawson, NetworKIN:
a resource for exploring cellular phosphorylation
networks, Cell 129 (2007) 1415–1426.