Clickable NAD analogues for labeling substrate proteins of poly(ADP-ribose) polymerases

Article abstract of DOI:10.1021/ja101588r

Poly(ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple adenine diphosphate ribose (ADP-ribose) units from nicotinamide adenine dinucleotide (NAD) to substrate proteins. There are 17 PARPs in humans. Several PARPs, such as PARP-1 and Tankyrase-1, are known to play important roles in DNA repair, transcription, mitosis, and telomere length maintenance. To better understand the functions of PARPs at a molecular level, it is necessary to know what substrate proteins PARPs modify. Here we report clickable NAD analogues that can be used to label PARP substrate proteins. The clickable NAD analogues have a terminal alkyne group which allows the conjugation of fluorescent or affinity tags to the substrate proteins. Using this method, PARP-1 and tankyrase-1 substrate proteins were labeled by a fluorescent tag and visualized on SDS-PAGE gel. Using a biotin affinity tag, we were able to isolate and identify a total of 79 proteins as potential PARP-1 substrates. These include known PARP-1 substrate proteins, including histones and heterogeneous nuclear ribonucleoproteins. About 40% of the proteins were also identified in recent proteomic studies as potential PARP-1 substrates. Among the identified potential substrates, we further demonstrated that tubulin and three mitochondrial proteins, TRAP1 (TNF receptorassociated protein 1), citrate synthase, and GDH (glutamate dehydrogenase 1), are substrates of PARP-1 in vitro. These results demonstrate that the clickable NAD analogue is useful for labeling, in-gel detection, isolation, and identification of the substrate proteins of PARPs and will help to understand the biological functions of PARPs.

Full text of DOI:10.1021/ja101588r

Published on Web 06/18/2010
Clickable NAD Analogues for Labeling Substrate Proteins of
Poly(ADP-ribose) Polymerases
Hong Jiang,† Jun Hyun Kim,† Kristine M. Frizzell,‡ W. Lee Kraus,‡,§ and
Hening Lin*,†
Department of Chemistry and Chemical Biology, Cornell UniVersity, Ithaca, New York 14850,
Department of Molecular Biology and Genetics, Cornell UniVersity, Ithaca, New York 14850,
and Department of Pharmacology, Weill Medical College of Cornell UniVersity,
New York, New York 10021
Received March 4, 2010; E-mail: hl379@cornell.edu
Abstract: Poly(ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple adenine diphosphate
ribose (ADP-ribose) units from nicotinamide adenine dinucleotide (NAD) to substrate proteins. There are
17 PARPs in humans. Several PARPs, such as PARP-1 and Tankyrase-1, are known to play important
roles in DNA repair, transcription, mitosis, and telomere length maintenance. To better understand the
functions of PARPs at a molecular level, it is necessary to know what substrate proteins PARPs modify.
Here we report clickable NAD analogues that can be used to label PARP substrate proteins. The clickable
NAD analogues have a terminal alkyne group which allows the conjugation of fluorescent or affinity tags
to the substrate proteins. Using this method, PARP-1 and tankyrase-1 substrate proteins were labeled by
a fluorescent tag and visualized on SDS-PAGE gel. Using a biotin affinity tag, we were able to isolate and
identify a total of 79 proteins as potential PARP-1 substrates. These include known PARP-1 substrate
proteins, including histones and heterogeneous nuclear ribonucleoproteins. About 40% of the proteins were
also identified in recent proteomic studies as potential PARP-1 substrates. Among the identified potential
substrates, we further demonstrated that tubulin and three mitochondrial proteins, TRAP1 (TNF receptor-
associated protein 1), citrate synthase, and GDH (glutamate dehydrogenase 1), are substrates of PARP-1
in vitro. These results demonstrate that the clickable NAD analogue is useful for labeling, in-gel detection,
isolation, and identification of the substrate proteins of PARPs and will help to understand the biological
functions of PARPs.
be required for DNA repair/genome maintenance5-7 and
transcriptional regulation of certain genes8-11 by poly(ADP-
Introduction
Poly(ADP-ribosyl)ation is the addition of multiple ADP-
ribosyl groups from NAD to substrate proteins.1-3 The first
ADP-ribosyl group is added to the carboxylate side chain of
Glu or Asp residues of the substrate proteins, followed by
addition of more ADP-ribosyl groups to the 2′-OH groups of
the two ribose rings (Figure 1), leading to long and branched
poly(ADP-ribose) chains (PAR) that can contain hundreds of
ADP-ribose units. The large size and the enormous negative
charges of PAR chains can affect protein structure and function
and thus regulate the biological processes in which the substrate
proteins are involved. The enzymes that catalyze poly(ADP-
ribosylation) are termed poly(ADP-ribose) polymerases, or
PARPs. Seventeen PARPs have been identified in humans.1,4
Of the 17 PARPs identified, only two of them, PARP-1 and
Tankyrase-1, are relatively well studied. PARP-1 is found to
ribosyl)ation of itself and many nucleosomal or nucleosome-
associated proteins. It is also responsible for the induction of
cell death under extreme stress (such as excessive DNA
damage)12,13 or pathological conditions (stroke, ischemia,
diabetes).14-16 Tankyrase-1 is known to be required for mitosis
and telomere length maintenance by modifying the nuclear
mitotic apparatus protein (NuMA)17-20 and a telomere-binding
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† Department of Chemistry and Chemical Biology, Cornell University.
‡ Department of Molecular Biology and Genetics, Cornell University.
§ Weill Medical College of Cornell University.
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Figure 1. Poly(ADP-ribosyl)ation reaction catalyzed by PARPs.
protein TRF1.21,22 The important biological functions of PARP-1
and Tankyrase-1 suggest that other PARPs could be likewise
important. However, very little is known about other PARPs.
To understand the biological function of different PARPs, it
is necessary to find out what substrate proteins are modified by
different PARPs, similar to the efforts that have been invested
in identifying kinase substrates.23,24 At present, 32P-NAD and
PAR antibodies are the major methods used to detect protein
poly(ADP-ribosyl)ation. For isolation and identification of
substrate proteins of PARPs, using PAR antibodies is the
preferred option because the use of 32P-NAD does not provide
an affinity tag for isolation and purification. Recently, macro
domain that can bind to ADP-ribose was used to isolate ADP-
ribosylated proteins.25 A major concern about using PAR
antibodies and macro domains to identify substrate proteins is
that the immunoprecipitation step is executed under native
conditions and thus some nonsubstrate proteins that interact with
a poly(ADP-ribosyl)ated protein will also be pulled down. This
will give false positive results. Thus, new and improved methods
to label and identify PARP substrate proteins are still needed.
Here, we report the use of clickable alkyne-tagged NAD
analogues for labeling and identifying PARP substrate proteins
(Figure 2). The terminal alkyne attached to the adenine ring of
NAD can be conjugated via click chemistry to many other
functional tags, such as fluorescent tags for in-gel visualization
and affinity tags for purification, while its small size will make
sure that the NAD analogue will still be accepted by PARPs as
the cosubstrate. We demonstrate that one of the clickable NAD
analogues, 6-alkyne-NAD, can be used to label the substrate
proteins of PARPs for in-gel visualization and identification.
In addition to known PARP-1 substrate proteins, over 70
unknown potential substrate proteins from cell lysate were
identified. Among these unknown potential substrate proteins,
we demonstrated that four of them are indeed PARP-1 substrates
in Vitro. These results demonstrate that the clickable NAD
analogue will be very useful for studying the biological function
of PARPs by facilitating the in-gel visualization, isolation, and
identification of substrate proteins.
Results and Discussion
Labeling of PARP-1 Automodification. The synthesis of the
alkyne-tagged NAD analogues has been reported.26 Here we
tested whether these alkyne-tagged NAD analogues can be
accepted by PARPs as a cosubstrate. We first tested the best
studied PARP family member, PARP-1. PARP-1 is a 110-kDa
protein consisting of three domains: an N-terminal DNA binding
domain, a C-terminal catalytic domain, and a central automodi-
fication domain. The DNA binding domain can bind to various
forms of DNA strand breaks, which stimulate its catalytic
activity leading to the poly(ADP-ribosyl)ation of PARP-1 itself
and other target proteins, including histones,27,28 DEK,29 and
p53.30-33
We expressed and purified full-length PARP-1 using bacu-
loviral expression system in Sf-9 cells.8 The purified protein
was incubated with 6-alkyne-NAD and 8-alkyne-NAD in the
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Clickable NAD Analogues for Labeling
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Figure 2. (A) Labeling of poly(ADP-ribosyl)ated proteins with NAD analogues. NAD analogues bearing an alkyne group will be used in PARP-catalyzed
reactions. An affinity tag can be added using click chemistry after the substrate protein is labeled. The labeled protein can then be affinity purified, separated
on 1D/2D protein gel, and then the sequence identified by MS. (B) Structure of 6-/8-alkyne-NAD, Rh-N3, and Biotin-N3, which were used in labeling
reactions.
Figure 3. Labeling of PARP-1 auto-(ADP-ribosyl)ation with 6- or 8-alkyne-NAD. The panel on the left shows the image of Rhodamine fluorescence and
the panel on the right is the same gel stained with Coomassie Blue. The arrow points at the position of unmodified PARP-1. All lanes contain PARP-1 (0.15
µM) and all lanes except 7 and 8 contain ssDNA (0.25 µg/µL). Lane 1, control without NAD; lane 2, control with normal NAD (100 µM); lane 3, 6-alkyne-
NAD (100 µM); lane 4, 8-alkyne-NAD (100 µM); lane 5, 6-alkyne-NAD (100 µM) and NAD (100 µM); lane 6, 8-alkyne-NAD (100 µM) and NAD (100
µM); lane 7, control with 6-alkyne-NAD (100 µM), NAD (100 µM), and no ssDNA; lane 8, control with 8-alkyne-NAD (100 µM), NAD (100 µM), and no
ssDNA. The fluorescence signal below 116 kDa in lane 5 and 6 is likely due to the hydrolysis of poly(ADP-ribose) chain resulting in poly(ADP-ribose)
polymers that are not covalently bound to proteins. The linkage between different ADP-ribose units is a relatively labile ester bond.
presence of salmon sperm DNA (ssDNA), an activator of
PARP-1 enzymatic activity. Next, click chemistry was initiated
to conjugate the fluorescent tag, Rh-N3, to the modified protein.
The reaction mixture was then resolved by SDS-PAGE and the
fluorescent labeling was detected. The fluorescent image and
the corresponding Coomassie Blue-stained gel are shown in
Figure 3. With 100 µM 6-alkyne-NAD, a strong fluorescent band
was observed at the top of the gel (Figure 3, lane 3), suggesting
that PARP-1 was extensively poly(ADP-ribosyl)ated. Cor-
respondingly, the intensity of the unmodified PARP-1 band was
decreased compared with the control in lane 1. When 100 µM
NAD was included in the labeling reaction with 6-alkyne-NAD,
in addition to the top fluorescent band, a smear of fluorescence
was also observed above the unmodified PARP-1 protein band
(Figure 3, lane 5). This smear is likely PARP-1 modified to a
lesser degree. A smear of fluorescence signal below 116 kDa
was also observed. This is likely due to the hydrolysis of
poly(ADP-ribose) chain resulting in poly(ADP-ribose) polymers
that are not covalently bound to proteins. The linkage between
different ADP-ribose units is a relatively labile ester bond. This
result suggests that the presence of normal NAD does not
decrease the labeling efficiency with 6-alkyne-NAD and may
in fact increase it. This is possibly because 6-alkyne-NAD is
not a good substrate for initiation of poly(ADP-ribosyl)ation,
but after a poly(ADP-ribose) chain is formed with the better
substrate NAD, then 6-alkyne-NAD becomes a better substrate
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Figure 4. Labeling of p53 and RAP74 subunit of TFIIF by PARP-1 using 6-alkyne-NAD (A) and 8-alkyne-NAD (B). The panel on the left shows the image
of Rhodamine fluorescence recorded by Typhoon 9400 Variable Mode Imager, and the panel on the right is the same gels stained with Coomassie Blue. In
addition to 6- or 8-alkyne-NAD (100 µM), the following were present in different lanes: 1 and 2, PARP-1 (0.15 µM); 3 and 4, PARP-1 (0.15 µM) and p53
(2.8 µM); 5 and 6, PARP-1 (0.15 µM) and RAP74 (0.59 µM); 7 and 8, p53 (2.8 µM); 9 and 10, RAP74 (0.59 µM). ssDNA was present in all lanes, and
normal NAD (100 µM) was present in lanes 2, 4, 6, 8, and 10. The fluorescence signal in the loading wells likely came from poly(ADP-ribosyl)ated PARP-1.
Labeling of Known Substrate Proteins of PARP-1 and
Tankyrase-1. It has been reported that PARP-1 can modify many
other proteins,34 even though the major substrate is PARP-1
itself.2 To make sure that 6-alkyne-NAD is a robust substrate
for PARP-1, we tested whether modification of other substrate
proteins can be detected. For this purpose, we chose to use p53
and TFIIF, both of which have previously been reported to be
PARP-1 targets.30-33,35-37 The RAP74 subunit of TFIIF or p53
was incubated with full-length PARP-1 and 6- or 8-alkyne-NAD,
followed by click chemistry to conjugate Rh-N3. The results
shown in Figure 4 demonstrate that p53 can be poly(ADP-
ribosyl)ated by PARP-1 when 6-alkyne-NAD was used, both
in the absence and presence of normal NAD (Figure 4A, lanes
3 and 4). The smear over p53 protein band is poly(ADP-
ribosyl)ated p53. The RAP74 subunit of TFIIF can be labeled
with 6-alkyne-NAD when normal NAD was present (Figure 4A,
lane 6), but not when normal NAD was not present (Figure
4A, lane 5). Comparing lane 6 to lane 2 in Figure 4A, the extra
smear on the top is poly(ADP-ribosyl)ated RAP74. With
8-alkyne-NAD, neither p53 nor RAP74 was labeled (Figure 4B).
for subsequent elongation of the poly(ADP-ribose) chain,
leading to higher labeling efficiency. Without ssDNA (lane 7),
the fluorescence signal is comparable to the negative controls
(lanes 1 and 2), consistent with the low activity of PARP-1 in
the absence of DNA strand breaks. The other analogue,
8-alkyne-NAD, can also label PARP-1 automodification. How-
ever, it is less efficient than 6-alkyne-NAD. Even in the presence
of normal NAD, the extensive poly(ADP-ribosyl)ation band at
the top of the gel is weak with 8-alkyne-NAD (Figure 3, lane
6). It is possible that attaching the alkyne group at position 8
of the adenine ring produces steric clash when it binds to PARP-
1.
To compare the labeling results with clickable NAD ana-
logues to that obtained with 32P-NAD, we carried out similar
labeling experiments with 32P-NAD (Supporting Information,
Figure S1). Comparing the results shown in Figure 3 and Figure
S1, the labeling results obtained with 6-alkyne-NAD is very
similar to that obtained with 32P-NAD, particularly when 100
µM normal NAD was present (Figure 3 lane 5 and Figure S1
lane 3). This result suggests that 6-alkyne-NAD can be used to
label and detect PARP-1 automodification.
We further demonstrated that two PARP-1 truncations, PARP-
1(374-524, the automodification domain) and PARP-
1(374-1014, the automodification domain plus the catalytic
domain), can be labeled with PARP-1 full-length and 6-alkyne-
NAD (Supporting Information, Figure S2). In the presence of
100 µM NAD and 6-alkyne NAD, the labeling of PARP-
1(374-1014) reached maximum levels in about 6 min (Sup-
porting Information, Figure S3). The labeling of as little as 2.5
pmol of PARP-1 (374-1014) can be detected (Supporting
Information, Figure S4).
To further confirm that 6-alkyne-NAD is a robust substrate
for labeling poly(ADP-ribosyl)ated proteins, we wanted to find
out whether substrate proteins of other PARPs can also be
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Figure 5. Labeling of TRF1 by tankyrase-1 using 6-alkyne-NAD. The panel on the left shows the image of Rhodamine fluorescence, and the panel on the
right is the same gel stained with Coomassie Blue. Lanes 1 and 2, tankyrase-1 suspension (0.09 µg/µL) with 6-alkyne-NAD (100 µM); 3 and 4, tankyrase-1
suspension (0.09 µg/µL) and TRF1 (1.1 µM) with 6-alkyne-NAD (100 µM); 5 and 6, TRF1 (1.1 µM) with 6-alkyne-NAD (100 µM); 7 and 8, suspension
of the insoluble fraction of noninfected SF9 cell lysate (0.15 µg/µL, negative control) and TRF1 (1.1 µM) with 6-alkyne-NAD (100 µM). Lanes 2, 4, 6, and
8 contained normal NAD (100 µM).
Table 1. Kinetics of PARP-1 and Tankyrase-1 with NAD, 6- and 8-Alkyne-NAD
PARP activity
NADase activity
PARPs
substrates
kcat (s-1
)
Km (µM)
kcat/Km (s-1 M-1
)
kcat (s-1
)
Km (µM)
kcat/Km (s-1 M-1
)
PARP-1
NAD
5.2 ( 0.1
0.73 ( 0.04
N/A
0.51 ( 0.04
N/A
97 ( 7
147 ( 23
N/A
1125 ( 133
N/A
5.4 × 104
5.0 × 103
0.51
453
122
0.99 ( 0.06
0.94 ( 0.1
N/A
0.54 ( 0.02
N/A
203 ( 33
1891 ( 273
N/A
607 ( 29
N/A
4.88 × 103
6-alkyne-NAD
8-alkyne-NAD
NAD
497
a
11.90
890
252
Tankyrase-1
6-alkyne-NADa
a
8-alkyne-NAD
N/A
N/A
0.29
N/A
N/A
3.41
a The reaction rate of PARP-1 or Tankyrase-1 with 6- or 8-alkyne-NAD was linear with the 6- or 8-alkyne-NAD concentration used (10 µM to 1
mM). Thus, kcat and Km could not be measured and only kcat/Km was obtained.
labeled. For this purpose, we used tankyrase-1, which can
regulate telomere length and mitosis by modifying TRF1 and
NuMA, respectively. Using the insect cell expression system
developed by de Lange and co-workers,21 we expressed and
purified tankyrase-1 and TRF1. The labeling reactions were then
carried out, similarly to those above. Consistent with the reported
results obtained with 32P-NAD,21 we observed efficient TRF1
modification in the presence of tankyrase-1 and 6-alkyne-NAD
(Figure 5A, lanes 3 and 4), but not in the absence of tankyrase-1
(Figure 5A, lanes 5-8). These results suggest that 6-alkyne-
NAD is a robust substrate, and may be applicable to other
PARPs.
Kinetics of PARP-1 and Tankyrase-1 Labeling with
6-Alkyne-NAD. To quantify how efficient 6-alkyne-NAD is as
a PARP substrate, we measured the steady state kinetics of
NAD, 6-alkyne-NAD, and 8-alkyne-NAD in the PARP-1 and
Tankyrase-1 automodification reactions. Previously, the kinetics
assays were typically done by measuring the PARP-1 auto-
modification using radiolabeled NAD.38,39 Recently, a chro-
mogenic NAD analogue was also reported.40 However, both
methods cannot be directly applied to the kinetic assay with
6-alkyne-NAD. Thus, we developed a HPLC-based assay to
measure the release of nicotinamide. To correct for the NADase
acitivity (hydrolysis of NAD to ADP-ribose and nicotinamide),
we also measured the release of ADP-ribose, 6-alkyne-ADP-
ribose, or 8-alkyne-ADP-ribose. The kcat and Km values for the
PARP activity and NADase activity are shown in Table 1.
Compared with NAD, the kcat/Km value of 6-alkyne-NAD is 12-
fold and 4-fold lower for PARP-1 and Tankyrase-1, respectively.
In contrast, 8-alkyne-NAD is a much worse substrate for both
PARP-1 and Tankyrase 1, consistent with the labeling results
(Figures 3 and 4). Although 6-alkyne-NAD has a lower catalytic
efficiency compared with NAD, the labeling efficiency will not
be affected significantly because in practice a much higher
6-alkyne-NAD concentration can be used, particularly when
comparing to 32P-NAD, the concentration of which in labeling
reactions is limited to a few micromolar by the commercially
available 32P-NAD.
Identification of Substrate Proteins of PARP-1 Using
6-Alkyne-NAD. Since 6-alkyne-NAD is a robust substrate for
PARP-1, we then tried to use 6-alkyne-NAD to identify substrate
proteins of PARP-1 from cell lysate. We used both MCF-7 wild-
type and PARP-1 knockdown (KD) cell lysate as the source of
the PARP-1 substrate proteins. After the lysate was incubated
with 6-alkyne-NAD, click chemistry was used to conjugate the
Rh-N3 fluoroscent dye. The fluorescence image shown in
Figure 6 suggests that incubation of the cell lysates with PARP-1
and 6-alkyne-NAD indeed led to the poly(ADP-ribosyl)ation
of proteins in the cell lysates.
To identify the proteins that are modified by PARP-1, an
affinity tag, Biotin-N3, was used for purification and isolation.
MCF-7 PARP-1 KD cell lysate was used as the source of the
PARP-1 substrate proteins. In the PARP-1 KD cells, because
PARP-1 level is decreased (Supporting Information, Figure
S5),41 fewer PARP-1 substrate proteins should be poly(ADP-
ribosyl)ated. Thus, more substrate proteins may be available
for labeling in the in Vitro reactions containing recombinant
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Jiang et al.
Figure 6. Labeling of MCF-7 wild-type and PARP-1 KD cell lysate with PARP-1 and 6-alkyne-NAD. The panel on the left shows the image of Rhodamine
fluorescence, and the panel on the right is the same gel stained with Coomassie Blue. PARP-1 (0.075 µM), MCF-7 wild-type cell lysate (2 µg/µL) and
PARP-1 KD cell lysate (2 µg/µL) were used in the labeling reactions. All lanes contain 6-alkyne-NAD (100 µM) and ssDNA (0.25 µg/µL). Lanes 6-10
contained normal NAD (100 µM).
PARP-1 and 6-alkyne-NAD. For negative controls, we used 100
µM PJ34 (a PARP-1 inhibitor) or leave out 6-alkyne-NAD in
the reactions.
a positive hit if the extracted ion chromatograms confirmed that
the protein was more abundant in experimental samples than
in control samples. The top 45 identified proteins are shown in
Table 2. Among the 45 proteins, 6 of them are known substrate
proteins of PARP-1, such as PARP-1 (automodification),
histones, and heterogeneous nuclear ribonucleoproteins. In
addition, 18 of the 45 proteins have been identified as potential
poly(ADP-ribosyl)ated proteins in a proteomic study using PAR
antibody.44 These results demonstrate that the strategy of using
6-alkyne-NAD to label and identify PARP-1 substrate proteins
works. Not all known substrate proteins of PARP-1 were
identified. One possibility is that these proteins have low
concentrations in cells and thus are difficult to identify.
Alternatively, these proteins may be conditional substrates that
can only be poly(ADP-ribosyl)ated under certain cellular
conditions.
Among the proteins identified, our attention is drawn to
several mitochondrial proteins. Although most known PARP-1
substrates are nuclear proteins, PARP-1’s mitochondrial local-
ization has been confirmed recently.45 This suggests that
PARP-1 potentially has more substrate proteins in mitochondria.
Consistent with this, several mitochondria proteins have been
identified as potential PARP substrates in a recent proteomic
study.46 To further confirm that identified potential substrate
proteins are indeed substrates of PARP-1, we chose tubulin and
three mitochondrial proteins TRAP1 (TNF receptor-associated
protein 1), citrate synthase, and GDH (glutamate dehydrogenase
1) to further validate the results. Tubulin (emPAI ratio 3.86) is
a structural protein and was previously identified as a potential
PARP substrate.44 TRAP1 (emPAI ratio 1.32) is a mitochondrial
After incubation of PARP-1 and 6-alkyne-NAD with MCF-7
PARP-1 KD cell lysate, click chemistry was carried out to
conjugate Biotin-N3. Controls were treated similarly. Following
the published protocol,42 labeled proteins were affinity purified
and isolated using streptavidin beads, and the samples were
prepared and analyzed by nanoLC-MS/MS. It should be noted
that proteins from the labeling reactions were denatured and
precipitated out with acetone, then solubilized with SDS buffer
for affinity purification with streptavidin beads. Under the
denaturing conditions, noncovalent interactions between
protein-protein or protein-DNA/RNA are minimized, unlike
the use of PAR antibodies or macrodomains that use native
conditions. Therefore, this affinity purification and identifica-
tion by nanoLC-MS/MS will give fewer false positive results,
making the results more reliable.
About 300 proteins were identified from each sample,
including negative controls. A protein is considered a potential
substrate proteins if more than 2 peptides are identified in g2
out of 3 samples and it is more abundant in the 3 experimental
samples than in the 3 negative controls. The quantification is
based on exponentially modified protein abundance index
(emPAI) values.43 Among the ∼300 proteins identified, 79
proteins with emPAI ratio (emPAI value in experimental sample/
emPAI value in negative control) greater than 1.20 were
identified as potential PARP-1 substrates (Table S1, Supporting
Information). For proteins with emPAI ratios smaller than 2.0,
up to three common peptide ions for each protein found in
experimental samples and control samples were compared using
extracted ion chromatograms.43 A protein was only considered
(44) Gagne, J.-P.; Isabelle, M.; Lo, K. S.; Bourassa, S.; Hendzel, M. J.;
Dawson, V. L.; et al. Nucleic Acids Res. 2008, 36, 6959–6976.
(49) Hassa, P. O.; Hottiger, M. O. Front. Biosci. 2008, 13, 3046–3082.
(45) Rossi, M. N.; Carbone, M.; Mostocotto, C.; Mancone, C.; Tripodi,
M.; Maione, R.; et al. J. Biol. Chem. 2009, 284, 31616–31624.
(46) Lai, Y.; Chen, Y.; Watkins, S. C.; Nathaniel, P. D.; Guo, F.; Kochanek,
P. M.; et al. J. Neurochem. 2008, 104, 1700–1711.
(42) Weerapana, E.; Speers, A. E.; Cravatt, B. F. Nat. Protoc. 2007, 2,
1414–1425.
(43) Schulze, W. X.; Usadel, B. Annu. ReV. Plant Biol. 2010, 61, 491–
516.
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Table 2. Potential Substrate Proteins of PARP-1 Identified from MS Using the Labeling Strategy with 6-Alkyne-NADa
potential PARP
substrates identified in
other proteomic studies
protein
accession name
known substrates
of PARP-1
description
MW (Da)
ATP5L_HUMAN
BAP31_HUMAN
CH10_HUMAN
COX41_HUMAN
DDX17_HUMAN
DDX5_HUMAN
DHE3_HUMAN
EF1G_HUMAN
ETFA_HUMAN
FUBP2_HUMAN
FUS_HUMAN
GLYM_HUMAN
GSTK1_HUMAN
H2B1C_HUMAN
H31T_HUMAN
HNRPL_HUMAN
HNRPM_HUMAN
HP1B3_HUMAN
HXK1_HUMAN
IDH3A_HUMAN
IDHP_HUMAN
IMB1_HUMAN
KU70_HUMAN
LETM1_HUMAN
MATR3_HUMAN
MPCP_HUMAN
NPM_HUMAN
PARP1_HUMAN
PHB_HUMAN
QCR2_HUMAN
RBM39_HUMAN
RL19_HUMAN
RL26L_HUMAN
RL7_HUMAN
RS14_HUMAN
RS2_HUMAN
RS24_HUMAN
RS3_HUMAN
SFRS1_HUMAN
SFRS6_HUMAN
SFRS7_HUMAN
TBB2C_HUMAN
TMED9_HUMAN
TR150_HUMAN
UBIQ_HUMAN
ATP synthase subunit g
B-cell receptor-associated protein 31
10 kDa heat shock protein
Cytochrome c oxidase subunit 4 isoform 1
Probable ATP-dependent RNA helicase DDX17
Probable ATP-dependent RNA helicase DDX5
Glutamate dehydrogenase 1
11421
28031
10925
19621
72953
69618
61701
50429
35400
73443
53622
56414
25594
13811
15613
64720
77749
61454
103561
40022
51333
98420
70084
83986
95078
40525
32726
113811
29843
48584
59628
23565
17246
29264
16434
31590
15413
26842
27842
39677
27578
50255
27374
108658
8560
reported in ref 44
reported in ref 44
reported in ref 44
Elongation factor 1-gamma
Electron transfer flavoprotein subunit alpha
Far upstream element-binding protein 2
RNA-binding protein FUS
Serine hydroxymethyltransferase
Glutathione S-transferase kappa 1
Histone H2B type 1-C/E/F/G/I
Histone H3.1t
Heterogeneous nuclear ribonucleoprotein L
Heterogeneous nuclear ribon ucleoprotein M
Heterochromatin protein 1-binding protein 3
Hexokinase-1
Isocitrate dehydrogenase [NAD] subunit alpha
Isocitrate dehydrogenase [NADP]
Importin subunit beta-1
ATP-dependent DNA helicase 2 subunit 1
LETM1 and EF-hand domain-containing protein 1
Matrin-3
Phosphate carrier protein
Nucleophosmin
Poly [ADP-ribose] polymerase 1
Prohibitin
Cytochrome b-c1 complex subunit 2
RNA-binding protein 39
60S ribosomal protein L19
60S ribosomal protein L26-like 1
60S ribosomal protein L7
40S ribosomal protein S14
40S ribosomal protein S2
40S ribosomal protein S24
40S ribosomal protein S3
Splicing factor, arginine/serine-rich 1
Splicing factor, arginine/serine-rich 6
Splicing factor, arginine/serine-rich 7
Tubulin beta-2C chain
Transmembrane emp24 domain-containing protein 9
Thyroid hormone receptor-associated protein 3
Ubiquitin
reviewed in ref 49
reviewed in ref 49
reported in ref 44
reported in ref 44
reported in ref 44
reviewed in ref 49
reviewed in ref 49
reported in ref 44
reported in ref44
reviewed in ref 49
reviewed in ref 49
reported in ref 44
reported in ref 44
reported in ref 44
reported in ref 44
reported in ref 44
reported in ref 44
reported in ref 44
reported in ref 44
reported in ref 44
reported in ref 44
a Top 45 potential substrate proteins of PARP-1 (emPAI ratio >1.50) were listed. References are given for known substrate proteins of PARP-1 and
potential PARP substrate proteins identified in other proteomic studies. Full list of identified potential substrate proteins of PARP-1 is given in
Supporting Information.
heat shock protein 75 with antioxidant and antiapoptotic
functions.47 It has been shown to protect cells from apoptosis
induced by DNA damaging reagents. GDH (emPAI ratio 2.47)
catalyzed the conversion of glutamate to R-ketoglutarate, which
feeds into the tricarboxylic acid (TCA) cycle for energy
production. Citrate synthase (emPAI ration 1.25) is an enzyme
in the TCA cycle. Tubulin and GDH were both identified in a
previous proteomic studies as potential PARP substrates but they
have not been confirmed in in Vitro enzyme assays.44 TRAP1
and citrate synthase have not been known to be PARP-1
substrates. TRAP1 can be expressed recombinantly,48 while
GDH, citrate synthase, and tubulin are commercially available.
Thus, we tested whether these proteins can be poly(ADP-
riboyl)ated by PARP-1 in Vitro using 6-alkyne-NAD. Comparing
lanes 3 and 4 with lanes 1 and 2 (Figure 7), it is clear that all
four proteins were poly(ADP-ribosyl)ated by PARP-1 when
normal NAD was present. GDH was modified even when no
normal NAD was present. These results further confirm that
the proteins we identified as PARP-1 substrates should be highly
reliable. Thus, the strategy of labeling, isolating, and identifying
PARP-1 substrate proteins with 6-alkyne-NAD is successful.
The substrate proteins that we identified for PARP-1 should be
very useful for understanding the biological function of PARP-1
and the strategy should be similarly applicable to other PARPs.
Conclusions
We have demonstrated that the clickable 6-alkyne-NAD is
an efficient substrate that can be used to label poly(ADP-
ribosyl)ated proteins. In contrast, 8-alkyne-NAD is not an
efficient substrate for PARPs. Using the alkyne functional group,
(47) Landriscina, M.; Amoroso, M. R.; Piscazzi, A.; Esposito, F. Gynecol.
Oncol. 2010, 117, 177–182.
(48) Felts, S. J.; Owen, B. A. L.; Nguyen, P.; Trepel, J.; Donner, D. B.;
Toft, D. O. J. Biol. Chem. 2000, 275, 3305–3312.
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Jiang et al.
Figure 7. Labeling of TRAP1 (A), GDH (B), citrate synthase (C), and tubulin (D) by PARP-1 using 6-alkyne-NAD. The panel on the left shows the image
of Rhodamine fluorescence, and the panel on the right is the same gels stained with Coomassie Blue. All lanes contain 6-alkyne-NAD (100 µM) and ssDNA
(0.25 µg/µL). Lanes 1 and 2, PARP-1 (0.05 µM); 3 and 4, PARP-1 (0.05 µM) with TRAP1 (1.0 µM, A) or GDH (3.1 µM, B) or citrate synthase (3.37 µM,
C) or tubulin (3.64 µM, D); 5 and 6, TRAP1 (1.0 µM, A), GDH (3.1 µM, B), citrate synthase (3.37 µM, C), or tubulin (3.64 µM, D). Lanes 2, 4, and 6
contained normal NAD (100 µM).
insect cells by FLAG M2 affinity chromatography following
published procedures.8 The PARP-1 truncation (374-524) was
PCR-amplified using primers 5′-CACCGCCTCGGCTCCTGCT-
GCT-3′ and 5′-TTATTTCATTCTCTTTTCAGATTTGTT-3′. The
PARP-1 truncation (374-1014) was PCR-amplified using primers
5′-CACCGCCTCGGCTCCTGCTGCT-3′ and 5′-TTACCACAGG-
GAGGTCTTAAAAT-3′. Amplified products were cloned using
TOPO and Gateway cloning technology (Invitrogen Corporation,
Carlsbad, CA) into pDEST-F1 (PARP-1(374-1014)) and pDEST-
566 (PARP-1(374-524)) for expression. PARP-1 (374-524) and
PARP-1 (374-1014) were expressed in Escherichia coli BL21
pRARE2 strain. Cells were first cultured in LB media at 37 °C
and 200 rpm to OD600 ∼0.5 in MaxQ 5000 shaker (Barnstead
International, Dubuque, IA). The temperature was then changed to
15 °C and the expression was induced by adding isopropyl ꢀ-D-
thiogalactoside (IPTG) to 0.1 mM. After incubation at 15 °C for
24 h, cells were harvested by centrifugation at 6370g for 10 min.
After resuspended in lysis buffer (20 mM Tris-HCl, 500 mM NaCl,
30 mM imidazole, pH 8.0) at 4 °C, cells were lysed using an
EmulsiFlex-C3 cell disruptor (AVESTIN, Inc., Canada), and cell
lysate was clarified by centrifugation at 48 400g for 30 min at 4
°C. Protein was purified by Bio-Rad BioLogic DuoFlow FPLC
using a HisTrap HP 5 mL column (GE Healthcare) with a linear
gradient of imidazole from 30 to 500 mM in elution buffer (20
mM Tris-HCl, 500 mM NaCl, pH 8.0). Fractions containing
PARP-1 proteins were collected and dialyzed to 25 mM Tris, pH
6.0, 50 mM NaCl (for PARP-1 (374-1014)) or to 25 mM Tris,
pH 8.0, 50 mM NaCl, 1 mM DTT (for PARP-1 (374-524)) at 4
°C. Protein concentrations were determined by Bradford assay or
by comparing Coomassie brilliant blue staining with BSA standards
after SDS-PAGE.
fluorescent or affinity tags can be conveniently installed on the
substrate proteins to facilitate in-gel visualization and identifica-
tion of the substrate proteins. Over 70 potential novel substrate
proteins of PARP-1 have been identified using the labeling
strategy with 6-alkyne-NAD, including many mitochondrial
proteins. Tubulin, TRAP1, citrate synthase, and GDH were
further proved to be PARP-1 substrate proteins in Vitro. We
believe 6-alkyne-NAD will greatly facilitate the identification
of the substrate proteins of the 17 PARPs in humans and help
to understand their biological functions.
Experimental Section
General Methods. Reagents were obtained from Aldrich in the
highest purity available and used as supplied. Kinetics experiments
were carried out on a SHIMADZU LCMS-QP8000R with a Sprite
TARGA C18 column (40 × 2.1 mm, 5 µm, Higgins Analytical,
Inc.) monitoring at 260 nm. Solvents were 50 mM ammonium
acetate pH 5.4 (buffer A) and 50% methanol in water (buffer B).
Rhodamine fluorescence signal from protein gel was recorded by
Typhoon 9400 Variable Mode Imager (GE Healthcare Life Sci-
ences) with setting of Green (532 nm)/555BP20 PMT450 V (normal
sensitivity), and analyzed by ImageQuant TL v2005. The syntheses
of 6-alkyne-NAD, 8-alkyne-NAD, Rh-N3, and N-(6-aminohexyl)-
azidoacetamide (for synthesis of Biotin-N3) have been reported.26,50
The synthesis of Biotin-N3 is given in the Supporting Information.
ssDNA was purchased from Sigma and used after sonication.
RAP74 (one subunit of TFIIF) was purchased from ProteinOne
(Bethesda, MD). GDH (from bovine liver, type II) and citrate
synthase (from porcine heart) were purchased from Sigma. Tubulin
(from bovine brain) was purchased from Cytoskeleton (Denver,
CO).
Expression of p53. GST-tagged human p53 in pGEX-2T vector
was transformed into E. coli BL21 pRARE2 strain. Cells were first
cultured in LB media at 37 °C and 200 rpm to OD600 ∼0.5. The
temperature was then changed to 15 °C and the expression was
induced by adding isopropyl ꢀ-D-thiogalactoside (IPTG) to 0.1 mM.
After incubation at 15 °C for 24 h, cells were harvested by
Expression of PARP-1 Full-Length and Truncations. FLAG-
tagged human PARP-1 (PARP-1-FLAG) was purified from SF9
(50) Jiang, H.; Congleton, J.; Liu, Q.; Merchant, P.; Malavasi, F.; Lee,
H. C.; et al. J. Am. Chem. Soc. 2009, 131, 1658–1659.
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centrifugation at 6370g for 10 min. Cells were resuspended in GST
binding buffer (4.3 mM Na3PO4, 1.47 mM K3PO4, 137 mM NaCl,
2.7 mM KCl, pH 7.3) at 4 °C, and lysed by using an EmulsiFlex-
C3 cell disruptor. Cell lysate was clarified by centrifugation at
48 400g for 30 min at 4 °C, and incubated with 1 mL of GST-
Bind Resin (Novagen) for 1 h at 4 °C. The resin was washed with
GST binding buffer thoroughly, and then incubated with 150 units
Thrombin (GE Healthcare) for 12 h at 4 °C. The supernatant was
collected and protein was dialyzed into 25 mM Tris-HCl, pH 8.0,
50 mM NaCl, and 1 mM DTT at 4 °C.
Expression of Tankyrase-1 and TRF1. His-tagged human
tankyrase-1 and TRF1 in baculovirus vector were provided from
Dr. de Lange, and proteins were purified from SF9 insect cells
following the published procedure.21 Recombinant tankyrase-1 was
found in the insoluble fraction during the purification. However,
the suspension of insoluble fractions contains active tankyrase-1
and was used in the labeling reactions. The suspension of insoluble
fraction from noninfected SF9 cells was used as the negative control.
Automodification of PARP-1 with 6- or 8-Alkyne-NAD.
PARP-1 (0.15 µM), ssDNA (0.25 µg/µL), and 6- or 8-alkyne-NAD
(100 µM) with or without NAD (100 µM) in 10 µL reaction buffer
(50 mM Tris-HCl, 4 mM MgCl2, 0.2 mM DTT, pH 8.0) were
incubated at 37 °C for 30 min. Control experiments were done
without ssDNA. Then, the click chemistry step was carried out
(described later).
Labeling of p53 and RAP74 by PARP-1 with 6- or 8-Alkyne-
NAD. PARP-1 (0.15 µM), p53 (2.8 µM) or RAP74 (0.59 µM),
ssDNA (0.25 µg/µL), and 6- or 8-alkyne-NAD (100 µM) with or
without NAD (100 µM) in 10 µL reaction buffer were incubated
at 37 °C for 30 min (without NAD) or 6 min (with NAD). Shorter
incubation time was used because if longer incubation time is used,
the protein will be extensively poly(ADP-ribosyl)ated and cannot
be distinguished from the automodified PARP-1. Control experi-
ments were done without PARP-1 or p53 or RAP74. Then, the
click chemistry step was carried out.
Labeling of TRAP1, GDH, Citrate Synthase, And Tubulin
by PARP-1 with 6-NAD. PARP-1 (0.05 µM), TRAP1 (1.0 µM,
overexpressed and purified according to published procedures48),
GDH (3.1 µM), citrate synthase (3.37 µM), tubulin (3.64 µM),
ssDNA (0.25 µg/µL), and 6-alkyne-NAD (100 µM) with or without
NAD (100 µM) in 10 µL reaction buffer were incubated at 37 °C
for 30 min (without NAD) or 6 min (with NAD). Control
experiments were done without PARP-1 or without TRAP1, GDH,
citrate synthase, or tubulin. Then, the click chemistry step was
carried out.
Labeling of TRF1 by Tankyrase-1 with 6- or 8-Alkyne-
NAD. Tankyrase-1 pellet suspension (0.09 µg/µL) or noninfected
SF9 cell pellet suspension (0.15 µg/µL), TRF1 (1.1 µM), and 6- or
8-alkyne-NAD (100 µM) with or without NAD (100 µM) in 10
µL reaction buffer were incubated at 37 °C for 30 min. Control
experiments were done without tankyrase-1 or TRF1. Then, the
click chemistry step was carried out.
Click Chemistry Condition to Conjugate Rh-N3 and
Detection of Poly(ADP-ribosyl)ation by Fluorescence. Rh-N3
(in DMF) was added to the above labeling reactions to a final
concentration of 200 µM, followed by the addition of Tris[(1-
benzyl-1H-1,2,3-triazol-4-yl)methyl]amine51 (in DMF, final con-
centration 600 µM), CuSO4 (in water, final concentration 1 mM),
and TCEP (in water, final concentration 1 mM).52 After the click
chemistry was allowed to proceed at room temperature for 15 min,
the reaction mixture was mixed with 10 µL of 2× protein loading
buffer and heated at 100 °C for 6 min. The samples were then
resolved by SDS-PAGE using 12% acrylamide gel. Before staining
with Coomassie blue, the fluorescence image of the gel was
recorded by Typhoon 9400 imager. The image of protein gel after
Coomassie blue staining was recorded with a digital camera (Canon
PowerShot S3).
Kinetics of PARP-1 with NAD, 6-Alkyne-NAD, and 8-Alkyne-
NAD as Substrates. PARP-1 (0.065 µM), ssDNA (0.05 µg/µL)
with different concentrations of NAD, 6-alkyne-NAD, or 8-alkyne-
NAD (from 10 µM to 1 mM) in 30 µL reactions (50 mM Tris-
HCl, 4 mM MgCl2, pH 8.0) were incubated at 25 °C for 1 min
(with NAD), 15 min (with 6-alkyne-NAD), or 60 min (with
8-alkyne-NAD). The reactions were quenched with 1 M HClO4,
and then neutralized with 3 M K2CO3 5 min later. After centrifuga-
tion, the supernatant was analyzed by a SHIMADZU LCMS-
QP8000R with a Sprite TARGA C18 column (40 × 2.1 mm, 5
µm, Higgins Analytical, Inc.) monitoring at 260 nm. Solvents were
50 mM ammonium acetate pH 5.4 (buffer A) and 50% methanol
in water (buffer B). For PARP-1 with NAD reactions, compounds
were eluted at a flow rate of 0.3 mL/min with 0% solvent B for 1
min, followed by a linear gradient of 0-3% solvent B over 14
min, and back to 0% solvent B over 2 min. Retention times of
ADP-ribose, nicotinamide and NAD were 1.89, 3.52, and 8.01 min,
respectively. For PARP-1 with 6-alkyne-NAD or 8-alkyne-NAD
reactions, compounds were eluted at a flow rate of 0.3 mL/min
with 0% solvent B for 1 min, followed by a linear gradient of 0-1%
solvent B over 5 min, then 1-50% solvent B over 5 min, and finally
50% solvent B for 1 min before equilibrating the column back to
0% solvent B over 2 min. Retention times of nicotinamide,
6-alkyne-ADP-ribose, 8-alkyne-ADP-ribose, 6-alkyne-NAD, and
8-alkyne-NAD were 3.52, 8.42, 6.98, 10.59, and 10.65 min,
respectively. Reaction progress was monitored by the formation
of nicotinamide, ADP-ribose, 6-alkyne-ADP-ribose, and 8-alkyne-
ADP-ribose. The quantification of nicotinamide, ADP-ribose,
6-alkyne-ADP-ribose, and 8-alkyne-ADP-ribose produced in the
reaction was analyzed by the integration of absorption peak
monitored at 260 nm comparing with the plot of nicotinamide, ADP-
ribose, 6-alkyne-ADP-ribose, and 8-alkyne-ADP-ribose standards.
The kcat and Km values were obtained by curve-fitting the V/[E] ∼
[S] plot by KaleidaGraph. NADase activity was obtained from the
formation of ADP-ribose, 6-alkyne-ADP-ribose, and 8-alkyne-ADP-
ribose. PARP activity was obtained from the formation of nicoti-
namide after deduction of ADP-ribose, 6-alkyne-ADP-ribose, and
8-alkyne-ADP-ribose.
Kinetics of Tankyrase-1 with NAD, 6-alkyne-NAD, and
8-alkyne-NAD as Substrates. Tankyrase-1 (0.03 µM) with dif-
ferent concentrations of NAD, 6-alkyne-NAD, or 8-alkyne-NAD
(from 10 µM to 1 mM) in 30 µL reactions (50 mM Tris-HCl, 4
mM MgCl2, pH 8.0) were incubated at 25 °C for 30 min (with
NAD), 2 h (with 6-alkyne-NAD), or 13 h (with 8-alkyne-NAD).
Then, the reactions were quenched and handled same as PARP-1
reactions for kinetics. NADase and PARP activities of tankyrase-1
were measured same as PARP-1 kinetics.
Cell Lysate of MCF-7 Wild-Type and PARP-1 KD Cells.
MCF-7 wild-type and PARP-1 KD cells41 from 10 10-cm plates
(90% confluency) were lysed by Dounce Homogenizer in 5 mL of
lysis buffer (25 mM Tris, 50 mM NaCl, 10% glycerol, pH 7.4)
with 100 µL of protease inhibitor cocktail (Sigma, Saint Louis,
MO). The cell lysate was then centrifuged at 2000g for 15 min at
4 °C. Under this condition, the pellet contained nuclei, mitochondria,
and other organelles. The pellet was solubilized in 4 mL of lysis
buffer with 1% NP-40 and 100 µL of protease inhibitor cocktail.
After centrifugation at 14 000g for 5 min at 4 °C, the supernatant
was collected as cell lysate for later labeling reactions with PARP-1
and 6-alkyne-NAD.
Labeling of MCF-7 Wild-Type and PARP-1 KD Cell
Lysate by PARP-1 with 6-Alkyne-NAD. PARP-1 (0.075 µM),
MCF-7 wild-type cell lysate (2 µg/µL), or PARP-1 KD cell lysate
(2 µg/µL), ssDNA (0.25 µg/µL), and 6-alkyne-NAD (100 µM) with
or without NAD (100 µM) in 10 µL of reaction buffer with 0.5%
NP-40 were incubated at 37 °C for 30 min. Control experiments
(51) Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.;
Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192–3193.
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Chem., Int. Ed. 2002, 41, 2596–2599.
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were done without PARP-1 or without cell lysates. Then, the click
chemistry step was carried out.
dependent acquisition (DDA) mode using FT mass analyzer for
one survey MS scan for precursor ions followed by MS/MS scans
on top 7 most intensive peaks with multiple charged ions above a
threshold ion count of 5000 in LTQ mass analyzer. MS survey
scans at a resolution of 60 000 (fwhm at m/z 400), for the mass
range of m/z 375-1400. Dynamic exclusion parameters were set
at repeat count 1 with a 20 s repeat duration, exclusion list size of
500, 30 s exclusion duration, and (10 ppm exclusion mass width.
Collision induced dissociation (CID) parameters were set at the
following values: isolation width 2.0 m/z, normalized collision
energy 35%, activation Q at 0.25, and activation time 30 ms. All
data are acquired under Xcalibur 2.1 operation software (Thermo-
Fisher Scientific).
Data Analysis. All MS and MS/MS raw spectra were processed
using Proteome Discoverer 1.1 (PD1.1, Thermo) and the spectra
from each DDA file are output as an MGF file for subsequent
database search using in-house license Mascot Deamon (version
2.2.04, Matrix Science, Boston, MA). The human protein sequence
database containing 20 349 sequence entries in the Swiss-Prot
database downloaded on January 21, 2010 was used for database
search and the search was performed to query to Swiss-Prot database
(taxonomy: human) with one missed cleavage site by trypsin
allowed. The peptide tolerance was set to 10 ppm and MS/MS
tolerance was set to 0.8 Da. A fixed carbamidomethyl modification
of cysteine, variable modifications on methionine oxidation and
deamindation of asparagine and glutamine were set. Data filtering
parameters were as follows: (a) the peptide identity probability is
99.9%CI with 35 peptide score cutoff; (b) only top matching
proteins with p < 0.001 (expectation value) were considered in the
analysis, and (c) at least two distinct peptides met above criteria
hit for each proteins as a finally identified protein list and used for
obtaining exponentially modified protein abundance index (emPAI)
number for each identified proteins. After filtering, the emPAIs
outputted directly from Mascot results for each identified proteins
were used for estimation of relative protein abundance in three
experimental samples versus the three negative control samples. A
protein is considered a potential PARP-1 substrate only if the emPAI
ratio (experimental/control) is more than 1.20. If the emPAI ration
is between 2.0 and 1.20, the emPAI-based relative protein quan-
tification results were also manually validated by averaging
precursor ion peak areas of up to three top score peptides (found
in both experimental samples and control samples) for each protein
using extracted ion chromatograms in PD1.1 software.
Pull-down of Substrate Proteins of PARP-1 Using
6-Alkyne-NAD. Cell lysate of MCF-7 PARP-1 KD cells (2 mg)
was incubated with PARP-1 (1.8 µg), 6-alkyne-NAD (10 nmol,
final concentration 10 µM), and ssDNA (20 µg) in 1 mL of reaction
buffer at 37 °C for 30 min with gentle rotating. Control experiments
were done with 100 µM PJ34 (a PARP-1 inhibitor) or without
6-alkyne-NAD. Then, the click chemistry step was carried out with
Biotin-N3 (in DMF, final concentration 16 µM), Tris[(1-benzyl-
1H-1,2,3-triazol-4-yl)methyl]amine (in DMF, final concentration
200 µM), CuSO4 (in water, final concentration 800 µM), and TCEP
(in water, final concentration 800 µM) at room temperature for 30
min. The reaction mixture was denatured and precipitated with 10
mL of cold acetone. After centrifugation at 14 000g for 5 min at 4
°C, the precipitate was further washed with 3 × 1 mL of cold
methanol to remove extra small molecule reagents. After solubi-
lizing the precipitate in 1 mL of 2% SDS in PBS with heating at
90 °C for 10 min and centrifugation at 14 000g for 5 min at room
temperature, the supernatant was diluted to 10 mL by adding 9
mL of PBS (final 0.2% SDS in PBS), and incubated with 100 µL
of streptavidin beads (Pierce Biotechnology, Rockford, IL) for 90
min at room temperature with gentle rotating. After centrifugation
at 1000g for 2 min at room temperature, the beads were further
washed with 3 × 1 mL 0.2% SDS in PBS, 3 × 1 mL PBS, 3 × 1
mL (20 mM Tris, 500 mM KCl, pH 7.4), 3 × 1 mL (20 mM Tris,
pH 7.4), and then incubated with 400 µL of 6 M urea in PBS with
9.5 mM TCEP for 20 min at 35 °C with gentle rotating.
Iodoacetamide (20 µL 400 mM in water) was added to the
suspension of beads with further incubation for 20 min at 35 °C
with gentle rotating. After dilution with 760 µL of PBS and removal
of supernatant, the beads were incubated with 2 µg of Trypsin
(Promega, Madison, WI) in 200 µL of 2 M urea in PBS and 1 mM
CaCl2 at 37 °C for 8 h with gently rotating. The supernatant was
collected and combined with washings of 2 × 300 µL water from
beads. Then, the digested peptides in above solution were purified
with Sep-Pak Vac C18 cartridge (Waters Corporation, Milford, MA)
and analyzed by nanoLC-MS/MS. Each experiment was done
individually three times. Proteins identified in the samples but not
in the control experiments were listed in Table 2.
Protein Identification by NanoLC/MS/MS Analyses. The
tryptic digest was reconstituted in 10 µL of 2% acetonitrile (ACN)
with 0.5% formic acid (FA) for nanoLC-ESI-MS/MS analysis,
which is carried out using a LTQ-Orbitrap Velos (Thermo-Fisher
Scientific, San Jose, CA) mass spectrometer equipped with nano
ion source. The Orbitrap is interfaced with an UltiMate3000 MDLC
system (Dionex, Sunnyvale, CA). The nanoLC was carried out by
Dionex UltiMate3000 MDLC system (Dionex, Sunnyvale, CA).
An aliquot of tryptic peptide (3.0 µL) was injected onto a PepMap
C18 trap column (5 µm, 300 µm × 5 mm, Dionex) at 20 µL/min
flow rate for online desalting and then separated on a PepMap C-18
RP nano column (3 µm, 75 µm × 15 cm), and eluted in a 90 min
gradient of 5-38% ACN in 0.1% FA at 300 nL/min., followed by
a 3-min ramping to 95% ACN-0.1% FA and a 5-min holding at
95% ACN-0.1% FA. The column was re-equilibrated with 2%
ACN-0.1% FA for 20 min prior to the next run. The eluted
peptides are detected by Orbitrap through nano ion source contain-
ing a 10-µm analyte emitter (NewObjective, Woburn, MA). The
Orbitrap Velos is operated in positive ion mode with nano spray
voltage set at 1.5 kV and source temperature at 175 °C. Either
internal calibration using the background ion signal at m/z
445.120025 as a lock mass or external calibration for FT mass
analyzer is performed. The instrument is performed at parallel data-
Acknowledgment. H.J. and H.L. thank Donald Ruhl and
Jhoanna Berrocal for help with protein expression in Sf9 cells. We
thank Dr. Jun-Lin Guan (University of Michigan Medical School)
for providing the p53 expression vector, Dr. Titia de Lange
(Rockefeller University) for providing the Sf9 expression cells for
Tankyrase-1 and TRF-1. This work is supported in part by Dreyfus
Foundation (New Faculty Award to H.L.), NIH R01 GM086703
(H.L.), and NIH R01 DK069710 (W.L.K).
Supporting Information Available: Labeling of PARP-1 with
32P-NAD, labeling of PARP-1 truncations by PARP-1, the time
course and the detection limit of the labeling reaction, confirma-
tion of PARP-1 KD in MCF-7 cells, synthesis of Biotin-N3,
the full list of identified potential PARP-1 substrate proteins.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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9372 J. AM. CHEM. SOC. VOL. 132, NO. 27, 2010