IEEE/ACM TRANSACTIONS ON COMPUTATIONAL BIOLOGY AND BIOINFORMATICS, VOL. 11, NO.

5, SEPTEMBER/OCTOBER 2014 915

Mining Conditional Phosphorylation Motifs

Xiaoqing Liu, Jun Wu, Haipeng Gong, Shengchun Deng, and Zengyou He

Abstract—Phosphorylation motifs represent position-specific amino acid patterns around the phosphorylation sites in the set of
phosphopeptides. Several algorithms have been proposed to uncover phosphorylation motifs, whereas the problem of efficiently
discovering a set of significant motifs with sufficiently high coverage and non-redundancy still remains unsolved. Here we present
a novel notion called conditional phosphorylation motifs. Through this new concept, the motifs whose over-expressiveness mainly
benefits from its constituting parts can be filtered out effectively. To discover conditional phosphorylation motifs, we propose an
algorithm called C-Motif for a non-redundant identification of significant phosphorylation motifs. C-Motif is implemented under the
Apriori framework, and it tests the statistical significance together with the frequency of candidate motifs in a single stage.
Experiments demonstrate that C-Motif outperforms some current algorithms such as MMFPh and Motif-All in terms of coverage
and non-redundancy of the results and efficiency of the execution. The source code of C-Motif is available at: https://sourceforge.
net/projects/cmotif/.

Index Terms—Phosphorylation motif, protein phosphorylation, frequent pattern, data mining

1 INTRODUCTION

P ROTEIN phosphorylation is one of the most frequent

post-translational modification events for the regula-
tion and maintenance of most biological processes. This
event plays vital roles in numerous key cellular processes
including metabolism, signal transduction, transcription,
translation, membrane transport, as well as the regulation
of cellular activities such as proliferation, migration, differ-
entiation, and death [1], [2], [3], [4]. The advent of high-
throughput methods has greatly enhanced the investiga-
tions into phosphorylation, typically the technique of
tandem mass spectrometry, which enables rapid and direct
discovery of large-scale phosphorylation sites in a single
experiment [5], [6], [7], [8].
Phosphorylation motifs represent common amino acids
aligned upstream and downstream of the phosphoryla-
tion sites. Phosphorylation motif discovery aims at find-
ing a set of motifs that occur with disproportionate
frequency in two coordinate sequence data sets: the
phosphorylated peptide set P and the unphosphorylated
peptide set N. In this context, P is regarded as the fore-
ground and N corresponds to the background. The identi-
fied interesting motifs are all “over-expressed” in the
foreground, that is, they appear more frequently in the
foreground than that in the background [9], [10]. Such
sets of reported phosphorylation motifs can also provide
information about the specificities of the kinases involved,
reveal the underlying regulation mechanism and facilitate
the prediction of unknown phosphorylation events.
Hence, achieving a rapid phosphorylation motif search

X. Liu, J. Wu, H. Gong, and Z. He are with the School of Software, Dalian
University of Technology, Dalian, China. E-mail: {eileenwelldone,
wujun.myway, haipengxf}@gmail.com, zyhe@dlut.edu.cn.

S. Deng is with the School of Computer Science and Engineering, Harbin
Institute of Technology, Harbin, China. E-mail: dsc@hit.edu.cn.
Manuscript received 9 Nov. 2013; revised 4 Apr. 2014; accepted 21 Apr. 2014.
Date of publication 30 Apr. 2014; date of current version 2 Oct. 2014.
For information on obtaining reprints of this article, please send e-mail to:
reprints@ieee.org, and reference the Digital Object Identifier below.
Digital Object Identifier no. 10.1109/TCBB.2014.2321400
1545-5963 ß 2014 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission.
See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
for gaining a comprehensive understanding of mecha-
nism of protein phosphorylation is an essential work.
The problem of discovering phosphorylation motifs has
been widely studied and several algorithms have already
been proposed. Motif-X [9] employs a greedy algorithm to
identify over-expressed motifs in an iterative manner. This
method is demonstrated to successfully uncover both
known and novel informative motifs. MoDL [10] makes an
attempt to optimize the expressiveness of a set of motifs
instead of quantifying the significance of a single motif. F-
Motif [11] borrows an idea from clustering and combines it
with an iterative greedy search derived from Motif-X. In
fact, the greedy approaches such as Motif-X and F-Motif
may miss some important motifs due to greedy choices and
foreground reduction, and MoDL leaves out a quantity of
significant motifs as well. MMFPh [12] extends Motif-X by
employing a more complete search and can report much
more statistically significant and sufficiently frequent motifs
than Motif-X. Motif-All [13] is two-step procedure: first
extracts a set of frequent motifs from the phosphorylated
peptide data set, and then evaluates the statistical signifi-
cance of frequent motifs using both phosphorylated and
unphosphorylated peptide data sets. Overall, both MMFPh
and Motif-All claim to guarantee the completeness of signif-
icant phosphorylation motifs under their corresponding
definitions [14], [15]. But the lack of consensus on the defini-
tion of significant motifs leads to a gap between the results
of these two methods. In general, Motif-All can ensure the
maximum level of coverage of the potentially interesting
results whereas both MMFPh and Motif-All involve some
redundant motifs.
Despite the significant progress achieved in the past
years, there are still some important issues that remain
unsolved. One challenging problem is how to filter out
those biased motifs whose over-expressiveness mainly
comes from its subsets so as to improve the accuracy of the
identified motif set. For example, compared to other algo-
rithms, experimental results demonstrate that Motif-All per-
forms relatively better by reporting the largest number of
916 IEEE/ACM TRANSACTIONS ON COMPUTATIONAL BIOLOGY AND BIOINFORMATICS,
significant motifs. However, no further statistical correction
is carried out in Motif-All, rendering an abundance of
motifs whose over-expressiveness mainly originates from
some portion of them in the result set. To remove these
redundant motifs, one method is to apply the permutation
test in statistics [16] to post-process the results. However, as
pointed out in [17], the standard permutation test procedure
may not fully address this issue. As a result, they utilize the
“sequential permutation test” method [18] to reduce the
effect of sub-motifs when calculating the statistical signifi-
cance of phosphorylation motifs. However, the permutation
test process is very time-consuming in practice. Another
alternative strategy is to choose a rigorous measure that can
remove the influence of subsets of motifs. Existing methods
such as Motif-X [9] and MMFPh [12] are examples of this
idea. They partially succeed in reducing the redundancy
with more stringent restrictions for pruning strategy. Nev-
ertheless, this is not the end of solution, and some addi-
tional motifs still remain in the results.
Another challenging problem is how to achieve a maxi-
mum coverage such that all frequent and statistically signif-
icant motifs are included in the result. Existing methods
such as Motif-X, F-Motif, MoDL and MMFPh will miss
some motifs that are both sufficiently large and significant
phosphorylation motifs due to their incomplete search or
overstrict requirements on target motifs.
Overall, the redundancy and coverage issues in phos-
phorylation motif discovery are only partially solved.
This paper addresses this problem and takes a further
step towards this direction by proposing a new problem
formulation for phosphorylation motif discovery: condi-
tional phosphorylation motif discovery. Owing to the
problem formulation, the motifs reported would be
“actual” motifs with more accurate statistical significance
scores. This new problem formulation is a non-trivial
extension and generalization of the motif assessment
strategy used in Motif-X and MMFPh. Different from pre-
vious methods, the key feature of our method is that, the
evaluation of significance with respect to one motif is
only relevant to its own inherent property rather than
whether it has significant constitute parts or not. Hence,
we achieve an ideal trade-off between the high coverage
and non-redundancy of significant motif set together with
running efficiency to some extent.
In the following, we will first illustrate the basic idea of
conditional phosphorylation motif and then show how this
concept can remove motifs whose over-expressiveness
mainly comes from their subsets. If a position in one motif is
specified by a certain fixed amino acid, then it is a so-called
conserved position. Otherwise, it is a wild position that can
match any arbitrary amino acid. One motif A is said to be a
k-motif if it has k conserved positions. If another motif B con-
tains only a subset of these k amino acids at the correspond-
ing positions in A, then B is a sub-motif of A. In fact, every
peptide that contains the motif must also contain its corre-
sponding sub-motifs, but the reverse is not true. So the set of
peptides that contain one motif must be a subset of the collec-
tion of peptides that contain its sub-motif. Notably there are
exactly k sub-motifs of size k1 for one k-motif. For each
sub-motif of size k1, we can generate a set of peptides in
which every peptide contains this sub-motif. On this new
VOL. 11, NO. 5, SEPTEMBER/OCTOBER 2014
data set, we can re-calculate the statistical significance of the
k-motif which is called the conditional (or local) significance.
Moreover, we define the statistical significance of a motif
based on the original data set according to the traditional def-
inition as the global significance. In this setting, this k-motif is
claimed as a conditional phosphorylation motif if it is not
only locally significant on all k sub-motif induced data sets
but also globally significant on the whole data sets. Hence,
there will be two parameters to measure the significance of
motifs: the global significance threshold and the local signifi-
cance threshold, respectively. As a result, the effects of its
sub-motifs in the evaluation of statistical significance about
the over-expressiveness are reduced.
To address the problem of conditional phosphorylation
motif mining, we present a new algorithm called C-Motif.
We implement C-Motif by utilizing a support constraint
together with statistical measures in the same mining pro-
cess. Here the support is defined as the percentage of
sequences that contain this motif. One motif is said to be
frequent if its support is no less than a given threshold.
Experiments on real data sets show that our algorithm is
efficient and effective in conditional phosphorylation
motif discovery.
The remainder of this paper is organized as follows:
Section 2 presents the details of C-Motif algorithm. Section 3
shows the experimental results on real data. Section 4 con-
cludes the paper.
2 METHODS
2.1 Basic Terminology
We describe a motif as a string with a single phosphorylated
residue that is denoted with an underlined character, e.g., S,
T or Y. We write the conserved positions of one motif as the
corresponding amino acids directly and its wild positions
are represented by ‘x’. For example, suppose a 2-motif has a
fixed ‘P’ one position downstream and a fixed ‘D’ two posi-
tions upstream as well as a wild position next to the cen-
tered phosphorylation site, this motif is thus represented as
‘PSxD’.
For a k-motif m, we use Supðm; P Þ to denote its support
(frequency) in the foreground data set P . Since the goal of
phosphorylation motif discovery is to find those motifs that
occur more frequently in the foreground data set against the
background data set, we usually only use the foreground to
assess the frequency of a motif.
Definition 1. m is a frequent motif if Supðm; P Þ is no less than
the user-specified threshold usup .
The set of candidate frequent motifs of size k is denoted
as Sk . To discover all frequent motifs, the level-wise search
strategy rooted from the Apriori algorithm [19] is widely
used in practice. It first accumulates the count for each 1-
motif and collects those motifs with larger support than the
given threshold usup to form the set of frequent 1-motifs F1 .
Subsequently, since a k-motif will not be frequent if one of
its sub-motifs of size k1 is infrequent, Fk1 is utilized to
generate Sk and those infrequent ones are pruned to gener-
ate Fk .
Owing to the fact that m consists of k fixed amino acids,
this motif has k sub-motifs of size k1. These motifs
LIU ET AL.: MINING CONDITIONAL PHOSPHORYLATION MOTIFS
subsumed by m are denoted as m1 ; m2 ; . . . ; mk , respectively.
The only difference between m and mi (1  i  k) is that the
ith fixed position in m is non-fixed in mi . And we describe
the sets of peptides in the foreground data P where these
sub-motifs occur as P ðm1 Þ; P ðm2 Þ; . . . ; P ðmk Þ, respectively.
Similarly, we use Nðmi Þ to denote the set of peptides in the
background data N that contain mi . We utilize Sigðm; P; NÞ
to denote the statistical significance calculation function for
each motif m, and it measures the over-expressiveness of m
in P against N. In fact, both the nonparametric measure-
ments such as odds ratio or relative risk and the binomial
probability model have been used in the assessment of
phosphorylation motif [13], [9], [12]. Note that the use of dif-
ferent evaluation methods will not change the nature of the
problem. Generally, these statistical significance assessment
functions are consistent with each other in practice. For the
ease of illustration and a fair comparison of different meth-
ods, here we use relative risk and odds ratio as the representa-
tives of statistical significance evaluation methods for
phosphorylation motifs. Both relative risk and odds ratio
describe a likelihood change of the occurrence of one motif
between the foreground and the background. Relative risk is
defined as the ratio of the supports of m in the two data
sets. When using relative risk to measure the statistical signif-
icance of m, it reads as:
Supðm; P Þ
Sigðm; P; NÞ ¼ : (1)
Supðm; NÞ
A relative risk of 1 indicates that the target motif under
study is equally likely to occur in both data sets. A relative
risk greater than 1 means that this motif is more likely to
occur in the foreground. In addition, the odds is the ratio
of the probability that the interesting event does happen
to the probability that it does not happen. The odds ratio
is defined as the ratio of the odds of an event occurring in
one group to the odds of it occurring in another group. In
the context of phosphorylation motif finding, if we adopt
odds ratio to measure the over-expressiveness, then the
significance of m is:
Supðm; P Þ=ð1  Supðm; P ÞÞ
Sigðm; P; NÞ ¼ : (2)
Supðm; NÞ=ð1  Supðm; NÞÞ
Odds ratio has the same characteristics as relative risk: only
those motifs whose odds ratio is greater than 1 have potential
to be statistically significant.
Particularly, if we consider P ðm1 Þ, P ðm2 Þ; . . . ; P ðmk Þ as
the new foreground data set instead of P and Nðm1 Þ,
Nðm2 Þ; . . . ; Nðmk Þ as the new background data set instead
of N when estimating the over-expressiveness, there are
exactly k different significance values for m, that are
Sigðm; P ðmi Þ; Nðmi ÞÞ where 1  i  k, respectively. With-
out loss of generalization, we assume that Sigðm; P; NÞ has
positive correlation with the over-expressiveness: the bigger
Sigðm; P; NÞ is, the more significant the motif m is. Under
this setting, we adopt the minimum value of Sigðm;
P ðmi Þ; Nðmi ÞÞ (1  i  k) as the local (or conditional) statis-
tical significance in the estimation of over-expressiveness of
each motif. Then, the problem of conditional phosphoryla-
tion motif discovery is to discover all frequent motifs from
P with sub-motif derived statistical significance values
917
passing the given threshold in addition to fulfilling the tra-
ditional definition.
Definition 2. Local statistical significance:
Sigl ðm; P; NÞ ¼ min Sigðm; P ðmi Þ; Nðmi ÞÞ:
1ik
Definition 3. Global statistical significance:
Sigg ðm; P; NÞ ¼ Sigðm; P; NÞ:
Overall, to identify all statistically significant, sufficiently
frequent conditional phosphorylation motifs, we assess at
least two aspects of each motif: frequency and statistical sig-
nificance including both the local significance and the global
significance:

Frequency. We impose the support constraint to
reduce the search space and prevent the generation
of random artifacts.

Statistical significance. Note that the statistical evalua-
tion of over-expressiveness for a motif can be done
in various ways. The statistical significance measures
such as relative risk and odds ratio are available to be
utilized interchangeably. The choice of significance
assessment measure will not change the perfor-
mance of underlying algorithms.
2.2 Problem Formulation
As shown above, we strengthen and optimize the defini-
tion of phosphorylation motif finding and try to conduct
an extensive and non-redundant (NR) discovery. The con-
ditional phosphorylation motifs are deemed to be true
positives with prominent over-expressiveness under no
subsets interplays. We impose a persuasive significance
constraint called the local or conditional significance on
each candidate motif that evaluates the statistical signifi-
cance of a phosphorylation motif with the sets of sequen-
ces induced from its sub-motifs. Furthermore, we also
perform the global significance evaluation using the origi-
nal data sets in the traditional way. Thus, there are two
parameters to measure the significance of motifs: the
global significance threshold ug sig and the local signifi-
cance threshold ul sig , respectively. That is, to ensure the
significance of one motif m over P against N, two criteria
must be satisfied simultaneously: Sigg ðm; P; NÞ  ug sig
and Sigl ðm; P; NÞ  ul sig . Hence, we can guarantee that
the conditional phosphorylation motifs are also signifi-
cant under the traditional definition.
However, there is one critical issue remaining for this
task. That is, whether the effect of the sub-motifs has really
been removed through the use of local statistical signifi-
cance? To address this issue, we employ a measure called
improvement proposed in [20] for justification. More pre-
cisely, the improvement is defined as the difference
between the statistical significance of one motif and that of
its sub-motifs. In general, the positive improvement indi-
cates that the over-expressiveness of one target motif comes
from the combinations of all its constituent amino acids
rather than just one of its subsets. We should prune those
redundant motifs that have no positive improvements: the
over-expressiveness of a motif is equal to or less than that
918 IEEE/ACM TRANSACTIONS ON COMPUTATIONAL BIOLOGY AND BIOINFORMATICS,
of its sub-motifs.1 To further clarify this issue, since we uti-
lize the local significance to get rid of sub-motif interplays,
we calculate the difference values between the conditional
phosphorylation kmotif and its corresponding sub-motifs
of k-1 with respect to the global significance. For the sim-
plicity of illustrations and mathematical derivations, here
we use relative risk as the significance measure as the exam-
ple measure in the remainder of this section.
Lemma 1. Each conditional phosphorylation motif possesses posi-
tive improvement if relative risk is used to measure the statis-
tical significance.
Proof. For a k-motif m in data set P and N, suppose one of
its sub-motifs mj of length k1 has the minimal signifi-
cance value, i.e., Sigl ðm; P; NÞ ¼ Sigðm; P ðmj Þ; Nðmj ÞÞ.
We set ul sig ¼ t, t > 1. Then the Lemma 1 can be formu-
lated as: if Sigl ðm; P; NÞ  t, then Sigg ðm; P; NÞ >
Sigg ðmi ; P; NÞ for any 1  i  k:
Supðm; P ðmi ÞÞ
Sigðm; P ðmi Þ; Nðmi ÞÞ ¼
Supðm; Nðmi ÞÞ
jP ðmÞkNðmi Þj
¼  Sigl ðm; P; NÞ
jNðmÞkP ðmi Þj
¼ Sigðm; P ðmj Þ; Nðmj ÞÞ  t;
(3)
Supðm; P Þ jNkP ðmÞj
Sigg ðm; P; NÞ ¼ ¼ ; (4)
Supðm; NÞ jP kNðmÞj
Supðmi ; P Þ jNkP ðmi Þj
Sigg ðmi ; P; NÞ ¼ ¼ : (5)
Supðmi ; NÞ jP kNðmi Þj
jP ðmÞj jP ðmi Þj
Since t is greater than 1, so jNðmÞj  jNðm i Þj
. As a result, we
can get that Sigg ðm; P; NÞ > Sigg ðmi ; P; NÞ by making
equation (4) minus equation (2), which returns a result
that is greater than 0. u
t sub-motifs as well. Therefore, we can infer that
To make the following description easier to follow, we
provide a precise problem definition of conditional phos-
phorylation motif discovery with clearly stated input and
output:

Input. The set of phosphorylated peptides (the fore-
ground data set P ) and the set of unphosphorylated
peptides (the background data set N), the support
threshold usup , the local significance threshold ul sig
and the global significance threshold ug sig .

Output. A set of conditional phosphorylation motifs
R, where each motif m 2 R satisfies: (1) Supðm; P Þ 
usup ; (2) Sigl ðm; P; NÞ  ul sig ; (3) Sigg ðm; P; NÞ 
ug sig .
2.3 Categorization of Existing Methods under the
New Formulation
Notice that Motif-X [9] and MMFPh[12] also measure the
over-expressiveness of one motif in a way that is similar to
our definition of local significance. More precisely, although
1. For justification, we also define some motifs as redundant ones if
their improvement is very small.
VOL. 11, NO. 5, SEPTEMBER/OCTOBER 2014
only ul sig is used in both Motif-X and MMFPh, they implic-
itly require that one sub-motif of the target motif should be
locally significant as well. In this section, we first show that
all the motifs reported by Motif-X and MMFPh are also
globally significant with high over-expressiveness as shown
in the lemma below.
Lemma 2. For a k-motif m in data set P and N, suppose we use
relative risk as the measure and set both ul sig and ug sig to be
t, t  1. Let mðrÞ represents one sub-motif of m of size r. If
SigðmðrÞ ; P ðmðr1Þ Þ; Nðmðr1Þ ÞÞ  t, for all 1  r < k, then
Sigg ðm; P; NÞ  t.
Proof.
SigðmðrÞ ; P ðmðr1Þ Þ; Nðmðr1Þ ÞÞ
SupðmðrÞ ; P ðmðr1Þ ÞÞ
¼
SupðmðrÞ ; Nðmðr1Þ ÞÞ (6)
ðrÞ ðr1Þ
jP ðm ÞkNðm Þj
¼ ;
jNðmðrÞ ÞkP ðmðr1Þ Þj
Sigðmðr1Þ ; P ðmðr2Þ Þ; Nðmðr2Þ ÞÞ
Supðmðr1Þ ; P ðmðr2Þ ÞÞ
¼
Supðmðr1Þ ; Nðmðr2Þ ÞÞ (7)
jP ðm ðr1Þ
ÞkNðm ðr2Þ
Þj
¼ ðr2Þ ðr1Þ
;
jP ðm ÞkNðm Þj
Supðm; P Þ jP ðmÞkNj
Sigg ðm; P; NÞ ¼ ¼ : (8)
Supðm; NÞ jP kNðmÞj
It is easy to see that SigðmðrÞ ; P ðmðr2Þ Þ; Nðmðr2Þ ÞÞ  t2
by multiplying Equations (6) and (7). That is, mðrÞ is sta-
tistically significant in the data sets derived from mðr2Þ .
This rule also applies to motif mðr2Þ so that mðr2Þ is sig-
nificant in the peptide sequences induced by one of its
Sigg ðm; P; NÞ  tk by iterating the multiplication process.
Since t is a positive number that is no less than 1, then
Sigg ðm; P; NÞ must be equal to or greater than t, too.
Thus, m is globally significant as well. u
t
Lemma 2 shows that Motif-X and MMFPh can find
motifs that are globally significant. However, two implicit
issues exist with regard to coverage and redundancy. The
first one is that they may miss some potentially meaningful
motifs under the conditional phosphorylation motif defini-
tion. This is because these methods investigate a certain can-
didate k-motif m on condition that it must contain at least
one both frequent and significant sub-motif of size k1. If
all constituent motifs mi s are not significant, there is no
chance to generate and evaluate this motif so that Motif-X
and MMFPh will not check m. Fig. 1 provides such an
example.
For illustration purpose, we adopt relative risk as the sig-
nificance measure here. Additionally, we set the significance
threshold usig ¼ ul sig ¼ ug sig ¼ 1:2 and the support thresh-
old usup ¼ 0:2. In this sample data set, we plant one frequent
and significant phosphorylation motif ‘KMS’ with relative
risk value larger than 1.2. There may be some other signifi-
cant motifs presented in this data set while we will
only focus our discussion on ‘KMS ’. ‘KMS’ consists of two
LIU ET AL.: MINING CONDITIONAL PHOSPHORYLATION MOTIFS
Fig. 1. A sample data set. Both the foreground data set and background
data set consist of 10 sequences.
sub-motifs, ‘KxS’ and ‘MS’, which are definitely insignifi-
cant with significance scores equal to 1.0. Thus, methods
like Motif-X and MMFPh have to filter out ‘KMS’ since it
lacks of frequent and significant constituent motifs. How-
ever, motifs of this type might also have great potential to
play valuable roles in biological research. So expanding the
scope of search and assimilating such kind of motifs as use-
ful ones is a considerable work for phoshphorylation motif
discovery.
An additional issue is that Motif-X and MMFPh may
involve some motifs that are not reported by C-Motif as
well. According to their definitions, one motif is deemed to
be significant as long as it can pass the significance test on
the reconstructed data sets induced from at least one sub-
motif. Such loose restriction makes it possible to report
some excess motifs whose over-expressiveness mainly
comes from their subsets. As a result, this kind of motifs
should be pruned according to our definition whereas
Motif-X and MMFPh will report them.
We will use one potential motif ‘GLxxxxSW’ in Fig. 1 as
an example for illustration. With the significance threshold
usig ¼ ul sig ¼ ug sig ¼ 1:2 and the support threshold
usup ¼ 0:2, its three sub-motifs ‘LxxxxSW’, ‘GxxxxxSW’ and
‘GLxxxxS’ are all frequent and (both locally and globally)
significant when relative risk is used for significance assess-
ment. So it is possible to generate ‘GLxxxxSW’ in three
ways in MMFPh and Motif-X. Moreover, ‘GLxxxxSW’
achieves three significance values on the subset induced
data sets: one is 1.0 induced by ‘GLxxxxS’, another is also
1.0 by ‘GxxxxxSW’ and the other is 1.5 by ‘LxxxxSW’.
According to MMFPh and Motif-X, ‘GLxxxxSW’ is a signifi-
cant motif with significance value 1.5 which is larger than
usig . However, ‘GLxxxxSW’ will be discarded by C-Motif
since its local significance value 1.0 is less than the local
significance threshold. Further investigation on the relation-
ship between the target motif and its sub-motifs shows
that ‘GLxxxxSW’ has equivalent global significance with
919
Fig. 2. The relationship of the sets of significant motifs discovered by dif-
ferent algorithms.
‘GLxxxxS’ and ‘GxxxxxSW’, rendering little improvement
of the over-expressiveness. As a result, ‘GLxxxxSW’ is a
redundant motif whose over-expressiveness possibly
mainly roots from ‘GLxxxxS’ and ‘GxxxxxSW’ other than
the integrity.
In summary, the algorithms that only adopt the global
significance such as Motif-All often return a set of phos-
phorylation motifs that contains lots of motifs whose over-
expressiveness mainly benefits from the subsets. Further-
more, MMFPh is able to find much more significant motifs
missed by Motif-X. This method decreases the number of
redundant motifs but excludes some ones with no “over-
expressed” sub-motifs and includes some redundant ones
under subsets interplays as well. In contrast, our formula-
tion and proposed algorithm use the global significance
together with the local significance and can discover as
many potential significant motifs as possible with non-
redundancy. The relationship of the motif sets reported by
the different algorithms is provided in Fig. 2.
2.4 The C-Motif Algorithm
C-Motif (Algorithm 1) is implemented in a single-stage
where the frequency and the statistical significance are
tested at the same time. In detail, when discovering the set
F1 from S1 in the first iteration, we calculate the local signifi-
cance as well as the global significance for each frequent
member of F1 . In fact, the global significance of 1-motifs is
identical to their local significance. Since one motif is statis-
tically significant on condition that its global significance
and local significance are not less than the given threshold
ug sig and ul sig , respectively. We filter out the insignificant
motifs and then add the rest to the result set. So after inves-
tigating every possible 1-motif, all the motifs in the result
set are significant conditional phosphorylation motifs of
size 1. With respect to the kth iterations, we perform the fol-
lowing operations:
1) Generate the set of potential frequent motifs of size k,
i.e., Sk , by joining Fk1 with itself naturally (Steps 5-
7). Let l1 and l2 be members of Fk1 . They are
assumed to be joinable if their first k2 amino acids
are in common with the same conserved positions.
To avoid duplications, we rank these two motifs
according to the positions of their last conserved
amino acids. Suppose l1 and l2 are a pair of joinable
920 IEEE/ACM TRANSACTIONS ON COMPUTATIONAL BIOLOGY AND BIOINFORMATICS,
motifs, then l1 < l2 if the last conserved amino acid
of l1 lies in the relative left of that of l2 . Line up these
identical amino acids and the left in l1 and l2 accord-
ing to their rank to compose one k-motif m.
2) Evaluate the frequency of each candidate in Sk by
their supports (Steps 8-10). Prune all the infrequent
ones and add all the left to Fk .
3) Test the local significance and the global significance
of each potential motif in Fk (Steps 11-18). For one k-
motif m, obtain all of its (k1)-sub-motifs. For each
sub-motif mi of m, construct its matching foreground
P ðmi Þ and background Nðmi Þ. Calculate the signifi-
cance value on the new data sets and choose the min-
imum one as the final local significance value. In
addition, we also obtain the global significance value
using the original data sets P and N in consistency
with traditional definition. Filter out the insignificant
motifs and save the both globally and locally signifi-
cant ones in R.
4) Repeat the above steps until no more frequent candi-
dates can be generated in Sk . Return R as the final
result.
2.5 Proofs of Completeness and Correctness
Theorem 1. The C-Motif algorithm is complete.
Proof. With respect to the definition of conditional phos-
phorylation motif, the completeness of C-Motif can be
shown by the following two facts. The first is that the
Apriori algorithm is complete, that is, it explicitly
VOL. 11, NO. 5, SEPTEMBER/OCTOBER 2014
enumerates and checks all frequent motifs in the mining
process. The second is that our implementation only
prunes those insignificant motifs with respect to both
global significance and local significance. u
t
Theorem 2. The C-Motif algorithm is correct.
Proof.. The correctness of the C-Motif algorithm can be
guaranteed by two facts. First, only frequent motifs are
generated. Second, both the local and global significance
values are exactly calculated and every motif with signif-
icance values that are lower than the user-specified
thresholds will be pruned. u
t
3 EXPERIMENTAL RESULTS
In order to demonstrate the efficacy and utility of our algo-
rithm, we conduct a series of tests with real data. In our
experiments, we compare our algorithm with the Motif-All
algorithm and the MMFPh algorithm with respect to effi-
ciency, coverage and non-redundancy. Note that several
motif-discovery methods have been proposed, the reason
why we choose Motif-All and MMFPh for comparisons
here is that they are representatives for algorithms that use
only global significance threshold and local significance
threshold, respectively. Furthermore, we use the same sig-
nificance measure in all algorithms so as to make their out-
puts comparable. More precisely, we choose relative risk to
measure the over-expressiveness so as to ensure the motifs
reported by MMFPh2 are also globally significant, which
can facilitate a fair comparison for our experiments.
In the experiments, we apply C-Motif, MMFPh and
Motif-All to both non-kinase-specific and kinase-specific
phosphorylation data sets with a fixed length of amino acids
upstream and downstream of the phosphorylated residues.
The details of these data sets are provided in the following
sections. In each experiment, we first present a brief descrip-
tion of the data and tune the thresholds so as to clarify the
comparison. Subsequently, we perform a general analysis
of the motifs discovered and then illustrate the superiority
of C-Motif against MMFPh and Motif-All.
3.1 Non-Kinase-Specific Phosphorylation Data
3.1.1 Data Description
We use Phospho.ELM (version 9.0) [21] and Swiss-Prot
(release 2011_11) [22] as the data sources to construct the
data for this experiment. To generate the set of phosphory-
lated peptides P, we directly extract the annotated phosphor-
ylated peptides from Phospho.ELM without considering
kinase information. To generate the set of unphosphorylated
peptides N, we first extract all the proteins annotated as
‘Homo sapiens’, and then follow Musite [23] to partition all
the proteins into different groups through BLAST-Clust in
BLAST [24] package version 2.2.19 with a sequence identity
threshold of 50 percent. We select the protein with the largest
number of known phosphorylation sites in each group to
2. Note that in the original MMFPh, it reports the maximal phos-
phorylation motifs that are not subsumed by motifs with more fixed
amino acids. In our implementation, we report all motifs that can pass
the significance threshold so as to generate more motifs for a fair com-
parison with respect to the size of motif set.
LIU ET AL.: MINING CONDITIONAL PHOSPHORYLATION MOTIFS
Fig. 3. The number of reported motifs of different sizes on non-kinase-
specific phosphorylation data sets. Here the support threshold usup is
0.005 and the significance threshold usig is 1.7.
form a non-redundant data set; if there are no phosphory-
lated proteins in the current group, we select the longest pro-
tein as the member of NR data set. In fact, there are at least
five kinds of methods to construct the set of unphosphory-
lated peptides in different way [25]. Here we employ the
method in [26] to construct the background data. More pre-
cisely, we generate the data by sampling 5,000 phosphory-
lated peptides and 5,000 unphosphorylated peptides from
NR data set. Here all the peptides have the fixed length 13
and they are aligned on the residue that lies in the center
position.
In the context of phosphorylation motif discovery, one
algorithm can report different result sets of motifs from dif-
ferent test data sets. Since our main objective in this paper is
to compare the performance of different algorithms in the
same data sets, we will not discuss the effects of the data
construction methods on motif discovery and just use some
existing methods for constructing both the foreground data
and the background data.
3.1.2 Results
In this experiment, we choose a lower value as the support
threshold and a smaller value as the significance threshold
so as to uncover a relatively larger number of significant
motifs for a more remarkable comparison.
For consistency, we set the support threshold usup ¼ 0:005
and the significance thresholds3 (both ug sig and ul sig ) to be
1.7 for all the algorithms under discussion. In fact, Motif-All
only uses the global significance ug sig and MMFPh only
adopts the local significance ul sig in their algorithms. The
questions we want to answer in this experiment are: How
many phosphorylation motifs of different sizes can be dis-
covered by the algorithms, respectively? Which methods
can detect more meaningful phosphorylation motifs? Which
methods can filter out more biased motifs with respect to
subsets interplays?
3. For the simplicity of notations, we use usig to denote both the
global significance threshold and local significance threshold when
they are same.
921
Fig. 4. The relationship of reported motifs of size 2 on non-kinase-specific
phosphorylation data sets. Here the rounded rectangle denotes the result
set of Motif-All, the circle presents that of C-Motif and the rectangle cor-
responds to that of MMFPh. Here the support threshold usup is 0.005 and
the significance threshold usig is 1.7.
Fig. 3 summarizes the number of phosphorylation motifs
discovered by C-Motif, MMFPh and Motif-All. Several
observations can be made from Fig. 3.
First, C-Motif is able to find more motifs than MMFPh in
general. Moreover, Motif-All reports much more motifs,
and all the reported motifs of C-Motif and MMFPh are
included in the result set of Motif-All.
Second, we find that all the algorithms present a same set
of interesting 1-motifs and there is visible difference in the
discovery of 2-motifs and 3-motifs. In Fig. 4, we describe the
relationship of the 2-motif sets reported by different algo-
rithms. Apparently, Motif-All reports a superset of the other
two methods, which can be divided into four parts accord-
ing to their distributions. Some considerable analysis upon
the four parts can be made as follows:
1) The motifs in the intersection of the results (the first
part with label ‘1’) reported by MMFPh, Motif-All
and C-Motif are all conditional phosphorylation
motifs.
2) The second part with label ‘2’ consists of motifs just
discovered by Motif-All and C-Motif. The common
characteristic of these motifs is that all of their sub-
motifs are insignificant on the subset induced data
sets. Accordingly, MMFPh fails to generate and
check these motifs even though they are both glob-
ally significant and locally significant.
3) The third part contains only one 2-motif ‘RxxSP’
detected by MMFPh and Motif-All. Since ‘RxxSP’ is
composed of two significant sub-motifs (‘RxxS’ and
‘SP’), which enables MMFPh to generate this motif
by extending either ‘RxxS’ or ‘SP’. Thus, ‘RxxSP’ can
achieve two significance values, 3.43 and 1.54, upon
the sub-motif induced data sets. MMFPh chooses the
first value and reports this motif as significant one
while C-Motif prunes it since the minimum value is
less than the given local significance threshold. In
fact, ‘RxxSP’ is quite dependent on ‘SP’ in the sense
that its global significance value of 6.6 is relatively
similar to that of 5.5 of ‘SP’. This indicates that the
discriminative power of ‘RxxSP’ mainly comes from
its subset ‘SP’. So regarding such kind of motifs as
redundant ones is reasonable and justifiable.
922 IEEE/ACM TRANSACTIONS ON COMPUTATIONAL BIOLOGY AND BIOINFORMATICS,
Fig. 5. The running time comparison of C-Motif, MMFPh and Motif-All on non-kinase-specific phosphorylation data.
4) The fourth part consists of motifs that are only
reported by Motif-All. Motifs in this category gener-
ally have at least one very significant sub-motif that
actually leads to the statistical significance of its
superset. For instance, ‘GxxRxxS’ is composed of
two sub-motifs: ‘GxxxxxS’ and ‘RxxS’. ‘GxxxxxS’ is
not a significant phosphorylation motif with both
local and global significance values of 0.99 while
‘RxxS’ is a significant one with the significance val-
ues of 2.48. Particularly, ‘GxxRxxS’ has the global
statistical significance values of 3.22, rendering little
improvement upon the over-expressiveness com-
pared to that of ‘RxxS’. In this regard, ‘GxxRxxS’ is
an insignificant motif according to our definition
although it can pass the significance threshold usig .
Similar analysis can also be made for the other motifs
in this part. Hence, these motifs are redundant in the
sense that their discriminative power mainly comes
from some sub-motifs that are already claimed as to
be statistically significant. Actually, filtering out this
kind of motifs is able to reduce the redundancy in
phosphorylation motif discovery.
We have similar observations on the motifs of size 3
reported by different algorithms. In general, Motif-All cov-
ers all the potentially significant motifs detected by MMFPh
and C-Motif but contains many redundant ones with no sig-
nificant improvements of statistical significance. The differ-
ence set between MMFPh and C-Motif includes several
potentially redundant motifs from MMFPh and some inter-
esting ones without any significant sub-motifs discovered
by C-Motif.
Note that the largest size of reported phosphorylation
motifs is 3 under the setting of usup ¼ 0:005 and usig ¼ 1:7.
There is more evident difference in the performance of C-
Motif and MMFPh as well as Motif-All especially when the
size of target motifs is larger than 2. That is, the bigger the
size is, the more different the results of the algorithms are.
Thus, we can infer that increasing the size of target motifs
will apparently highlight the advantage of C-Motif over
MMFPh and Motif-All.
To further check if this is true, we also perform phos-
phorylation motif mining at the support threshold of 0.1
with significance threshold of 4.0. C-Motif obtains an
VOL. 11, NO. 5, SEPTEMBER/OCTOBER 2014
identical set of motifs of size 1 with MMFPh and a slightly
different set from Motif-All under this setting. If we
change the support threshold to 0.001 with the significance
threshold equivalent to 1.5, C-Motif reports 1,530 motifs,
whereas MMFPh reports 515 motifs and Motif-All reports
4,236 motifs. Accordingly, there exists a bigger gap
between their result sets. On one hand, MMFPh misses a
number of interesting motifs as well as includes several
potentially redundant motifs whose over-expressiveness
mainly derives from their sub-motifs. On the other hand,
Motif-All also reports many motifs whose sub-motifs are
included in the result as well. This demonstrates that C-
Motif not only can find more significant motifs than
MMFPh but also is more qualified than Motif-All and
MMFPh to achieve non-redundancy in a flexible manner.
In conclusion, it has been illustrated that MMFPh is par-
tially useful and effective in presenting meaningful motifs
and reducing redundant motifs than Motif-All, although it
would miss some statistically significant motifs. Further-
more, Motif-All acquires higher coverage at the cost of
including many motifs whose over-expressiveness is
rooted from sub-motifs. In contrast, C-Motif makes a rea-
sonable trade-off between redundancy and coverage.
Hence, the empirical comparison shows that C-Motif out-
performs the other methods like MMFPh and Motif-All,
by discovering more meaningful and non-redundant
phosphorylation motifs.
Fig. 5 presents the running time of different algorithms.
For this data set, C-Motif and Motif-All spend similar execu-
tion time in the mining process under the same parameter
setting. Due to the specific motif candidate generation pro-
cedure, MMFPh needs more time when the significance
threshold is very low and is more efficient when the signifi-
cance threshold is increased. Overall, C-Motif is relatively
efficient and is comparable with other algorithms in terms
of running efficiency.
3.2 CDK-Specific Phosphorylation Data
3.2.1 Data Description
A protein kinase phosphorylates the substrates by transfer-
ring phosphate from adenosine triphosphate or guanosine
triphosphate to specific amino acids (serine, threonine and
LIU ET AL.: MINING CONDITIONAL PHOSPHORYLATION MOTIFS
Fig. 6. The number of reported motifs of different sizes on CDK-specific
phosphorylation data sets. Here the support threshold usup is 0.015 and
the significance threshold usig is 2.0.
tyrosine). Cyclin-dependent kinases (CDK) is a major class
of enzymes involved in the regulation of the cell cycle. This
kind of kinases is activated alternatively along with the cell
cycle, and phosphorylates the corresponding substrates so
as to make the cell cycle proceed in an orderly manner.
Similar to generating the non-kinase-specific phosphory-
lation data, we first extract the phosphorylated peptides
from phosphorylated proteins of the kinase CDK to com-
pose the foreground data set. We use the method in [27] to
construct the nonphosphorylation data. There are approxi-
mately 200 sequences (i.e., 13-mers) in both the foreground
data and background data.
3.2.2 Results
For this data set, we first set the support threshold usup ¼
0:015 for all the algorithms and the significance threshold
usig ¼ 2:0 for Motif-All and MMFPh. Particularly, we set an
identical threshold for global significance and local signifi-
cance for C-Motif with usig ¼ 2:0. That is, both ug sig and
ul sig are equal to 2:0. Fig. 6 presents the number of the sig-
nificant motifs found by C-Motif, Motif-All and MMFPh
under this setting. Accordingly, C-Motif presents a set of
phosphorylation motifs whose size is less than that of
Motif-All while it is greater than that of MMFPh.
Motif-All also succeeds in finding all the phosphoryla-
tion motifs reported by C-Motif, whereas including some
controversial ones that need to be further discussed. For
this reason, we have to re-conduct significance tests for
those motifs only found by Motif-All. We first extract them
to form a new set, and then check the statistical significance
of each motif together with that of its component parts.
Most of these motifs have something in common that they
consist of significant parts and insignificant parts, and their
significant parts contribute to the over-expressiveness of the
whole motifs. According to our definition, these motifs
should be considered as redundant motifs. The result set of
Motif-All may also contain some motifs that have similar
over-expressiveness compared to their subsets. Therefore,
due to the weaker pruning of the global significance in the
assessment of over-expressiveness, Motif-All often fails to
get rid of subsets interplays and returns a larger number of
motifs with lots of undesired noise.
923
Specially, we find that almost all the motifs found by
MMFPh also occur in the result sets reported by the other
algorithms in addition to some special cases. The motifs
that are reported by C-Motif but missed by MMFPh are
both globally and locally significant according to our def-
inition. For instance, ‘PxVxxxSxxK’ is a 3-motif reported
by C-Motif and Motif-All but missed by MMFPh. With
respect to the global significance, this motif is indeed a
significant motif with prominent over-representation in
the foreground data set. In contrast, its sub-motifs
‘VxxxSxxK’, ‘PxxxxxSxxK’ and ‘PxVxxxS’ are all insignifi-
cant on their sub-motif induced data sets with lower
over-expressiveness. As shown in the former section,
since there are no frequent and significant sub-motifs for
‘PxVxxxSxxK’, so there is no chance to generate and eval-
uate the target motif in MMFPh, and thus MMFPh
prunes it for certain. In this sense, MMFPh fails to dis-
cover some underlying interesting motifs because of its
restrictive definition. In addition, we also find that most
of those motifs missed by MMFPh are of larger sizes.
Particularly, the motifs discovered by MMFPh while fil-
tered out by C-Motif are some exceptions whose over-
expressiveness mainly roots from their significant parts.
‘STPxxxxR’ is such one special case with three significant
sub-motifs: ‘STP’, ‘STxxxxxR’ and ‘TPxxxxR’. ‘STPxxxxR’
can be extended from any sub-motif so that it has three
possible significance values to measure the over-expressive-
ness, which are 1.5, 2.17 and 1.0, respectively. So the target
motif is significant only if we evaluate it relying on its sec-
ond sub-motif induced data sets. In addition, ‘STPxxxxR’
has an identical global significance value with that of its
sub-motif ‘TPxxxxR’. That whether ‘TPxxxxR’ is significant
or not greatly affects the significance degree of ‘STPxxxxR’.
As a result, motifs like this are biased ones that should be
removed according to our definition. In conclusion, MMFPh
still includes some redundant motifs even though they have
made some contributions to redundancy reduction.
For illustration purpose, we also study the performance
of the different algorithms under different statistical sig-
nificance standards and support levels. We gradually
change the significance threshold and the support thresh-
old to obtain various cases. The results are summarized in
Table 1. Under any setting, it is easy to see that Motif-All
always reports the most motifs and MMFPh returns the
least quantitatively. If we lower the support threshold
with unchanged significance threshold, or lower the sig-
nificance threshold with unchanged support threshold,
there will be much more motifs of different sizes to be
reported. In this situation, more redundant motifs are con-
sidered as significant ones by Motif-All while more poten-
tial ones are missed by MMFPh. It indicates that the
smaller the support threshold and the significance thresh-
old are, the more motifs of the larger sizes are found, and
the more obvious difference between the results of the
algorithms is visible. This is because if one motif has larger
size, then this motif contains more sub-motifs, and the
subset interplays play more important role and are more
potential to affect the whole combination with respect to
over-expressiveness, especially compared to the motif of
smaller size. Hence, it increases the probability for both
Motif-All and MMFPh to regard the target motif as an
924 IEEE/ACM TRANSACTIONS ON COMPUTATIONAL BIOLOGY AND BIOINFORMATICS, VOL. 11, NO. 5, SEPTEMBER/OCTOBER 2014

TABLE 1
The Number of Reported Motifs of Different Sizes on the CDK Phosphorylation Data Sets by Tuning
the Thresholds usup and usig Gradually

usig indicates identical threshold for ug sig and ul sig in C-Motif.

interesting one even its over-expressiveness is derived
from some subsets, or just for MMFPh to filter one motif
out if it has no frequent and significant sub-motifs
although this motif is globally significant. On the contrary,
more motifs will be pruned and less ones can be discov-
ered with higher thresholds. This will reduce the perfor-
mance gap among these algorithms. In this sense, Motif-
All and MMFPh may be more appropriate for discovering
those motifs of smaller size. To conclude, C-Motif is a bet-
ter algorithm with respect to coverage as well as non-
redundancy in phosphorylation motif discovery.
Fig. 7 depicts the running time of the algorithms under
different parameters. Despite of the fluctuation of MMFPh,
it is apparent that C-Motif needs similar running time as
Motif-All and a little more than MMFPh to finish the mining
process. Meanwhile, we would like to point out that the per-
formance gap between our algorithm and other methods is
not significant. Therefore, C-Motif is a relatively efficient
algorithm for the task of finding phosphorylation motifs.
3.3 Protein kinase A (PKA)-Specific
Phosphorylation Data
3.3.1 Data Description
Protein kinase A is a class of cAMP-dependent enzymes. It
plays important role in gene regulatory protein phosphory-
lation and activating transcription of specific genes. We also
use Phospho.ELM and Swiss-Prot databases to generate
phosphorylated peptides and unphosphorylated peptides
with PKA-specific phosphorylation sites, which is similar to
the construction of CDK-specific phosphorylation data. In
the generated data, the number of phosphorylated peptides
is roughly equivalent to that of unphosphorylated peptides.
3.3.2 Results
In this experiment, we further validate our approach using
the PKA-specific phosphorylation peptides with parameters
as usup ¼ 0:01 and usig ¼ 2:0. For a fair comparison, the sig-
nificance thresholds for C-Motif are specified as: usig ¼ ul sig
¼ ug sig ¼ 2:0. Under this setting, the results of all the motif
extraction algorithms are shown in Fig. 8.
As shown in Fig. 8, in addition to the gap on the total
number of motifs, MMFPh fails to find any motifs of size
larger than 2 while Motif-All reports motifs of size even up
to 9. C-Motif returns a result set of motifs whose sizes are
not larger than 3.
To further describe the difference, here we use several
identified motifs as examples for illustration. ‘SxxSVT’ is a
statistically significant motif with both global significance
and local significance values larger than the given threshold
usig . However, MMFPh ignores this motif since it lacks fre-
quent and significant sub-motifs. The significance values of
its three 2-sub-motifs (‘SxxSV’, ‘SxxSxT’ and ‘SVT’) are all
less than usig . On the other hand, ‘GEKxxxS’ reported by
Motif-All is composed of a significant part ‘GxKxxxS’ with
local significance value of 3.6 and an insignificant part
‘ExxxxS’ with local significance value of 0.5 with the assess-
ment in C-Motif. In addition, ‘GEKxxxS’ has a global signifi-
cance value of 5.14 that has no significant improvement

Fig. 7. The running time comparison of C-Motif, MMFPh and Motif-All on CDK-specific phosphorylation data.
LIU ET AL.: MINING CONDITIONAL PHOSPHORYLATION MOTIFS
Fig. 8. The number of reported motifs of different sizes on PKA-specific
phosphorylation data sets. Here the support threshold usup is 0.01 and
the significance threshold usig is 2.0.
over that of ‘GxKxxxS’ (the global significance value of
‘GxKxxxS’ is 4.11). As a result, the significant sub-motif
‘GxKxxxS’ will directly enhance the statistical significance
of ‘GEKxxxS’ to some extent. Hence, motifs like this are
unmeaningful ones since its over-expressiveness does
induce from its component parts other than the whole com-
bination. Owing to the less-stringent pruning criterion,
Motif-All reports ‘GEKxxxS’ as an interesting one improp-
erly. Similarly, ‘KRxxxxS’ reported by MMFPh and Motif-
All possesses similar global significance value of 5.14 as that
of 4.52 of its sub-motif ‘RxxxxS’, which results in that
‘KRxxxxS’ is a redundant motif with little improvement
with respect to the over-expressiveness.
The detailed investigation on all the motifs reported by
C-Motif, MMFPh and Motif-All demonstrates two crucial
points. The first point is that the motifs identified by C-
Motif generally have no globally significant sub-motifs. Fur-
thermore, these motifs survive after the pruning stage due
to their inherent properties of the combination of each part
rather than some subsets. The second point is that a propor-
tion of the motifs reported by Motif-All and several ones
reported by MMFPh are motifs whose over-expressiveness
mainly benefits from their constituent parts. Consequently,
though Motif-All has much success in returning the highest
coverage of the significant motifs and MMFPh achieves the
Fig. 9. The running time comparison of C-Motif, MMFPh and Motif-All on PKA-specific phosphorylation data.
925
ability of identifying conditional phosphorylation motifs at
the cost of excluding some underlying ones, they suffer
from failure to filter out those potential false positives
whose over-expressiveness comes from their sub-motifs.
Overall, C-Motif outperforms Motif-All and MMFPh
with a trade-off between coverage and non-redundancy of
the final significant motif set. Next, we will concentrate our
discussion on the efficiency of the motif discovery algo-
rithms. Fig. 9 presents the running time of C-Motif, MMFPh
and Motif-All on PKA-specific phosphorylation data sets by
changing the parameters gradually. We first fix the support
threshold usup ¼ 0:015 and then increase the significance
threshold usig from 1.0 to 10.0. Subsequently, we keep the
significance threshold usig remain unchanged as 2.0 and
increase the support threshold usup from 0.01 to 0.1. As
shown in Fig. 9, our method has comparable running effi-
ciency with the other two algorithms. In addition, we have
the following remarks.
First, there is an obvious gap between MMFPh and
the other two methods in most cases. MMFPh seems
like the most ‘inefficient’ approach before a critical point
and the most ‘efficient’ approach after that point. This is
because both Motif-All and C-Motif conduct the discov-
ery under an Apriori framework, which is very efficient
in frequent pattern mining. However, MMFPh generates
candidates with enumeration method and includes lots
of duplications, which is relatively time-consuming espe-
cially when there are plenty of significant ones to extend.
Moreover, MMFPh would ignore some globally signifi-
cant motifs if the motifs lack significant and frequent
sub-motifs, while C-Motif and Motif-All would not. So
C-Motif and Motif-All have to spend more time to test
those candidate motifs. In addition, C-Motif also pro-
vides global significance assessment like Motif-All for
justice. As a result, MMFPh runs more effectively when
the significance threshold is relatively large.
Second, the running time of Motif-All and C-Motif is
almost the same. Though there is a little difference between
these two methods in some special situations, they are still
in the same magnitude and the gap can be negligible in the
range of the errors permitted. This is because when detect-
ing one potential significant motif, C-Motif has to investi-
gate every peptide in the original data and check if this
926 IEEE/ACM TRANSACTIONS ON COMPUTATIONAL BIOLOGY AND BIOINFORMATICS,
peptide contains one of its sub-motifs. Then it generates a
new data set instead for calculating the local significance
value. In contrast, Motif-All only adopts global significance
to evaluate one candidate, rendering that it obtains much
reduction of computation and enumeration for generating
the new data. While Motif-All has to spend additional run-
ning time on checking more candidates than C-Motif.
Third, considering C-Motif is better at achieving an over-
all balance of the coverage and non-redundancy of signifi-
cant phosphorylation motifs compared to Motif-All and
MMFPh, it is reasonable to consider C-Motif as an efficient
algorithm for mining phosphorylation motifs.
4 CONCLUSION
This paper formally proposes the notion of conditional
phosphorylation motif and presents the problem of condi-
tional phosphorylation motif discovery. Additionally, we
also propose a new algorithm called C-Motif for this task
and make an attempt to conduct a non-redundant
discovery. Experiments on both non-kinase-specific phos-
phorylation data and kinase-specific phosphorylation data
demonstrate that C-Motif is able to uncover more interest-
ing phosphorylation motifs than MMFPh and retains less
false positives than Motif-All under the same given parame-
ter setting. Moreover, it is very fast so that it is able to find
hundreds of significant motifs from large data sets. As a
result, C-Motif outperforms the existing phosphorylation
motif discovery algorithms with respect to efficiency, cover-
age as well as non-redundancy.
For the future work, there are several possible directions
that need further investigations.
Firstly, although methods such as C-Motif and MMFPh
can reduce the number of phosphorylation motifs returned
to the biologists, there are still many statistically significant
motifs that remain. As a result, it is still not an easy task
for people to find really biologically meaningful motifs
from this pool. From the computational and statistical per-
spective, we still need to develop effective algorithms and
rigorous statistical testing procedures for further reducing
the number of reported motifs. The concept of “maximal
motif” in MMFPh and the application of permutation test
in [17] are research efforts towards this direction. However,
that is still not sufficient and further investigations should
be conducted.
Secondly, existing motif discovery algorithms fulfill the
mining task merely with the sequence data around the phos-
phorylation site as input. This may prevent us to find really
biologically interesting motifs to derive useful scientific
insights. One possible remedy for this limitation is to con-
duct motif search on expanded data sets that include supple-
mentary information such as the 3D protein structures.
Finally, it is highly necessary to collect biologically vali-
dated motifs to build a public repository as the reference
database for performance assessment and comparison.
ACKNOWLEDGMENTS
This work was partially supported by the Natural Science
Foundation of China under Grant No. 61003176 and No.
61073051, the Fundamental Research Funds for the Central
Universities of China (DUT14QY07).
VOL. 11, NO. 5, SEPTEMBER/OCTOBER 2014
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Xiaoqing Liu received the BS degree in software
engineering from Dalian University of Technol-
ogy, China, in 2013. She is currently working
toward the MS degree in the School of Software
at Dalian University of Technology. Her research
interests include bioinformatics and data mining.
Jun Wu received the BS degree in software engi-
neering from Dalian University of Technology,
China, in 2013. He is currently working toward
the MS degree in the School of Software at
Dalian University of Technology. His research
interests include bioinformactics and data mining.
927
Haipeng Gong received the BS degree in soft-
ware engineering from Dalian University of Tech-
nology, China, in 2011. He is currently working
toward the MS degree in the School of Software
at Dalian University of Technology. His research
interests include data mining and computational
proteomics.
Shengchun Deng received the PhD degree in
computer science from Harbin Institute of Tech-
nology in 2002. He is currently a professor at
Harbin Institute of Technology. His research
interests include database, software engineer-
ing, and software interoperability.
Zengyou He received the BS, MS, and PhD
degrees in computer science from Harbin Insti-
tute of Technology, China, in 2000, 2002, and
2006, respectively. He was a research associate
in the Department of Electronic and Computer
Engineering at the Hong Kong University of Sci-
ence and Technology from February 2007 to
February 2010. Since March 2010, he has been
an associate professor in the School of Software
at Dalian University of Technology. His research
interests include computational proteomics and
biological data mining.
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