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Journal of Protein Chemistry, Vol. 22, No. 4, May 2003 (© 2003)

Application of Pseudo Amino Acid Composition for

Predicting Protein Subcellular Location: Stochastic
Signal Processing Approach

Yu-Xi Pan,1,3 Zhi-Zhou Zhang,1 Zong-Ming Guo,1 Guo-Yin Feng,1 Zhen-De Huang,1
and Lin He1,2

January 27, 2003

The function of a protein is closely correlated with its subcellular location. With the success of
human genome project and the rapid increase in the number of newly found protein sequences
entering into data banks, it is highly desirable to develop an automated method for predicting the
subcellular location of proteins. The establishment of such a predictor will no doubt expedite the
functionality determination of newly found proteins and the process of prioritizing genes and pro-
teins identified by genomics efforts as potential molecular targets for drug design. Based on the con-
cept of pseudo amino acid composition originally proposed by K. C. Chou (Proteins: Struct. Funct.
Genet. 43: 246–255, 2001), the digital signal processing approach has been introduced to partially
incorporate the sequence order effect. One of the remarkable merits by doing so is that many ex-
isting tools in mathematics and engineering can be straightforwardly used in predicting protein sub-
cellular location. The results thus obtained are quite encouraging. It is anticipated that the digital
signal processing may serve as a useful vehicle for many other protein science areas as well.

KEY WORDS: Quasi-sequence order effect; covariant-discriminant algorithm; Mahalanobis distance; Chou’s
invariance theorem; low/high-pass Butterworth filter; bioinformatics; proteomics.

1. INTRODUCTION is time consuming and costly to acquire this kind of

As a result of the development of the high-throughput
sequencing technology with its improving efficiency and
decreasing cost, the data in various biological database
has been increasing at an unprecedented speed in the
recent years (Chou and Elrod, 1999a, 1999b). The
explosion of biological data challenges biologists’ and
computer scientists’ ability and speed of analyzing these
data. The function of a protein is closely correlated with
its subcellular location (Chou, 2000b). Although the sub-
cellular location of a protein can be determined by con-
ducting various locational determination experiments, it
1
Bio-X Life Science Research Center, Shanghai Jiao Tong University,
Shanghai, China.
2
Shanghai Institutes for Biological Sciences, Chinese Academy of
Science, Shanghai, China.
3
To whom correspondence should be addressed at Bio-X Life Science
Research Center, Shanghai Jiao Tong University, Shanghai, 200030,
China. E-mail: wzhong@chartermi.net
395
knowledge solely based on experimental measures. Con-
fronted with the enormous data sets in the areas of
genome and proteome, it turns out to be highly desirable
to develop an efficient method to predict the subcellular
location of a new protein so as to expedite the process
of deducing its function. Many efforts were made in this
regard (Cai and Chou, 2000; Cai et al., 2000, 2002a,
2002b; Cedano et al., 1997; Chou, 2000a, 2000b; Chou
and Cai, 2002; Chou and Elrod, 1998, 1999a, 1999b;
Nakashima and Nishikawa, 1994; Reinhardt and
Hubbard, 1998; Zhou and Doctor, 2003). Actually, a
new branch in proteomics, the so-called “Prediction of
Protein Cellular Attributes” (Chou, 2002) has emerged.
It should be pointed out that most of the existing algo-
rithms were based on the amino acid composition alone
(Cedano et al., 1997; Chou and Elrod, 1998, 1999a,
1999b; Nakashima and Nishikawa, 1994; Reinhardt and
Hubbard, 1998; Zhou and Doctor, 2003). Although this
is a reasonable approximate method and did yield some
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396 Pan, Zhang, Guo, Feng, Huang, and He

encouraging result (Chou, 2000b), the sequence order in-
formation should also be taken into account as a logical
step for further development in this area. However,
owing to the extreme variation in both the sequence
order and the sequence length, it is very difficult to for-
mulate a prediction algorithm that can incorporate the
where xi is the numerical code for Ri (i = 1, 2, . . . , N ).
Thus, we can immediately generate five characteristic
parameters as formulated below. The first one is the
signal mean value, given by
1
N

sequence order effects as well. An important advance in p1 = M(x) = x(i) (3)

N i=1
this regard is the introduction of the concept of pseudo
amino acid composition as recently proposed by Chou
The second parameter is called standard deviation
(2001). The essence of pseudo-amino-acid-composition
given by:
is: on one hand, it bears the main feature of amino acid
composition; but on the other, it also contains some
1
N
elements to reflect the sequence order effects. p2 = SD(x) = |x(i) − M(x)| (4)
This study was initiated in an attempt to use the N i=1
concept of pseudo amino acid composition and the
stochastic signal processing approach to develop a new The SD reflects how a signal is dispersed around its
method for predicting protein subcellular location. mean value. The smaller the SD is, the more centralized
the signal is toward its mean value. The third parameter
is the variance as defined by
2. METHOD
1
N

A protein sequence is composed of a series of amino p3 = var(x) = [x(i) − M(x)]2 (5)

acids represented by characters as A, C, D, E, F, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W, and Y. As lingual sym-
bols, the sequence composed by these characters cannot
participate in any form of mathematical computation in
computers because each element in this sequence does not
have its corresponding numerical value at all. To describe
a protein sequence in a quantitative way, what kind of
code should be used to represent a certain amino acid?
Actually, any numerical code would work as long as it can
distinguish one amino acid from another. For simplicity,
let us just assign: A = 10, C = 20, D = 30, E = 40, F =
50, G = 60, H = 70, I = 80, K = 90, L = 100, M = 110,
N = 120, P = 130, Q = 140, R = 150, S = 160, T = 170,
V = 180, W = 190, and Y = 200. Through the above en-
coding procedure a protein sequence is transformed to a
serial of digital signals. Thus, all the existing tools in the
area of digital signal processing (DSP) can be straightfor-
wardly used for the current study.
DSP is defined as a process of sampling, transform-
ing, synthesizing, estimating, and recognizing of signals
through numerical computing with computers and some
other special equipment so as to extract useful informa-
tion from the signals. Given a protein sequence
R1R2R3R4R5R6R7 · · · RN (1)
where R1 is the 1st residue, R2 the 2nd, and so forth. To
utilize the technique of DSP, let us first represent the
protein sequence by a stochastic digital signal, i.e.,
{xi }, 1≤i ≤N (2)
N i=1
The fourth and fifth characteristic parameters are the
low-pass and high-pass Butterworth filter contents (Candy,
1988; Jones, 1982; Tretter, 1990) as represented by
cov(x, yL )
p4 = corrcoef(x, yL ) = (6)
std(x) · std( yL )
where yL is the output signal of the low-pass Butterworth
filtering process with the input signal of {xi }. Thus, yL
represents the low-frequency component of the digital
signal {xi }. The low-pass filtering process can be given
by the following expression


26
yL (n) = − a(k) yL (n − k + 1)
k=2


26
+ b(k)x(n − k + 1) (7)
k=1
where {a(i)} and {b(i)} are two sets of parameters of the
low-pass digital filter with their values listed in Table 1.
The filtering process expressed by Eq. 7 and how the dig-
ital filter’s parameters of {a(i)} and {b(i)} are deter-
mined could be easily found in the aforementioned ref-
erence books. In Eq. 6, corrcoef(x, yL ) represents the
function of correlation coefficient of the two digital sig-
nals of x and yL . This function reflects the correlative ex-
tent of the two signals. In addition, cov(x, yL ) represents
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Application of Pseudo Amino Acid Composition 397

Table 1. Parameters of {a(i)} and {b(i)} for the Low-Pass Butterworth Filter in this Work (cf. Eq. 7)

Index: i 1 2 3 4 5 6 7 8 9 10 11 12 13
Values of {a(i)} 1.00 5.00 15.11 32.49 54.80 75.51 87.53 86.78 74.49 55.78 36.63 21.16 10.77
Values of {b(i)} 1.68e-5 4.21e-4 5.05e-3 3.87e-2 0.21 0.89 2.98 8.09 18.2 34.38 55.01 75.01 87.51

Index: i 14 15 16 17 18 19 20 21 22 23 24 25 26

Values of {a(i)} 4.82 1.90 0.65 0.20 0.05 1.13e-2 2.11e-3 3.27e-4 4.08e-5 3.95e-6 2.79e-7 1.27e-8 2.83e-10
Values of {b(i)} 87.51 75.01 55.01 34.38 18.20 8.09 2.98 0.89 0.21 0.04 5.05e-3 4.21e-4 1.68e-5

the covariance of the two signals of x and yL , and the ex- where yH is the output signal of the high-pass Butterworth
pression is given by filtering process with the input signal of {xi }. Similarly,
{c(i)} and {d(i)} are the parameters for the high-pass dig-
1
N
ital filter and their values are given in Table 2. Again, the
cov(x, yL ) = (x(i) − M(x))( yL (i) − M( yL ))
N − 1 i=1 filtering process expressed by Eq. 10 and how the digital
(8) filter’s parameters of {c(i)} and {d(i)} are determined
could be easily found in the same reference books.
where N is the length of the amino acid sequence. It can Thus, by following exactly the same procedure as
be seen from the above equations that the characteristic described by Chou (2001), a protein can be expressed by
parameter p4 incorporates some sequence order effect. a vector or a point in a 25-D (dimensional) space, i.e.,
Similarly, we have  
x1
 .. 
cov(x, yH )  . 
p5 = corrcoef(x, yH ) =  
std(x) · std( yH )
(9)  x20 
X=   (12)

 x20+1 
 .. 
where  . 
x20+5


26
yH (n) = − c(k) yH (n − k + 1) where
k=2 



fk
(1 ≤ k ≤ 20)
26

 ,
+ d(k)x(n − k + 1) (10) 

20 5

 fi + wj pj
k=1  i=1 j=1
xk = (13)

 wk pk
and 
 , (21 ≤ k ≤ 25)

 20 5

 fi +
cov(x, yH )  wj pj
i=1 j=1
1
N
= (x(i) − M(x))( yH (i) − M( yH )) (11) where f k is the normalized occurrence frequency of
N − 1 i=1 the 20 amino acid in the protein X, pi the ith additional

Table 2. Parameters of {c(i)} and {d(i)} for the High-Pass Butterworth Filter in this Work (cf. Eq. 10)

Index: i 1 2 3 4 5 6 7 8 9 10 11 12 13

Values of {c(i)} 1.00 −5.00 15.11 −32.49 54.80 −75.51 87.53 −86.78 74.49 −55.78 36.63 −21.16 10.77
Values of {d(i)} 1.68e-5 −4.21e-4 5.05e-3 −3.87e-2 0.21 −0.89 2.98 −8.09 18.20 −34.38 55.01 −75.01 87.51

Index: i 14 15 16 17 18 19 20 21 22 23 24 25 26

Values of {c(i)} −4.82 1.90 −0.65 0.20 −0.05 0.01 −2.11e-3 3.27e-4 −4.08e-5 3.95e-6 −2.79e-7 1.27e-8 −2.83e-10
Values of {d(i)} −87.51 75.01 −55.01 34.38 −18.20 8.09 −2.98 0.89 −0.21 3.87e-2 −5.05e-3 4.21e-4 −1.68e-5
468706.qxd 7/31/03 1:38 PM Page 398
398
characteristic parameter derived from digital signal pro-
cessing theory (see Eqs. 2–11), and wi the weight factor
for the ith parameter pi . In this work, we chose the
weight factors given by
    data set contains 12 subcellular locations, much more
w1 0.5
 w   0.015 
 2  
 w  =  0.005  (14)
 3  
 w4   0.5 
w5 0.5
The purpose of using the weight factors is to re-
arrange each of these five characteristic parameters and
limit their magnitude within the same region as that of
the 20 amino acid composition components of the pro-
tein. According to our preliminary computed results, the
values of the 5 characteristic parameters (p1, p2, p3, p4,
p5) are distributed in quite different ranges. Among the
25 pseudo amino acid components in Eq. 12, a compo-
nent with an oversize magnitude might hide the contri-
bution from all the others. Therefore, before exactly
knowing how strong role each of the five characteristic
parameters plays, it would be wise to use the weight
factor as shown in Eq. 14 to adjust their magnitudes and
make them fall within the range as the corresponding
20 amino acid composition components do. As we can
see from the Eqs. 12–13, the 25-D vector of a protein
contains the information of not only the amino acid com-
position but also some of the sequence order effects.
Now we can directly use the augmented covariant-
discriminant algorithm developed by Chou (2000a) to
conduct the prediction. The covariant-discriminant
algorithm is a combination of Mahalanobis distance
(Mahalanobis, 1936; Pillai, 1985) and Chou’s invariance
theorem (Chou, 1995; Zhou and Doctor, 2003). The
details about the algorithm can be found in some recent
papers (Chou, 2000a, 2001) and hence there is no need
to repeat here. It is instructive, however, to point out
that, since the normalization condition imposed by
Eq. 13, the 20 + 5 components of the pseudo amino
acid composition are not independent. Therefore, a
dimension-reduced operation by leaving out one of the
components and making the rest completely independent
is needed when using the augmented covariant discrim-
inant algorithm, i.e., a protein should be defined in a
24-D space instead of 25-D space. Otherwise, a diver-
gence difficulty will occur. However, which one of the
25 components should be removed? Any one. The rea-
son is that according to Chou’s Invariance Theorem
(Chou, 1995), the values of the covariant discriminant
function will remain the same regardless of which one of
the 25 components is left out.
Pan, Zhang, Guo, Feng, Huang, and He
3. RESULTS AND DISCUSSION
For testing our approach, the data set constructed by
Chou and Elrod (1999b) was adopted here since their
complete and practical than those constructed by the
other investigators. However, as mentioned in Chou
(2001), due to the change of code names, the sequences
for some proteins could no longer be retrieved from the
SWISS-PROT databank. Of the 2319 proteins originally
listed in Appendix A of Chou and Elrod (1999b), 2191
protein sequences were retrieved. They form the data set
S12, which consists of 145 chloroplast proteins, 571
cytoplasm, 34 cytoskeleton, 49 endoplasmic reticulum,
224 extracellular, 25 Golgi apparatus, 37 lysosome, 84
mitochondria, 272 nucleus proteins, 27 peroxisome, 699
plasma membrane, and 24 vacuole. For the convenience
of those readers who are not trained as a cellular biolo-
gist, a schematic illustration is given in Fig. 1 to show
the 12 different subcellular locations of proteins.
The prediction quality was examined by two meth-
ods, the self-consistency test and the jackknife test. In the
self-consistency test, the subcellular location for each of
the proteins in a given data set was in turn identified
using the rules derived from the same data set, the so-
called training data set. The predicting rate thus obtained
for the 12 subcellular locations of the 2191 proteins is
summarized in Table 3, from which we can see that 1779
proteins were correctly predicted for their subcellular lo-
cations and 412 proteins were incorrectly predicted. The
overall success rate is 81.5%, indicating a quite high self-
consistency. However, it should be pointed out that,
during the process of the re-substitution test, the rule
parameters derived from the training data set include the
information of the query protein sequence later plugged
back in the test. This will certainly underestimate the
error and enhance the success rate because the same pro-
teins are used to derive the rule parameters and to test
themselves. Accordingly, the success rate thus obtained
represents some sort of optimistic estimation (Cai, 2001;
Chou, 1995; Chou and Zhang, 1994; Zhou, 1998; Zhou
and Assa-Munt, 2001; Zhou and Doctor, 2003). Never-
theless, the re-substitution test is absolutely necessary
because it reflects the self-consistency of a prediction
method, especially for its algorithm part. A prediction
algorithm certainly cannot be deemed as a good one if
its self-consistency is poor. In other words, the re-
substitution test is necessary but not sufficient for evalu-
ating a prediction method. As a complement, a cross-
validation test for an independent testing data set is
needed because it can reflect the effectiveness of a pre-
diction method in practical application. This is important
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Application of Pseudo Amino Acid Composition 399

Fig. 1. Schematic illustration to show the twelve subcellular locations of proteins: chloroplast, cytoplasm, cytoskeleton, endoplasmic
reticulum, extracell, Golgi apparatus, lysosome, mitochondria, nucleus, peroxisome, plasma membrane, and vacuole. Note that the
vacuole and chloroplast proteins exist only in a plant. [Reproduced from Fig. 2 of Chou (2001) with permission.]

especially for checking the validity of a training data set:
whether it contains sufficient information to reflect all
the important features concerned so as to yield a high
success rate in practical application.
As is well known, the independent data set test, sub-
sampling test and jackknife test are the three methods
often used for cross-validation in statistical prediction.
Among these three, however, the jackknife test is deemed
as the most effective and objective one; see, e.g., a rele-
vant review (Chou and Zhang, 1995) for a comprehensive
discussion about this and a monograph (Mardia et al.,
1979) for the mathematical principle. During jackknifing,
each protein in the data set is in turn singled out as a
tested protein and all the rule parameters are calculated
based on the remaining proteins. In other words, the sub-
cellular location of each protein is identified by the rules
derived using all the other proteins except the one that is
being identified. During the process of jackknifing both
the training data set and the testing data set are actually
open, and a protein will in turn move from one to the
other. The results of jackknife test thus obtained for
the 2191 proteins are also given in Table 3, from which
we can see that 1492 proteins were correctly predicted for
their subcellular locations whereas 699 proteins were in-
correctly predicted. The overall success rate is 67.7%.
Moreover, as a demonstration of practical applica-
tion, predictions were also conducted for an independent
data set on the rule parameters derived from the 2191
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400
Table 3. Overall Success Prediction Rates for the 12 Subcellular Locations of Proteins by Different Algorithms and Test Methods
Algorithm Input
Least Hamming distance Conventional
(Chou, 1989) amino acid
composition
Least Euclidean distance Conventional
(Nakashima et al., 1986) amino acid
composition
ProtLock (Cedano et al., Conventional
1997) amino acid
composition
Augmented covariant- Pseudo amino
discriminant (Chou, 2001) acid
composition
generated by
digital signal
processing
a
Using the training data set taken from (Chou, 2001; Chou and Elrod, 1999b).
b
Using the independent data set taken from (Chou, 2001; Chou and Elrod, 1999b).
proteins in the training data set. The independent data set
was also adopted from Chou and Elrod (1999b). How-
ever, for the same reason as mentioned above, of the
2591 independent proteins originally studied in (Chou
and Elrod, 1999b), only 2494 proteins were retrieved.
They are: 112 chloroplast proteins, 761 cytoplasm, 19
cytoskeleton, 106 endoplasmic reticulum, 95 extracellu-
lar, 4 Golgi apparatus, 31 lysosome, 163 mitochondria,
418 nucleus proteins, 23 peroxisome, 762 plasma mem-
brane. None of these proteins occurs in the training data
set. The predicted result thus obtained for the 2494
proteins in the independent data set are summarized in
Table 3 as well, from which we can see that 1874 pro-
teins were correctly predicted for their subcellular loca-
tions and only 620 proteins were incorrectly predicted.
The overall success rate is 73.9%.
Furthermore, to facilitate comparison, the results
obtained by some other algorithms using the conven-
tional amino acid composition (Cedano et al., 1997;
Chou, 1989; Nakashima et al., 1986) on the same data
sets are also listed in Table 3. From the table we can see
that the success rates obtained by the current approach
are remarkably higher than those by the approaches
based on the conventional amino acid composition alone,
i.e., without taking into account the sequence order ef-
fects at all. Although the success rates obtained here are
still lower than those by Chou using different ranks of
sequence-order-correlated factors to define the pseudo
amino acid composition (Chou, 2001), the current
approach has a unique feature that might be of use to
Pan, Zhang, Guo, Feng, Huang, and He
Test method
Self-consistencya Jackknifea Independent data setb
1067
2191 = 48.7% 1033
2191 = 47.2% 1151
2494 = 46.2%
1096
2191 = 50.0% 1063
2191 = 48.5% 1197
2494 = 48.0%
1023
2191 = 46.7% 971
2191 = 44.3% 1018
2494 = 40.8%
1785
2191 = 81.5% 1483
2191 = 67.7% 1842
2494 = 73.9%
complement Chou’s approach. The five pseudo amino
acid components introduced here have a “global feature”
regardless of the length of proteins considered. However,
in Chou’s approach, the sequence-order-correlated rank
must be smaller than the length of the shortest protein
chain in the data set considered. Therefore, when a pro-
tein data set contains many short chains, the advantage
of the present approach will become more remarkable.
Furthermore, as is well known for those who are work-
ing on the area of statistical prediction, an approach,
which has yielded a higher success rate than the other for
a given data set, will not necessarily so when used to a
different data set, particularly when the prevailingness is
not overwhelming (Chou and Zhang, 1995).
The goal of this study is not to determine the possi-
ble upper limit of the success rate for the prediction of
protein subcellular location, but to propose a different
approach to incorporate the sequence order effect. The
results obtained in this study suggest that the stochastic
signal processing approach is quite promising, at least
it may play a complementary role to the other existing
methods.
4. CONCLUSION
The development of the algorithm for predicting
protein subcellular location generally consists of two
cores: one is how to give a mathematical expression to
effectively represent a protein and the other is how to
468706.qxd 7/31/03 1:38 PM Page 401
Application of Pseudo Amino Acid Composition
find an operational equation to effectively perform the
prediction. The process in expressing a protein from the
classical 20-D amino acid composition vector (Chou,
1980; Chou and Zhang, 1993, 1994; Nakashima et al.,
1986) to the (20 +
)-D pseudo amino acid composition
vector (Chou, 2000a, 2001, 2002) and to the functional
domain approach (Chou and Cai, 2002) reflects the de-
velopment of defining a protein in terms of different
mathematical representations. The process in conducting
prediction using from the simple geometry distance al-
gorithm (Chou, 1980, 1989; Nakashima et al., 1986), to
the Mahalanobis distance algorithm (Cedano et al., 1997;
Chou, 1995; Chou and Zhang, 1994), to the covariant
discriminant algorithm (Chou et al., 1998; Chou and
Elrod, 1999a, 1999b; Liu and Chou, 1998; Zhou, 1998),
and to the current SVM algorithm (Chou and Cai, 2002)
reflects the development of computation by means of dif-
ferent mathematical operations. In this study, we have
proposed a different approach, the digital signal process-
ing approach, to define the pseudo amino acid composi-
tion for a protein, followed by using the augmented
covariant discriminant algorithm to predict its subcellu-
lar location. Our results have further confirmed that the
pseudo amino acid (Chou, 2001) is quite a promising ve-
hicle for incorporating the sequence order effect, and that
the augmented covariant discriminant algorithm (Chou,
2000a) is indeed a powerful technique in performing the
task of prediction. One of the remarkable advantages of
introducing the digital signal processing approach is that
many well-established sophisticated mathematical and
engineering tools can be directly applied to various sub-
areas of biology. For example, the current approach can
be directly used to improve the prediction quality of var-
ious protein attributes, such as G-protein-coupled recep-
tor types (Chou and Elrod, 2002; Elrod and Chou, 2002)
and enzyme-family classes (Chou and Elrod, 2003).
ACKNOWLEDGMENTS
The authors would like to express their gratitude to
the anonymous referees whose constructive comments are
very helpful for improving the presentation of this paper.
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