Analytica Chimica Acta 767 (2013) 35–43

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Analytica Chimica Acta

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Identification of discriminatory variables in proteomics data analysis

by clustering of variables夽
Sadegh Karimi a,b , Bahram Hemmateenejad a,b,∗
a
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
b
Chemistry Department, Shiraz University, Shiraz, Iran

h i g h l i g h t s g r a p h i c a l a b s t r a c t

◮ A new method was suggested for A new method based on the clustering of variables (CLoVA) was proposed for identification of discrimi-
identification of discriminatory vari- natory variables in proteomic data analysis.
ables.
◮ The method works based on the clus-
tering of variables (CLoVA).
◮ CLoVA was used as an efficient
method in proteomics data analysis.
◮ The method was applied successfully
in cancer detection.

a r t i c l e i n f o a b s t r a c t

Article history: This article presents a data analysis method for biomarker discovery in proteomics data analysis. In fac-
Received 5 August 2012 tor analysis-based discriminate models, the latent variables (LV’s) are calculated from the response data
Received in revised form measured at all employed instrument channels. Since some channels are irrelevant and their responses
24 December 2012
do not possess useful information, the extracted LV’s possess mixed information from both useful and
Accepted 28 December 2012
irrelevant channels. In this work, clustering of variables (CLoVA) based on unsupervised pattern recog-
Available online 8 January 2013
nition is suggested as an efficient method to identify the most informative spectral region and then it is
used to construct a more predictive multivariate classification model. In the suggested method, the instru-
Keywords:
Classification
ment channels (m/z value) are clustered into different clusters via self-organization map. Subsequently,
Proteomics the spectral data of each cluster are separately used as the input variables of classification methods such
Clustering of variables as partial least square-discriminate analysis (PLS-DA) and extended canonical variate analysis (ECVA).
Cancer The proposed method is evaluated by the analysis of two experimental data sets (ovarian and prostate
Discriminant analysis cancer data set). It is found that our proposed method is able to detect cancerous from healthy samples
Self-organization map with much higher sensitivity and selectivity than conventional PLS-DA and ECVA methods.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Ovarian cancer is the most common cancer and it causes more

夽 Paper presented at the XIII Conference on Chemometrics in Analytical Chemistry
(CAC 2012), Budapest, Hungary, 25–29 June 2012.
∗ Corresponding author at: Chemistry department, Shiraz University, Shiraz, Iran.
Tel.: +98 711 613 7360; fax: +98 711 228 6008.
E-mail address: hemmatb@sums.ac.ir (B. Hemmateenejad).
deaths than any other cancer of the female reproductive system [1].
This occurs most often in women aged 50–79; over 70% of the can-
cers occur after age 50. With early detection and quick treatment,
the overall survival rate increases to 95%. However, early stage diag-
nosis is unusual. At the present time, only 25% of all ovarian cancers
are found at an early stage [2], this fact confirms the importance of
methods which can improve the early detection of ovarian cancer.

0003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aca.2012.12.050
36 S. Karimi, B. Hemmateenejad / Analytica Chimica Acta 767 (2013) 35–43
Prostate cancer is one of the most widespread malignancies in
men and the second leading cause of death from cancer. Currently,
prostate-specific antigen (PSA) is the best tumor marker available
for the early detection of prostate cancer. But, PSA lacks specificity
and is also detectable in benign cases as well as it can be elevated
in who with cancer [3].
Since the development of large mass spectrum profiling; includ-
ing huge large number of biomarkers, modern statistical research
has been focused on selecting the important biomarkers related to
diagnosable symptoms such as prostate and ovarian cancers [4].
Analyzing of such large data sets has various difficulties, one of
the most general problem is about the high-throughput in mass
spectrometry methods like SELDI-TOF (surface enhanced laser des-
orption/ionization time-of-flight) and MALDI-TOF (matrix-assisted
laser desorption and ionization time-of-flight) [5]. Each generated
spectrum by these instruments contains tens of thousands inten-
sity measurements representing an unknown number of protein
peaks. The problem of both methods for such studies is that the
ratio of spectral variable number to available samples may be larger
than 1000 [6], which increases the chance correlation. In addition,
the unknown complexity of the relationship(s) between the mea-
sured mass spectrum profiles and the observed disease states is
another problem which should be concerned. On the other hand,
these techniques efficiently investigate a biological sample to verify
the relative abundance of many protein/peptide sequences simul-
taneously [7]. Moreover, at the present, these methods are the most
popular techniques applied for detecting quantitative or qualitative
changes of proteins in body fluids including serum, plasma, urine
etc. [8].
For finding the potential biomarkers and revealing differences
in complex mass spectral patterns of proteins with rather low
signal-to-noise ratio, computational analysis is necessary [9]. In
this context, pattern recognition analysis has been found to play
a considerable role in proteomics for biomarker discovery. Recent
publications on cancer classification using MALDI or SELDI-TOF
data sets have been focused on identifying biomarkers in serum to
distinguish between cancer, benign and normal samples [10–18].
Until now, different feature selection and learning algorithms have
been reported in cancer studies (Prostate [11,18] and Ovarian
[10,12,15] cancers). Genetic algorithm (GA)[10], AUC (area under
receiver operating characteristic curve) [11,18], ANOVA [12] and
statistic tests [14] have been widely used in feature selection.
Statistical methods such as t-test simply give an estimate of the
significant of variables in a regression models. However, in prac-
tical applications, they are associated with some limitations, e.g.,
the significance of a variable in a model, as measured by t-test,
would be dependent on the absence or presence of the other vari-
ables in the model [19]. In this context, new and efficient methods
for assessing the significant of variables are demanding [20]. As
learning algorithms, different clustering and classification methods
such as self-organizing map [10,11] decision tree [11,18], discrim-
inant analysis [15], and support vector machine [12] are often
employed. In each study, the best m/z values, denoted as discrim-
inatory patterns or proteomics biomarkers, have been identified
to categorize cancer samples with very high sensitivity and speci-
ficity. However, finding a few numbers of variables from the pool
of thousands of instrumental channels is a difficult task. Further-
more, from these studies, has been found that some identified
discriminatory patterns with high sensitivity and specificity are not
biologically significant [10]. In addition; the lack of reproducibility
of discriminatory patterns by different analysis methods is also a
problem [12].
Very recently, we have suggested a segmented principal com-
ponent analysis and regression (named SPCAR) approach in
multivariate calibration and QSAR studies [21–23]. In those stud-
ies, instead of application of PCA on whole data set, the variables
were first clustered into different parts and then PCA was applied
on each part, separately. So, the main object was improvement the
modeling ability of principal component regression. In the present
research, we investigated the applicability of this method for vari-
able selection in proteomics data analysis based on classification
methods. Similar to SPCAR, the basic principle of the method is
clustering of variables, before doing regression or classification
analyses. So, we would like to call it “clustering of variable” with
CLoVA abbreviation. It should be noted that CLoVA is a new exten-
sion of SPCAR for using in variable selection. In the present study,
CLoVA has been successfully applied for identification of the most
significant variables (m/z values) in discrimination and then for
obtaining more discriminating models. As classification methods,
partial least square-discriminate analysis (PLS-DA) and extended
canonical variates-discriminant analysis (ECVA-DA) [24] have been
employed. Successful application of the suggested method for can-
cer detection based on proteomics data confirms the superiority of
our method with respect to the existing ones.
2. Theory
2.1. Notations
The standard chemometrics notations will be used. Capital
and lowercase letters in boldface demonstrate matrix and vec-
tor, respectively. Lowercase italic letters denote the scalars. The
response data matrix (X) is denoted by an (m × n), where m and
n are the number of objects (samples) and variables (m/z), respec-
tively, each row is a mass spectrum of a studied sample. The column
vector of class information (predictor variable) is denoted by y. The
response data matrix is divided into different sub-matrices (clus-
ters), each sub-matrix is denoted by Xi and the symbol ‘|’ is used
to show clustering of X so that X = [X1 |X2 |. . .|Xq], where q is the
number of clusters.
2.2. CLoVA algorithm
From the classification point of view, the X-variables can be
partitioned into two parts (i) those having information about the
class variable (cancerous or healthy samples) and (ii) those are
irrelevant and their information is not related to the class infor-
mation. Various efforts have been made to propose an algorithm
for selecting the useful part of data which is suitable for classifica-
tion purposes. But, it is difficult to separate these parts from each
other completely. On the other hand, the informative variables,
which possess high correlation with y-variable, are also correlated
with each other and can be considered as collinear variables. The
irrelevant variables that are not correlated with the class informa-
tion can form another sets of collinear variables. Nevertheless, if the
PCA is applied on the original data matrix including all variables,
the extracted PCs, which are used as input of discriminant model,
will possess mixed information from both irrelevant and relevant
variables. The CLoVA attempts to find clusters of variables based on
interrelation between them and to use variables of each cluster as
input of separate classification models.
The CLoVA method is composed of two main steps:
(1) Clustering of variables (here m/z values) into q clusters (sub-
matrices) using unsupervised clustering method such as PCA, self-
organizing map and so on:
X = [X1 |X2 | . . . |Xq ] (3)
Each sub-matrix Xi is composed of a subset of variables clustered
in one cluster. The number of clusters should be optimized through
model development.
(2) Using the variables of each cluster to build a separate classi-
fication model by e.g., PLS-DA or ECVA.
 S. Karimi, B. Hemmateenejad / Analytica Chimica Acta 767 (2013) 35–43
Thus, in the CLoVA, a clustering method is followed by a regres-
sion or a classification method. Here, self-organizing map has
been used as clustering method, PLS-DA and ECVA-DA have been
employed as classification methods. For more details about this
algorithm, the interested readers can refer to our previous publica-
tions [21–23]. All the chemometrics methods which have been used
in this work are well known, however in order not to prolong the
manuscript the descriptions of these algorithms have been done in
supporting information.
Self-organizing map (SOM) [25] is an interactive and iterative
technique used to map the multivariate data. The maps, which are
consisting of layer of neuron arranged in two-dimensional grid,
should be able to reflect the relationship that exists between sam-
ple points in the original space. The interesting point of view is
that, during the training step, the network adapt itself in a way
that similar input (here variables) are associated with the topolog-
ical close neuron in the network. This method can be considered as
unsupervised clustering method. Self-organizing map projects the
input data into a bi-dimensional regular array (grids) of nodes size
(p × p), where p is defined as network size. For each neuron in the
grid, we have a weight vector which is in the same dimensions as
the pattern of input data set. The input vector is passed to each neu-
ron (in the created network) and the neuron whose weight is the
most similar to pattern vector of input is selected as winner. So for
the winning neuron its weight is adjusted in such way to be much
more similar to the pattern of input vector. Those neurons which
are neighbors to winner neuron also adjusted its weight as well but
in different degree. Finally, when the process is completed, similar
input vectors (variables) are clustered in the space based on similar-
ities. Note that we have applied self-organizing map to cluster the
variables not the objects. Hence, the variables in the original data
matrix, including similar information (in our case similar spectral
information regarding the sample classes), are projected into one
node or neighboring nodes. During the computation of the map, the
variables are projected onto the self-organizing map’s array and the
model vectors adapted according to the learning rule. The output of
self-organizing map is a pattern distribution of variables in a map
of (p × p) nodes. Among the original variables in X matrix, the vari-
ables in each node can be collected to form a sub-matrix Xi . Thus,
the number of clusters is equal to power of 2 of node number in
self-organizing map.
2.3. Selectivity ratio (SR)
Among the different criteria suggested for identification of the
most important discriminatory variables, selectivity ratio (SR) plot
has been reported recently by Rajalahti et al. as a promising method
[6,20]. SR is closely connected to the ratio of inter- to intra-group
variation and it is a measure of variable’s performance to separate
groups. SR can reveal regions in spectral profiles with both high
explanatory and high predictive significance for the investigated
response. Explained vexpl,j and residual (unexplained) vres,j variance
for each variable j in the target projection model can be calculated
from Eq. (1).
X = X̂TP + ETP = tTP pTTP + ETP
The ratio between explained and residual (Eq. (2)) variance
defines a selectivity ratio SR for each variable:
vexp l,j
SRi = , j = 1, 2, 3, . . .
vres,j
A high SR value means that the variable in question has a strong
(predictive) correlation to the response, that is, the variable is
highly selective. The limit between spectral regions with marker
candidates and less important regions is chosen by user. In this
37
work, the F-test criterion has been chosen to select the marker
candidate.
3. Experimental
3.1. Data sets
To illustrate the performance of the proposed algorithm, two
cancer data sets (ovarian and prostate) have been analyzed.
Both data sets were downloaded from the Clinical Proteomics
Program Data bank (http://home.ccr.cancer.gov/ncifdaproteomics/
ppatterns.asp). Using the data set on this website, for the ovar-
ian data set we have analyzed the third ovarian dataset (8-7-02).
It is composed of the low resolution MALDI-TOF mass spectra
proteomic profiles of blood serum for 162 biopsy-proven ovarian
cancer patients and 91 unaffected women (controls). In order to
have more balance data set, only first 100 numbered patients (of
the cancer group) have been selected. Each proteomic profile, or
spectrum, consists of 15,154 distinct m/z values ranging from 0 to
20,000.
The prostate data set is composed of mass spectral profiles
consisting of 15154 SELDI-TOF m/z ratios from 321 samples (69
patients diagnosed with malignant prostate cancer, 189 patients
with benign prostate hyperplasia and 63 controls) [26].
3.2. Computation
Data Analyses have been performed in MATLAB environment
(Math works, Inc., Natick, MA, USA, version 7.2). PLS-DA cali-
bration is based on the PLS Toolbox version 4 from Eigenvector
Research. ECVA has been performed with the MATLAB models
available at http://www.models.life.ku.dk. The self-organizing map
Toolbox, provided by Todeschini and Ballabio was downloaded
from the website of Milano Chemometrics and QSAR research group
(http://michem.disat.unimib.it/chm/download/kohoneninfo.htm).
3.3. Training and test set selection
These data sets have been divided into training and external
test sets by sample set partitioning based on joint x–y distances
(SPXY) algorithm, so that about 30% of each group has been selected
as test set and the remaining have been used as training to build
the classification model. This algorithm, which considers both dis-
tances in the dependent and independent variable spaces, has
been explained in details by Galvao et al. [27]. This procedure
ensures that representative samples, internal to the data domain,
are selected as test objects.
3.4. Preprocessing
PCA, PLS-DA and ECVA are scale-dependent methods. In addi-
tion, variable clustering by self-organizing map would be also
affected by scaling method since self-organizing map does not use
(1) simple correlation to measure the similarity between variables. The
disadvantage of auto-scaling in optical spectroscopy data is about
the exaggeration of the import of noisy wavelength regions, which
may mask the interested effects. Pareto scaling [28], has become
prevalent for NMR and MS data sets. It can be regarded as method
(2) between the extremes of no scaling and unit variance scaling and
involves dividing each spectral variable by the square root of its
standard deviation after first centering. In our study Pareto scaling
has been applied to these data sets prior to the multivariate data
analyses. We have also applied the orthogonal signal correction in
each cluster, before application of discriminate analysis methods.
38 S. Karimi, B. Hemmateenejad / Analytica Chimica Acta 767 (2013) 35–43
4. Results and discussion
The MALDI–TOF spectra of the ovarian data set and SELDI-TOF
spectra of prostate samples are represented in Fig. S1 and S2 of
supporting information, respectively. One can observe that all sam-
ples of each data set exhibit similar spectra, so that, discrimination
between healthy and cancerous samples by visual inspection of the
spectra is impossible. In this section, the results of the analysis for
these data sets by CLoVA method followed by discriminant analysis
methods (PLS-DA and ECVA) are explained separately and then they
are compared with the results of conventional discriminant analy-
sis methods. In the proceeding sections, we will show how CLoVA
helps us to discriminate the healthy and cancerous samples and to
determine the most significant variables for biomarker discovery.
As clustering method in CLoVA, we used self-organizing map in
this study since in previous study [21] we found that self-organizing
map over-performed with respect to PCA. The latter is considered
as linear clustering method whereas self-organizing map is a non-
linear one. Thus, variables having nonlinear relationships which
others (such as quadratic, logarithmic, etc. relationships) are con-
sidered as neighboring variables in the self-organizing map pattern.
In addition, according to a parallel study in our group on the com-
parison of the performance of 5 different clustering methods in
CLoVA (the results of which will be published elsewhere) it was
found that self-organizing map usually resulted in better modeling
power. So, in this study we preferred to use self-organizing map as
inner clustering method in CLoVA.
4.1. Data set 1 (ovarian cancer dataset)
4.1.1. CLoVA analysis of ovarian data set
The first step in CLoVA analysis is to find clusters of variables.
Indeed the self-organizing map is similarly unsupervised as PCA,
but it is a non-linear method on the contrary to PCA which both
of them can be employed in CLoVA [21–23]. Moreover the results
of self-organizing map depend on initial given weights. Previously,
we have shown that self-organizing map as a nonlinear cluster-
ing method is more efficient than PCA. So that, in present study,
we used self-organizing map to find clusters of variables. Self-
organizing map produces a distribution map of variables in an
(p × p) array of nodes. Subsequently, the total number of clusters
is p2 . The number of clusters in CLoVA should be optimized. The-
oretically, the number of clusters can be changed between 1 and
nV , where nV is the number of variables. If number of cluster sets
to 1, this means that all variables are used as input of classifi-
cation method, which is the conventional discriminant analysis
method. On the other hand, the number of clusters of nV resem-
bles the stepwise variable selection method, where all variables
are examined separately. In real applications, the number of clus-
ters could be optimized by sequential increasing in p followed by
discriminant analysis to find a model with the desired level of
acceptability (the least prediction error). Here, we have examined
the number of nodes from 2 to 9. The distribution of variables in the
self-organizing map of network size (n = 2) is presented for ovarian
data set as an example (Fig. S3) in the supporting information. The
clusters in self-organizing map are arranged in a bi-dimensional
(matrix-like) pattern. Each cluster is denoted as Si,j , where i and j are
the row and column numbers of the cluster in the self-organizing
map, respectively. The variables of each cluster have been used as
input of separate discriminant analysis models (PLS-DA or ECVA-
DA).
4.1.2. PCA of ovarian data set
PCA [29] has been performed to obtain an overview of the data.
The spectra span the mass range from 0 to 20,000 Da (15,154m/z
channels). The results of application of PCA on spectral data matrix
of ovarian samples (whole region) are given in Table (S1) for the
first 10 principal components (PCs). The two-dimensional scores
plot of the first and second PCs, which explain respectively 30.39%
and 28.56% of total spectral variations is shown in Fig. S4a. This
figure indicates the relative position of samples with each other
based on the similarity between their MALDI-TOF spectra. Due to
high similarity between the MALDI-TOF spectra of the ovarian sam-
ples, there is no evidence of separation between two classes along
the first two principal axes and the groups are severely overlap-
ping. In other words, much of the spectral variation is unrelated to
class differences and the model is not practical. Extraction of further
principal component (PCs) did not provide any separation either.
However, by CLoVA variable analysis before PCA, better class
separation has been observed in two-dimensional spaces of fac-
tor scores. In Fig. S4b one representative plot is shown. The
scores of this plot have been calculated from cluster S1,2 of the
self-organizing map model of p = 2. Accordingly, a much better
separation of classes is observed. This suggests that the vari-
ables clustered in cluster S1,2 possess better spectral information
regarding the ovarian cancer biomarkers.
4.1.3. Classification by PLS-DA
Firstly, PLS-DA has been run on all m/z variables. To reduce the
number of components resulting from the large variations in X
which are not related to the Y space, the orthogonal signal correc-
tion (OSC) algorithm [30,31] suggested by Wold et al. has been used.
The number of significant components has been determined using
leave-many-out cross-validation (LMO-CV) with 1/8th of the data
being excluded during each round. The changes in the classification
error as a function of PLS latent variables are shown in supporting
information (Fig. S5). This yielded a five PLS-components model as
the optimum one. Then, it has been used to predict the class variable
of the prediction samples. Fig. 1a shows the two-dimensional score
plot of PLS-DA on whole data set. It is observed that PLS-DA presents
a partial discrimination between normal and cancerous samples
with some degrees of overlapping. The classification parameter
results for calibration and prediction samples are shown in the first
row of Table 1. From the results in Table 1 it is evident that conven-
tional PLS-DA failed to completely differentiate between healthy
and cancerous persons. Similar to PCA, it can be concluded that all
parts of the used m/z channels do not possess useful information
about the class of ovarian samples. On the other hand, many parts
of the MALDI-TOF data are highly collinear and containing simi-
lar spectral information. Therefore, CLoVA analysis of variables has
been performed before PLS-DA running.
Eight self-organizing map networks with node size of 2–9 were
checked (see Fig. (S3) from supporting information section as an
example). The numbers in the clusters which present the m/z values
of the MALDI-TOF spectra are shown in Fig. S1. The best clas-
sification results (lower number of misclassified samples) have
been obtained when the node number is 2 and hence the results
of this network size will be presented and explained. The shown
self-organizing map pattern (Fig. S3) indicates that the pattern dis-
tribution of these m/z values is not homogeneous. Obviously, some
clusters (S2,1 , S2,2 of 2 × 2 network) contain a high population of m/z
values whereas some others (S1,1 and S1,2 of 2 × 2 network) include
limited numbers. The interesting aspect of clustering method such
as self-organizing map is that m/z value with similar information
are collected into some specified cluster. The m/z values of each
cluster form a sub-matrix (Xi ) of the original variable data matrix
(X). Each sub-matrix has been separately subjected to PLS-DA to
find the most suitable m/z values, which are useful for classifica-
tion. Some statistical parameters of the PLS-DA models obtained
from variables of each cluster are listed in Table 1. In addition,
the two-dimensional PLS score plots of different clusters are given
in Fig. 1b–e. Apparently, result of cluster S2,1 leads to superior
 S. Karimi, B. Hemmateenejad / Analytica Chimica Acta 767 (2013) 35–43 39

(a) (b)

20

PLS component 2

PLS component 2
50

0 0

-50
-20
-450 -400 -350 -150 -140 -130
PLS component 1 PLS component 1
(c) (d)
40
PLS component 2

PLS component 2
20
20
0 0
-20
-20
-40
-280 -260 -240 -220 -200 -180 -130 -120 -110 -100
PLS component 1 PLS component 1
(e)
PLS component 2

40

20

-20

-300 -280 -260 -240

PLS component 1

Fig. 1. Distribution pattern of the ovarian samples in the two-dimensional PLS-DA based factor space of their MALDI-TOF spectra (a) whole spectral region and (b–e) CLoVA-
based PLS-DA of different clusters of (2 × 2) self-organizing map model: (b) cluster S1,1 , (c) cluster S2,1 , (d) cluster S1,2 and (e) cluster S2,2 . (, 䊉) cancer, (, ) control. Filled
marker is calibration and open marker is prediction.

discrimination information than the full data. Interestingly, a very
clear separation is observed between healthy and cancerous sam-
ples from the score plot of S1,2 , which suggests that variables of this
cluster own the largest discriminating information. PLS-DA model
of this cluster has non-error rate of 1 for both calibration and test
samples (see Table 1), which means that this model has perfect
prediction for all samples.
In addition to model production of a zero misclassification error,
similar to other variable selection method, CLoVA is associated
with two other interesting features: (1) using much lower num-
ber of variables (902m/z variables in cluster S1,2 compared to total
number of 15,154 variables) it is possible to make faster pattern
recognition model and (2) it identifies m/z channels that are rele-
vant for the investigated classes of samples.

Table 1
Number of m/z value (Ni ). Number of latent variable used in each model (LV)a , non-error rate (NER), i.e., percentage of correctly assigned samples achieved for ovarian cancer
data set. Sensitivity (Sn)d and specificity (Sp)e achieved for ovarian cancer data set.

Method N LVa NERcalb NERprec Normal Cancer

Snd Spe Sn Sp

Cal Pred Cal Pred Cal Pred Cal Pred

PLS-DA 15,154 5 0.8557 0.8617 0.9000 0.9512 0.8085 0.8077 0.8085 0.8077 0.9512 0.9000
C CLoVA based-PLSDA(S1,1 ) 233 2 0.8247 0.7979 0.8200 0.9024 0.7898 0.7308 0.7898 0.7308 0.9024 .8200
CLoVA based-PLSDA(S1,2 ) 907 2 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
CLoVA based-PLSDA(S2,1 ) 2943 3 0.9381 0.9149 0.9800 0.9756 0.8936 0.8846 0.8936 0.8846 0.9756 0.9800
CLoVA based-PLSDA(S2,2 ) 11,076 5 0.8765 0.8723 0.8800 0.9024 0.8723 0.8654 0.8723 0.8654 0.9024 0.8800
a
Number of PLS latent variables.
b
NER for calibration set.
c
NER for prediction set.
d
Class sensitivity (Sn) describes the model ability to correctly recognize samples belonging to the gth class, i.e. if all the samples belonging to g are correctly assigned, Sn
is equal to 1.
e
Class specificity (Sp) describes the model ability to reject samples of all the other classes from class g, i.e. if samples not belonging to g are never assigned to g, Sp is equal
to 1.
40 S. Karimi, B. Hemmateenejad / Analytica Chimica Acta 767 (2013) 35–43
Fig. 2. SR plot for identification of discriminatory variables of ovarian data set: (a)
PLS-DA of whole spectral region and (b) CLoVA-based PLS-DA (cluster S1,2 of (2 × 2)
self-organizing map model.
Once the classification of cancerous and healthy samples has
been achieved by CLoVA-based PLS-DA, it has been investigated to
identify the most significant variables, which is of special impor-
tance in biomarker discovery. Fig. 2 compares the SR plot of the
PLS-DA analysis of whole spectral region with spectral variables
included in cluster S1,2 of (2 × 2) self-organizing map network.
Noticeably, the number of significant variables identified by SR
plot for CLoVA–based PLS-DA (15) (Table (S2)) is much lower than
those have been found for conventional PLS-DA (872). The sim-
pler pattern of the SR plot obtained by CLoVA-based PLS-DA is
more convenient for biomarker discovery. Selection of important
variables based on SR plot and CLoVA-based PLS-DA approach
(Fig. 2b) provides one main m/z region for biomarker candidates
(25–1000). It should be noted that since we have 97 samples
in calibration section, the critical F-value with 0.95 probability
level is 1.41. Thus, with this criterion many false biomarker can-
didates have been removed. Actually, the SR combines predictive
power (regression coefficients) with explanatory power (vari-
ance/covariance between spectral variables). Noticeably, many of
the largest coefficients associated with m/z values lies below 1000,
which is in good agreement with previous findings [32].
4.2. Data set 2 (prostate cancer data set)
This data set is more complex than the ovarian data set. The
number of samples in this case is much more than the previous
one. In addition, here we have 3 classes including 63 normal, 69
cancer and 189 benign samples which cause to more complex-
ity of prostate dataset in comparison with ovarian ones dataset.
Thus, finding the subset of m/z values which are related to the class
information property is not so simple.
In the same manner as in the ovarian data set study, the m/z
variables have been subjected to CLoVA analyses by self-organizing
map. The network sizes of 2–9 have been investigated and the
same convention has been applied to denote the clusters in the
self-organizing map.
Table 2
Non-error rate (NER), i.e., percentage of correctly assigned samples achieved with
different classification methods for prostate data set.
Methods LVa NERcalb NERprec
PLS-DA 8 0.7634 0.7320
CLoVA based-PLSDA(S3,2 of p = 3) 4 0.9141 0.8763
CLoVA based-ECVA(S3,3 of p = 5) 26 1.000 0.9287
a
Number of PLS latent variables.
b
NER for calibration set.
c
NER for prediction set.
4.2.1. PCA of prostate data set
The results of PCA application in the prostate samples are given
in Table (S3). The PCA has been applied on whole region of spectral
data matrix and the results are presented for the first 10 principal
components (PCs). The eigenvalue (EV) of each PC, the percent of
variances in the data explained by each PC (PV) and the cumulative
percent of variances (CPV) are reported. Table (S3) reveals that the
first two principal components explain about 60% of the total spec-
tral variations. In other words by projecting 15,154-dimensional
spectra into two-dimensional factor space, about 60% of informa-
tion is retained. In accordance with Fig. S6a, a severe overlapping
between classes is observed. PCA is an unsupervised method and
the PCs in PCA are calculated only from the data matrix (X) and
do not use the class information (Y). Subsequently, they might not
necessarily be the components relevant for discrimination. In addi-
tion, whole region has been used for extracting the PC’s, and as it is
shown, the information of whole regions is not useful for discrim-
inations. However, better separation of data has been achieved by
application of CLoVA concept. As it is shown in Fig. S6b, by applying
the PCA on the variables, which is in cluster S3,2 from network size
3, one can see some degrees of improvement in the scatter plot of
PCs.
4.2.2. Classification by PLS-DA
The PLS-DA model has been developed using selected 224
training samples selected by SPXY algorithm. The changes in the
classification error (utilizing 10-segment contiguous block CV) as a
function of number of PLS latent variables are depicted in suppor-
ting information (Fig. S7). The lowest minimum misclassification
error of cross-validation has been obtained at 8 numbers of PLS
latent variables and at this latent variable the errors of calibra-
tion and cross-validation are very similar. Thus, an 8-component
PLS model; has been used for class prediction of the test set com-
pounds. The non error rate (NER, % of correctly assigned samples)
for calibration and prediction samples are shown in the first row
of Table 2, also in Table 3 specificity and sensitivity achieved on
all samples are shown. The distribution of samples in the three-
dimensional PLS score space is shown in Fig. 3a. Whilst the class
separations are better than that we observed in the score space of
PCs, the classification quality is not high at all. There is a high degree
of overlapping between benign and cancerous samples.
In the next step, CLoVA method has been employed to increase
the degree of classification by selecting the most informative m/z
variables. PLS-DA analysis of the size 3 network clusters has been
produced the best classification results for both calibration and pre-
diction. By analyzing different clusters of 3 × 3 self-organizing map,
the cluster S3,2 (possessing 1893m/z variables out of 15,154 vari-
ables; about 90% reduction in the number of variables) is resulted in
better sensitivity and selectivity. The distribution of sample points
in the three-dimensional PLS score space of the S3,2 variables is
presented in Fig. 3b. Clearly, there is an improvement in sample
discrimination with respect to conventional PLS-DA (Fig. 3a). Also,
the statistical parameters in the second row of Table 2 confirm the
superiority of CLoVA-based PLS-DA model in predicting class vari-
ables of prostate data set. However, the reported data in Table 2
 S. Karimi, B. Hemmateenejad / Analytica Chimica Acta 767 (2013) 35–43 41

Table 3
Specificity (Sp)a and sensitivity (Sn)b achieved by different classification methods for prostate data set.

Subset Method Normal Benign Cancer

Sn Sp Sn Sp Sn Sp

Cal PLS DA 0.8611 0.9181 0.7391 0.8720 0.7600 0.7931

CLoVA based-PLSDA(S3,2 of p = 3) 1.000 0.9894 0.9348 0.9302 0.8400 0.9483
CLoVA based-ECVA(S3,3 of p = 5) 1.000 1.000 1.000 1.000 1.000 1.000
Pred PLS DA 0.8889 0.9086 0.7255 0.7391 0.5263 0.7846
CLoVA based-PLSDA 0.9630 0.900 0.8627 0.8913 0.7895 0.8620
CLoVA based-ECVA 1.000 1.000 0.9020 0.9565 0.8947 0.9359
a
Class sensitivity (Sn) describes the model ability to correctly recognize samples belonging to the gth class, i.e. if all the samples belonging to g are correctly assigned, Sn
is equal to 1.
b
Class specificity (Sp) describes the model ability to reject samples of all the other classes from class g, i.e. if samples not belonging to g are never assigned to g, Sp is equal
to 1.

explain that in spite of significant improvement in classification
accuracy achieved by CLoVA, the suggested model is associated
with small misclassification errors (especially for test set samples)
due to complexity of the investigated data set.
4.2.3. Classification by ECVA
It has been shown previously that ECVA-DA performs better
than PLS-DA in different classification problems [33–35]. So, to
improve the classification result for prostate data set, the com-
bination of CLoVA and ECVA has been performed. In CLoVA step,
among the different number of nodes which is examined in self-
organizing map network (2–9 nodes), the number of node of 5 led
to the best classification results (the least classification error). It
should be noted the number of PLS component, which is used as
an inner relation in ECVA, has been obtained by 10-segment con-
tiguous block CV, similar to that used for PLS-DA (see Fig. S8 of
supporting information). By ECVA-DA analysis of the variables in
different clusters of (5 × 5) self-organizing map, the cluster S3,3
(a)
200
PLS component 3
resulted in zero misclassification error for calibration and cross-
validation. The variables of this cluster were also optimum for test
samples and the least misclassification error has been obtained for
these samples.
The distribution of the samples in the two-dimensional space of
extended canonical variates space (calculated from the variables of
cluster S3,3 of 5 × 5 self-organizing map) is shown in Fig. 4. A com-
parison between PLS-DA and CLoVA-based PLS-DA confirms larger
separations between data points of different groups which have
been obtained by CLoVA-based ECVA-DA. The statistical parame-
ters of suggested CLoVA-based ECVA-DA model are given in the last
row of Tables 2 and 3. The reported data suggest that this model
has the best sensitivity and selectivity with respect to PLS-DA and
CLoVA-based PLS-DA. However, some misclassification errors are
still observed for the test samples. All normal samples have been
correctly assigned but a few benign and cancerous samples have
been predicted interchangeably.
Interestingly, a close look at the extended canonical variates
shows how these variables results in such class prediction. The
number of canonical directions is always one less than the num-
ber of groups in the data set. For 3-groups case, this means that the
solution is two-dimensional. As it is shown in Fig. S9, the canon-
ical variates in the first direction clearly show the discrimination

100 of normal samples from other groups whereas those in the sec-
ond direction shows the discrimination of cancer and benign. For
0 example, those have positive variates in the first direction and
small negative variates in the second direction are normal and
-100 those have negative variates in both directions are the most prob-
able benign samples. On the other hand, those samples that their
300 200 400 respective canonical variates in the first and second directions are
100 200
0-100 0
PLS component 2 PLS component 1
0.4

(b) 0.3
50
PLS component 3

0.2
ECV# 2

0 0.1

0
-50
-0.1

-0.2
0 -50
-50 0
-100 50
-150 100 -0.3
PLS component 2 -0.2 0 0.2 0.4 0.6 0.8
PLS component 1
ECV# 1
Fig. 3. Distribution pattern of the prostate samples in the three-dimensional PLS-
DA based factor for SELDI-TOF spectra: (a) whole region and (b) cluster S3,2 . (, ) Fig. 4. Distribution pattern of the prostate cancer samples in the two-dimensional
control, (夽, ⋆) benign and (▽, ) cancer: filled and open markers denote calibration extended canonical variate (ECVA) space of their SELDI-TOF spectra for cluster (S3,3 )
and prediction samples, respectively. of network size (n = 5). The markers are the same as describe in Fig. 3.
42 S. Karimi, B. Hemmateenejad / Analytica Chimica Acta 767 (2013) 35–43
(a)
0
Selectivity Ratio
-1
-2
-3
-4
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
M/Z value
(b) 3
Selectivity Ratio
2
1
0 Financial support of this project by Shiraz University Research
-1
-2
0 2000 4000 6000 8000 10000 12000 14000 16000 18000
M/Z value
Fig. 5. The selectivity ratio plot of CLoVA-ECVA model of SELDI-TOF spectra of
prostate data set for (a) cluster S3,3 of network size of 5 and (b) whole region.
positive can be considered as the most probable cancerous sam-
ple.
Now, it is the time to identify the discriminating m/z vari-
ables for prostate cancer. We give our discussion on this issue
for the CLoVA-based ECVA-DA, from which the least classi-
fication errors have been obtained. In the same manner as
ovarian data set, SR plot and F-ratio statistics have been used
to identify the significant variables. As it was mentioned pre-
viously, ECVA uses a PLS model in the inner relation in order
to solve the collinearity in data matrices. We used the regres-
sion vector of this PLS model for calculating SR plot. The
selectivity ratio plot for whole region of prostate samples SELDI-
TOF spectra and selected cluster (S3,3 ) are shown in Fig. 5.
Based on the SR criteria of cluster (S3,3 ) it is observed that
three main regions (450–460, 504–505 and 7160–7384m/z)
are responsible for class separation of prostate samples. Crit-
ical F-value (1.25) is shown that 172m/z channels (out of
1832m/z channels of cluster S3,3 and 15,154m/z of total vari-
ables) have been selected as the most significant discriminatory
variables. The selected m/z values are reported in Table (S4) of
supporting information.
5. Conclusion
The presented results in the current study, demonstrate that
it is possible to split the information in the scores and canoni-
cal variates of PLS-DA and ECVA into informative and irrelevant
variables. Then by selecting the informative parts, a multivariate
pattern recognition model is obtained which has higher predic-
tion ability with respect to conventional PLS-DA and ECVA. In
this method, called CLoVA based PLS-DA and CLoVA based ECVA,
the original variables are clustered into different clusters by self-
organizing map and then PLS-DA and ECVA is performed on data
variables of each cluster separately. The advantage of presented
approach is that it used the small part of m/z value (based on clus-
tering of self-organizing map) instead of entire proteomics profile
(15,154) m/z value for analysis. The performance of this method
has been validated by analysis of the MALDI-TOF spectra of ovar-
ian data set (for classification of normal from cancerous samples)
and prostate data set (for classification of normal versus benign
and cancerous samples). For first data set (ovarian cancer) CLoVA
based PLS-DA, reveals better results than conventional PLS-DA
whilst for prostate dataset which is more complex than first ones,
CLoVA based ECVA exhibits better prediction result than PLS-DA,
and CLoVA based PLS-DA. Identification of the most informative m/z
regions by CLoVA based PLS-DA and CLoVA based ECVA are simpler
and more straightforward than conventional pattern recognition
methods because we can easily focus on selected regions instead
of whole region. The proposed method can be considered as an
alternative to variable selection method for PLS-DA and ECVA-
DA.
Acknowledgment
Council is appreciated.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.aca.2012.12.050.
References
[1] F. Kong, C. Nicole White, X. Xiao, Y. Feng, C. Xu, D. He, Z. Zhang, Y. Yu, Oncology
100 (2006) 247–253.
[2] J.M. Schildkraut, W.D. Thompson, Am. J. Epidemio. 128 (1988) 456–466.
[3] D.L. Meany, Z. Zhang, L.J. Sokoll, H. Zhang, D.W. Chan, J. Proteome Res. 8 (2008)
613–619.
[4] D. Donald, T. Hancock, D. Coomans, Y. Everingham, Chemom. Intell. Lab. Syst.
82 (2006) 2–7.
[5] L. Chen, Chemom. Intell. Lab. Syst. 84 (2008) 123–130.
[6] T. Rajalahti, R. Arneberg, F.S. Berven, K.M. Myhr, R.J. Ulvik, O.M. Kvalheim,
Chemom. Intell. Lab. Syst. 95 (2009) 35–48.
[7] N. Jeffries, Bioinformatics 21 (2005) 3066–3077.
[8] R. Tibshirani, T. Hastie, B. Narasimhan, S. Soltys, G. Shi, A. Koong, Q.T. Le, Bioin-
formatics 20 (2004) 3034–3044.
[9] E.F. Petricoin, L.A. Liotta, Clin. Chem. 49 (2003) 533–534.
[10] Iii.E.F. Petricoin, A.M. Ardekani, B.A. Hitt, P.J. Levine, V.A. Fusaro, S.M. Stein-
berg, G.B. Mills, C. Simone, D.A. Fishman, E.C. Kohn, Lancet 359 (2002)
572–577.
[11] Y. Qu, B.L. Adam, Y. Yasui, M.D. Ward, L.H. Cazares, P.F. Schellhammer, Z. Feng,
O.J. Semmes, G.L. Wright Jr., Clin. Chem. 48 (2002) 1835–1843.
[12] G. Mor, I. Visintin, Y. Lai, H. Zhao, P. Schwartz, T. Rutherford, L. Yue, P. Bray-Ward,
D.C. Ward, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 7677–7682.
[13] T.C.W. Poon, T.T. Yip, A.T.C. Chan, C. Yip, V. Yip, T.S.K. Mok, C.C.C.Y. Lee, T.W.T.
Leung, S.K.W. Ho, P.J. Johnson, Clin. Chem. 49 (2003) 752–760.
[14] A. Valerio, D. Basso, S. Mazza, G. Baldo, A. Tiengo, S. Pedrazzoli, R. Seraglia, M.
Plebani, Rapid Commun. Mass Spec. 15 (2001) 2420–2425.
[15] W. Zhu, X. Wang, Y. Ma, M. Rao, J. Glimm, J.S. Kovach, Proc. Natl. Acad. Sci. U.S.A.
100 (2003) 14666–14671.
[16] Y. Hu, S. Zhang, J. Yu, J. Liu, S. Zheng, Breast 14 (2005) 250–255.
[17] T.D. Veenstra, D.R.A. Prieto, T.P. Conrads, Drug Discov. Today 9 (2004) 889–897.
[18] B.L. Adam, Y. Qu, J.W. Davis, M.D. Ward, M.A. Clements, L.H. Cazares,
O.J. Semmes, P.F. Schellhammer, Y. Yasui, Z. Feng, Cancer Res. 62 (2002)
3609–3614.
[19] D.L. Massart, B.G.M. Vandeginste, L.M.C. Buydens, Handbook of Chemometrics
and Qualimetrics, Elsevier Science, Amesterdam, 1997.
[20] T. Rajalahti, R. Arneberg, A.C. Kroksveen, M. Berle, K.M. Myhr, O.M. Kvalheim,
Anal. Chem. 81 (2009) 2581.
[21] B. Hemmateenejad, S. Karimi, J. Chemometr. 25 (2011) 139–150.
[22] B. Hemmateenejad, M. Elyasi, Anal. Chim. Acta 646 (2009) 30–38.
[23] B. Hemmateenejad, R. Miri, M. Elyasi, J. Theor. Biol. 305 (2012) 37.
[24] L. Nørgaard, G. Sölétormos, N. Harrit, M. Albrechtsen, O. Olsen, D. Nielsen, K.
‘Kampmann, R. Bro, J. Chemometr. 21 (2007) 451–458.
[25] B.K. Lavine, C.E. Davidson, D.J. Westover, J. Chem. Inf. Comp. Sci. 44 (2004)
1056–1064.
[26] E.F. Petricoin, D.K. Ornstein, C.P. Paweletz, A. Ardekani, P.S. Hackett, B.A. Hitt,
A. Velassco, C. Trucco, L. Wiegand, K. Wood, J. Natl. Cancer Inst. 94 (2002)
1576–1578.
[27] R.K.H. Galvão, M.C.U. Araujo, G.E. José, M.J.C. Pontes, E.C. Silva, T.C.B. Saldanha,
Talanta 67 (2005) 736–740.
[28] Z. Ramadan, D. Jacobs, M. Grigorov, S. Kochhar, Talanta 68 (2006) 1683–1691.
 S. Karimi, B. Hemmateenejad / Analytica Chimica Acta 767 (2013) 35–43 43

[29] A.N. Zira, S.E. Theocharis, D. Mitropoulos, V. Migdalis, E. Mikros, J. Proteome
Res. 9 (2010) 4038–4044.
[30] H. Yu, J.F. MacGregor, Chemom. Intell. Lab. Syst. 73 (2004) 199–205.
[31] S. Wold, H. Antti, F. Lindgren, J. Öhman, Chemom. Intell. Lab. Syst. 44 (1998)
175–185.
[32] O.P. Whelehan, M.E. Earll, E. Johansson, M. Toft, L. Eriksson, Chemom. Intell.
Lab. Syst. 84 (2006) 82–87.
[33] C.L. Hansen, F. den Berg, M.A. Rasmussen, S.B. Engelsen, S. Holroyd, Chemom.
Intell. Lab. Syst. 104 (2010) 243–248.
[34] H. Winning, N. Viereck, T. Salomonsen, J. Larsen, S.B. Engelsen, Carbohyd. Res.
344 (2009) 1833–1841.
[35] N. Mobaraki, B. Hemmateenejad, Chemom. Intell. Lab. Syst. 109 (2011)
171–177.