A Novel Pipeline Method for the Preprocessing of Mass

Spectrometry Proteomics Data

Maria Anna v. Rapsomaniki, Panagiotis G. Zerefos, Konstantinos A. Theofilatos,
Spiridon D. Likothanassis, Athanasios K. Tsakalidis and Seferina P. Mavroudi

Abstract- Mass spectrometry based methodologies allow us thousands of features. In addition to this high dimensionality
to analyze complex mixtures of proteins from biological - small sample size problem, each spectrunI contains a great
samples in order to identify important proteomic patterns and amount of noise and artifacts, mostly due to the high
discover novel disease biomarkers. However proteomics data
sensitivity of the instrunIent, sample contamination and
are high-dimensional and complex and need to undergo a
electrical noise. Another common problem is the
number of preprocessing steps so that they can be further
analyzed. This procedure involves various steps that interact in
miscalibration of the spectra that makes the data impossible
complex ways and is an open research area, as it has been to compare. For all those reasons, it is more than obvious
shown that it has a big effect on the final results. In this work that in order to extract knowledge about the true underlying
we propose a novel pipeline method for the preprocessing of biological differences in the proteome, various preprocessing
MS-based data. Our proposed strategy addresses some of the
steps need to be applied.
major problems when dealing with proteomics data, like
The main goal of preprocessing is to come up with a
miscalibration, noise and redundancy. Our method exploits the
advantages of using the mean spectrum, ensuring at the same
matrix of important features (i.e. peaks) and their
time higher sensitivity. In order to illustrate the utility of our corresponding intensity values, which can be further
proposed method we experimented on data coming from analyzed using a variety of computational methods. To
samples of patients with bladder cancer or benign disease. The achieve this, one must first remove noise, artifacts and
results were quite encouraging, as 31 statistically important
systematic bias without loss of information and then detect
peaks were identified, some of which are not detected by
and quantify a set of peaks. Preprocessing involves various
existing methods.
steps that are highly interrelated and it has been shown that
I. INIRODUCTION if those steps are not applied carefully, it will be difficult to
extract meaningful conclusions about the underlying disease
he rapid developments in mass spe�trometry �M �) �d
T the introduction of new expenmental 10ntzatlOn
[7]. For each step, a number of methods have been proposed
making the decision about the best combination of methods
methods, like matrix-assisted laser desorption ionization
a very challenging task. Furthermore, it is difficult to
(MALDI) and surface-enhanced laser desorption ionization
evaluate the performance of each method and come up with
(SELDI), has made it possible to study protein expression
a standard strategy, as for each dataset a different set of
levels in complex mixtures of proteins from various
methods appear to be more effective.
biological samples, like serum [1] plasma [2] and urine [3].
This paper proposes a new method for the preprocessing
The data generated from these technologies can be used to
of proteomics data that deals with the problematic
identify proteomic patterns that can successfully separate
characteristics of the data and exploits the advantages of
states (e.g. normal versus disease) and possibly discover
various existing methods. More specifically, our proposed
novel disease biomarkers. Those patterns have high
strategy focuses on three main problems: correcting the
diagnostic significance, as they can be used for early
miscalibration of the mass spectra, detecting the peaks in a
diagnosis, prognosis, monitoring disease progression or
sensitive yet robust manner and extracting the true intensity
therapeutic response [4]. This strategy has already been used
values that correspond in each peak. For the peak fmding
in various types of cancer, like ovarian [1] [5], breast [2] and
step, we used a method based on the mean spectrunI
prostate cancer [6], giving interesting results.
approach, where we first fmd the peaks per category, then
However, the complex nature of proteomics data makes
apply certain criteria to ensure their reproducibility and then
their analysis a challenging task, as the initial raw data are
combine them in a single peak list. Instead of working with
very difficult to handle. More specifically, the data retrieved
peak locations, we propose the use of peak intervals, to
after an MS experiment contain hundreds of samples (i.e.
ensure that the small shifts present in the data do not
mass spectra), and in each sample correspond tens of
interfere with the fmal results. Our proposed pipeline was
applied in a MALDI MS dataset, obtained by the Proteomics
Manuscript received June 2010
Maria Anna v. Rapsomaniki (rapsoman@ceid.upatras.gr), Konstantinos Research Unit of the Biomedical Research Foundation,
A. Theofilatos (theofilk@ceid.upatras.gr), Spiridon D. Likothanassis concerning patients with bladder cancer (high or low grade)
(likothan@ceid.upatras.gr), Athanasios K. Tsakalidis (tsak@ �i.gr� and and benign bladder disease. After the preprocessing, a
Seferina P. Mavroudi (mavroudi@ceid.upatras.gr) from Umverslty of
Patras, Greece. Panagiotis G. Zerefos (pzerefos@bioacademy.gr) from classification step was applied, which achieved extremely
Biomedical Research Foundation,Academy of Athens,Athens, Greece

978-1-4244-6561-3/101$26.00 ©2010 IEEE

high performance. Furthermore, 31 statistically important
peaks were identified, some of which are not detected by
existing methods.
Our method is programmed in MATLAB R2007b. All
source codes are available upon request.
II. BACKGROUND
Before proceeding in the next section that describes our
proposed pipeline method, we first briefly review the
separate steps usually involved in MS data preprocessing.
The first preprocessing step is usually denoising, where one
tries to get rid of noise without loss of information. Usual
approaches involve variations of the Wavelet Transform,
and because of its time-shift invariance, the Undecimated
Discrete Wavelet Transform (UDWT) [8] is becoming the
standard choice in this task.
The next preprocessing step is usually baseline
correction, where the aim is to remove from the signal the
exponential baseline curve that is present because of the co­
ionization of the matrix molecules. Various methods are
available, that are mainly based on iteratively fitting a curve
to the local minima of the signal [9].
The existence of systematic error is another common
characteristic that makes the comparison of different spectra
problematic. Global normalization of intensity values helps
get rid of the experimental variations and is usually
performed according to the Total Ion Current (TIC), which
describes the total amount of protein present in the sample.
The peak finding and peak quantification steps are
crucial in dimension reduction, as from a spectrum of tens of
thousands of mlz points we end up with a single matrix
consisting of some hundreds of peak locations and their
corresponding intensity values (matrix of protein expression
levels). The aim is to detect peak positions as local maxima
and find corresponding intensity values, based on a peak's
height or area, for every spectrum.
Finally, miscalibration and measurement errors cause the
peaks to appear in slightly different mlz locations and thus
peak alignment is used to correct those minor shifts.
Commonly used algorithms try to fmd a common set of mlz
values that represent all the samples, so that the same peaks
appear at identical mlz locations. In some of them each
signal is shifted and scaled, so that the maximum alignment
among spectra is achieved, while in others the peaks are
clustered so that the ones that represent the same protein
belong to the same bin. The latter category is often referred
as peak matching and a common approach includes the use
of hierarchical clustering [10].
There are two main approaches available concerning the
fmal two steps: the first one proposes to detect and quantify
peaks in individual spectra, which gives n peak lists (where
n the number of samples) that need to be matched afterwards
[11]. This method is very sensitive, as it detects all peaks,
but requires the application of peak matching. Common
problems are that the results are often redundant, as even the
insignificant or random peaks are detected, and that the same
peak is sometimes detected twice, because of mistakes in the
peak matching step. The second approach suggests peak
detection using the mean spectrum and then peak
quantification by finding the corresponding intensities in
individual spectra [4]. This method is becoming increasingly
popular, as there is no need to match peaks afterwards and
also the peaks detected are statistically important. However,
a common problem with this approach is that quite often
small peaks that may be of biological importance disappear
in the mean. It has also been shown that if the spectra are
misaligned, the peaks are not detected correctly [12].
III. METHODOLOGY
Dataset: Our proposed method was tested on a MALDI
dataset, composed of urine samples that were collected from
approximately 200 patients with Bladder Cancer (High or
Low Grade) or benign bladder disease. The samples were
run through the spectrometer during 9 days, giving 9 subsets
(batches) of mass spectra. For each batch, a set of control
samples were also provided. Each sample was run 5 times,
giving 5 replicates. The preprocessing of this dataset was
very challenging because apart from the aforementioned
problems we had to deal with some additional ones. More
specifically, the calibration of the samples was not done
systematically, which resulted in incomparable spectra.
Furthermore, as most urine sample spectra, the spectra were
significantly contaminated and contained a great amount of
irrelevant peaks [13]. All those reasons necessitated the
application of extra preprocessing steps.
Proposed Pipeline Method: Our proposed method is
organized in two main steps: fmding the optimal peak list
that describes all samples and accurately quantifying the
peaks in all spectra. For the first step, we used the strategy
shown in Fig. 1. First, we applied a replicate selection step
by computing the pairwise Euclidean distances between each
replicate and their mean and then selecting the one with the
minimum distance, as we considered it to be the most
representative. In that way we obtain a more manageable
sample size without loss of information, as all 5 replicates
actually refer to the same sample. We also applied a
resampling step, in order to homogenize the mlz vectors and
be able to compare different samples under the same
reference. To achieve that, each signal was decimated into a
more manageable mlz vector and at the same time an
antialias filter was applied, so that high-frequency noise is
prevented from folding into lower frequencies.
One of the biggest problems we had to correct was the
miscalibration, which caused the presence of shifts in the
spectra, in some cases bigger than 100 mlz units. For that
reason we used an iterative process that involved first the
alignment of the control (reference) samples, then the
correction of the shifts (linear transformation) in individual
spectra using the control alignment and fmally the global
alignment using a vector of reference peaks.
In order to fmd the most appropriate peak list, we propose
a strategy based on the idea of using the mean spectrum for
peak detection. As mentioned, this method results in a peak interval. In that way we ensure that the minor shifts do not
list with statistically important peaks, as random peaks that cause mistakes in peak quantification.
appear only in individual samples are usually not detected.
However, there is no way to tell if a small yet persistent
peak, present in the majority of the samples of one category
and thus a possible biomarker, is detected or is disappeared
in the mean. On the other hand it is possible that sometimes
random peaks with high intensity values "survive" in the
mean.
For all those reasons, we used the mean spectrum per
category in order to combine the robustness of peak fmding
through the mean spectrum with higher sensitivity. We also Fig. 2: Peak quantification
applied a SNR cutoffcriterion, discarding the peaks that their
Signal-to-Noise Ratio (SNR) was under a certain threshold. The fmal matrix of peak expression levels was further
We introduced a consistency criterion, counting the times analyzed using an approach (Fig. 3) that involved a simple
each peak was detected and discarding the ones with a filtering feature extraction method (two-way t-test) followed
frequency lower than 35%. Finally, a peak matching by a Support Vector Machine (SVM) classifier with a
algorithm was used in order to combine the separate peak quadratic kernel function [14]. The details of this analysis
lists into a final one. are beyond the aim of this paper but are available upon
request.

Feature Selection
and SVM learning

Fig. 3: Final Analysis

IV. RESULTS
We applied our proposed method in the dataset described
earlier in order to detect and quantify the peaks. In Table I
we provide the results in the peak finding step for each
category, before and after the application of the cutoff
criteria. As we can see, each step reduces significantly the
dimensionality of the problem. The application of the peak
matching algorithm combined these 3 peak lists in a vector
of 456 peak bins, which are the features that will be further
used in the analysis.

TABLE I
NUMBER OF PEAKS DETECTED PER CATEGORY

Benign Low Grade High Grade

initial set 593 580 478

after SNR cutoff 477 451 393
after Consistency cutoff 416 415 338
Fig. I: Pipeline method for peak detection
The feature extraction and classification process was
In order to quantify the peaks in individual spectra, we repeated 100 times, to ensure reproducibility of the results,
used the method shown in Fig. 2. More precisely, first we and each time the classifier was built using a different
denoised the spectra using the UDWT. Then, we computed feature set. The average values of the evaluation
the baseline in multiple shifted windows and regressed it performance measures for our method are provided in Table
using a shape-preserving piecewise cubic interpolation. II.
Finally, we normalized the intensity values by standardizing TABLE II
the Area under Curve (AUC) to the group median. Since CLASSIFICAnON PERFORMANCE
small shifts still appear in the spectra, we used peak Accuracy Sensitivity Specificity
intervals of fixed width, centered at each peak's position,
and searched for the maximum intensity value inside each 0.9903 0.9887 0.9911

In order to compare our results with existing methods, we
also performed peak finding using the overall mean
spectrum [4], which gave us a matrix of 484 peaks and
corresponding intensities. The feature extraction step failed
to identify statistically important features among those 484
peaks.
A further investigation of the results revealed that using
our method a certain subset of 31 features is selected in
every run of the feature selection algorithm. This indicates
that those features have high discriminatory power and thus
could be possible biomarkers. We also observed that the
mean spectrum method failed to identify 7 of those features.
In Fig. 3 we show one of these features, which as we can see
is far more abundant in cancer (both low and high grade)
than benign samples.
Mean Bening Spectrum
10000 r----,-----,---�
-�
� 5000
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In this paper we proposed a novel pipeline method for the and Proteomics, 2007,pp. 79-102.
[13) P. Zerefos,1. Prados,S. Kossida,A Kalousis,and A Vlahou,
preprocessing of MS-based proteomics data, which deals
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development of a web-based tool, which will implement the [14) M. Hilario and A Kalousis, "Approaches to dimensionality
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number of steps and parameters involved in preprocessing, [15) R Armananzas, Y. Saeys,I. Inza,M. Garcia-Torres,C. Bielza,
our tool will propose optimal combinations of steps and Y.V.D. Peer,and P. Larranaga, "Peakbin Selection in Mass
Spectrometry Data Using a Consensus Approach with Estimation of
optimal parameter values according to the dataset each time Distribution Algorithms," IEEE/ACM Transactions on
provided, and in that way will ease the preprocessing job for Computational Biology and Bioinformatics,vol. 99,2010.
the unfamiliar user.

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