Review

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Bioinformatics approaches in
clinical proteomics
Eric T Fung†, Scot R Weinberger, Ed Gavin and Fujun Zhang

Protein expression profiling is increasingly being used to discover, validate and

characterize biomarkers that can potentially be used for diagnostic purposes and to aid in
pharmaceutical development. Correct analysis of data obtained from these experiments
CONTENTS requires an understanding of the underlying analytic procedures used to obtain the data,
statistical principles underlying high-dimensional data and clinical statistical tools used to
Analytic approaches to
clinical proteomics determine the utility of the interpreted data. This review summarizes each of these steps,
with the goal of providing the nonstatistician proteomics researcher with a working
Data preprocessing
understanding of the various approaches that may be used by statisticians. Emphasis is
Data mining
placed on the process of mining high-dimensional data to identify a specific set of
Biomarker identification biomarkers that may be used in a diagnostic or other assay setting.
Expert commentary
Expert Rev. Proteomics 2(6), 847–862 (2005)
& five-year view
Key issues Clinical proteomics is an emerging field dedi- have proven to be implausible. Each algorithm
References cated to the discovery, validation and charac- comes with its own assumptions and biases;
terization of biomarkers that can address a consequently, a certain level of agnosticism is
Affiliations
variety of clinical questions. Generally, most recommended when it comes to choosing data
clinical proteomics studies involve fractionat- analysis approaches. A generic workflow of the
ing a set of clinical samples from a case group clinical proteomics process is shown in FIGURE 1.
and a set of clinical samples from a control Several papers discuss issues such as study design
group, and then analyzing the fractions by mass and sample acquisition which, albeit critical, are
spectrometry (MS) or 2D gel electrophoresis. beyond the scope of this review [1,5]. Rather, this
The data streams resulting from these processes review starts with the data analysis steps, com-
are then subjected to complex pattern recogni- mencing with acquisition of the data, which are
tion analysis, resulting in the conclusion that a acquired primarily using time-of-flight (TOF)
set of biomarkers has been generated. The MS, for example, using surface-enhanced laser
strength of that conclusion is dependent on a desorption/ionization (SELDI) TOF-MS or
careful approach to each step of the discovery matrix-assisted laser desorption/ionization
process, involving appropriate study design, (MALDI) TOF-MS. This is followed by a dis-
attention to data acquisition protocols and a cussion of data preprocessing; for example, base-
† conservative approach to data mining. Perhaps line subtraction, intensity normalization, peak
Author for correspondence
Ciphergen Biosystems, Inc., naively, initial forays in this field were met with alignment and peak detection. Once these steps
6611 Dumbarton Circle, Fremont, overexuberance, with the expectation of quick are performed, the data are mined with multi-
CA 94555, USA answers to long-standing questions, such as the variate analysis tools. Selected validated biomar-
Tel.: +1 510 505 2242 early detection of cancer. A retrospective assess- kers are then purified and identified so that
Fax: +1 510 505 2101
ment of these early promises reveals that errors assays can be made. This review will discuss
efung@ciphergen.com
in seemingly obvious parameters of study design various approaches to multivariate analysis and
KEYWORDS: and execution can have devastatingly misleading strategies with which to implement them. It will
biomarker discovery, clinical
proteomics, clinical statistics, results when complex multivariate analysis tools conclude with a discussion of protein identifica-
data analysis, expression profiling, are applied [1–4]. Furthermore, statements that tion, the final step in biomarker discovery, using
mass spectrometry, pattern
recognition, time-of-flight one or another particular data mining algorithm peptide mass fingerprinting or tandem MS
mass spectrometry was superior to others have also been made, but (MS/MS) and searching of databases.

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Fung, Weinberger, Gavin & Zhang
Analytic approaches to clinical proteomics
No discussion of clinical proteomics would be complete with-
out background on the analytic approaches taken to generate
the data that will be the subject of this review. Basic analytic
approaches can be coarsely grouped into two different schemes:
those that directly analyze nascent proteins as they present
themselves in living systems (top-down) and those that directly
examine their proteolytic fragments (bottom-up).
2D gel electrophoresis
The most widely used top-down discovery approach is 2D
gel electrophoresis MS [6]. While 2D gels do not directly
study proteins by MS, they do provide a means to qualita-
tively and quantitatively generate protein profiles of authen-
tic samples. Briefly, proteins are first separated by their iso-
electric point via immobilized pH gradients, and then further
fractionated using sodium dodecyl sulfate (SDS) polyacryl-
amide gel electrophoresis (PAGE). PAGE slabs are then
stained, creating a 2D array of spots. Slabs are digitally
Data acquisition (10,000 –1,000,000s datapoints)
Baseline subtraction
Calibration
Normalization
Alignment
Peak detection
Data mining (100–1000s input features)
Unsupervised learning
Feature selection
Supervised learning
Cross-validation/permutation testing
Independent validation
Selected biomarkers (1–10s validated biomarkers)
Biomarker purification
Biomarker identification
Assay development
Clinical assay
Expert Review of Proteomics
Figure 1. Generic clinical proteomics workflow. Clinical samples taken
from the comparison groups are analyzed using any of a variety of
proteomics technologies. For the purposes of this discussion, these
technologies almost uniformly require sample fractionation followed by
analysis by mass spectrometry. Thus, each sample generates tens of
thousands to millions of data points. The data generated by the mass
spectrometer are subjected to preprocessing steps, and it is the processed
data that are submitted to data mining techniques. Once data mining is
completed, a best-set of validated biomarkers is derived. The biomarkers are
then identified so that high-throughput clinical assays can be constructed.
848
imaged using an optical scanner, and scanned images can be
differentially compared, with particular focus upon spot loca-
tion and stain intensity. As such, 3D patterns can be generated
for each sample, resolving isoelectric point (pI), molecular
weight and abundance.
Early attempts at interpreting 2D gel profiles were facilitated
by using artificial intelligence and machine learning programs.
One particular program, the Medical Electrophoresis Analysis
Interactive Expert System (MELANIE) was created to auto-
matically classify 2D profiles using heuristic clustering analysis
and hierarchical classification [7,8]. The overall goal of this
work was to create a means to determine disease-associated
patterns with the intent of creating a computer-based diagnos-
tic regimen. It was successfully applied towards the diagnosis of
liver cirrhosis and the distinction of a variety of cancer types
from cancerous biopsies [9]. Later, the work was extended
towards the comparative analysis of plasma/serum obtained
from apparently healthy individuals and from patients with a
few selected, known diseases. Despite their apparent complex-
ity, the patient electropherograms revealed readily detectable
modifications of the reference protein profile for the selected
diseases. Several disease-associated spot patterns were eluci-
dated from patients with monoclonal gammopathies, hypo-
gammaglobulinemia, hepatic failure, chronic renal failure and
hemolytic anemia [10].
While initially successful, the 2D approach failed to translate
to the clinic because it was inherently troubled in terms of its
limited reproducibility, restricted dynamic range of detection
and laboriously slow throughput. More recently, a new 2D
approach termed difference in-gel electrophoresis (DIGE) has
been introduced as a possible means to improve reproducibility
and throughput [11]. DIGE is a modification of the classic 2D
approach in which multiple samples can be analyzed within the
same gel, thus allowing for the simultaneous analysis of experi-
mental and control cohorts. DIGE is performed by fluores-
cently tagging multiple samples with different amine-reactive
dyes, running them on the same 2D gel, and then performing
post-run fluorescence imaging of the gel, allowing for direct
superimposition of groups. In this manner, the number of gels
to be processed and imaged is reduced. Furthermore, the effects
of gel-to-gel irreproducibility are somewhat minimized. DIGE-
based analysis has been successfully performed in the study of
colorectal cancer [12,13].
While 2D gel patterns can be instructive, ultimately, the
identity of the unique markers must be established. Towards
this end, 2D gel analysis has been married with electrospray
ionization (ESI) and MALDI-MS. Protein spots of interest are
typically excised from the gel and then destained. The excised
gel plugs are then digested using proteases with specific proteo-
lytic activity, such as trypsin, endo-Lys-c and V-8 protease.
Liberated peptides diffuse out of the gel plugs, lending them-
selves to subsequent MS analysis. An excellent overview of this
process, along with suggested protocols, is provided by
Corthals and coworkers [14]. The specific details of MS-based
protein identification are discussed later in this review.
Expert Rev. Proteomics 2(6), (2005)

To address the reproducibility and throughput issues asso-
ciated with classic 2D gel analysis, several researchers com-
bined the direct analysis of polyacrylamide gels with
MALDI-MS, eliminating the SDS-PAGE step [15]. Ultrathin
(<10 µm when dry) gels were soaked in MALDI matrix solu-
tion and then directly analyzed in a MALDI-TOF mass
spectrometer. Initial spectra were acquired from isoelectric
focusing, native and SDS gels. Virtual 2D gels were created by
MALDI scanning isoelectric gels. Virtual 2D gels were
extended to the study of the Escherichia coli proteome [16].
When compared with classic 2D studies of the same
proteome, virtual 2D analysis allowed for the postulation of
protein identities (<50 kDa) based upon improved molecular
mass determination and pI (±0.3 pH units). Putative identi-
fications were further confirmed by MALDI in-source decay
of the intact protein or by peptide mass mapping following
gel-wide chemical cleavage. Post-translational modifications,
such as fatty acid acylation, were detected. In total, approxi-
mately 250 different proteins (2–120 kDa) were discovered
in the 5.7 to 6.0 pI range. Data reduction and display algo-
rithms were created to allow for facilitated viewing and study
of virtual 2D results [17]. In its most advanced state, virtual
2D gel analysis demonstrated high sensitivity (analogous to
silver-stain detection limits) and improved throughput, reso-
lution and mass accuracy when compared with classic 2D
analysis. However, complications associated with interfacing
gels with MALDI-MS systems fundamentally limited
broader adoption of this approach. In some cases, gels were
fractured or ruptured during the sample introduction pump-
down process, often polluting the MS system with acrylamide
particles and dust. Furthermore, while superior to 2D gel
analysis, direct MALDI-MS analysis of gels resulted in com-
promised analytic sensitivity, mass accuracy and mass-resolving
power when compared with traditional MALDI or SELDI
measurements. As such, only a few investigators continue to
use this approach.
Mass spectrometry
Top-down MS-based approaches to biomarker discovery
include SELDI-TOF-MS [18], liquid chromatography (LC)-
MS [19] and Fourier-transform ion cyclotron resonance
(FTICR) MS [20]. As expected, data generation for each
approach is technology-dependent. For the most part, top-
down MS studies are performed using either FTICR or TOF-
MS detection. In FTICR analysis, the detector output is a
time-dependent image current that represents the coulombic
charge of all orbiting ions. The angular velocity of each orbiting
ion is related to its mass-to-charge (m/z) ratio. To deconvolve this
complex signal, a Fourier transform is performed to convert the
signal from the time to the frequency domain. Since ions of dif-
ferent m/z values demonstrate different angular velocities, they
will generate m/z-specific frequencies whose amplitude is
roughly dependent on ion abundance. The system is calibrated
by analyzing well-characterized analytes and calibrating the
observed frequency to the expected m/z.
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Bioinformatics approaches in clinical proteomics
Almost universally, current TOF-MS systems operate on the
constant energy principle. As such, ions are accelerated by the
device to a final kinetic energy, and achieve final velocities that
are dependent upon their respective m/z values. Direct signal
output of a TOF-MS system is a time-dependent current gen-
erated by the impact of charged particles upon an ion-to-elec-
tron converting detector. Assuming the absence of detector
saturation, current amplitude can be taken to represent the
number of incident particles over the sampling period. Gener-
ated current is either directly monitored or converted to a
time-dependent voltage prior to converting to a digital signal.
Digitization is achieved using either a time interval recording
approach, such as time-to-digital converter (TDC), or via time
array recording devices, such as high-speed analog-to-digital
converters (ADCs). With the exception of some orthogonal
TOF systems, most TOF devices use ADCs to capture gener-
ated data in a digital form. In terms of data processing, the
temporal resolution of the device is dependent upon the ana-
log bandwidth of the detector and associated analog electron-
ics as well as the data acquisition rate of the analog/digital con-
verter. Modern TOF systems employ analog bandwidths
ranging from 0.5 to 2 GHz with data acquisition rates typi-
cally ranging from one to five gigasamples per second. For the
purpose of m/z determination, calibration is performed by
measuring the TOF for a number of well-characterized sam-
ples and then correlating observed TOF to known m/z. A
classic calibration function uses a second-order polynomial to
convert observed TOF to ion m/z:
2
m/z = a ( TOF ) + b
Values for a and b are empirically determined during the
calibration process.
Data preprocessing
The general objective of data preprocessing of spectra is to
arrive at a peak list that can be used for downstream data min-
ing (although some investigators prefer to use m/z values
directly). Most analysis packages include some combination of
background correction, filtering, noise estimation and peak
detection algorithms. All systems must include some method to
transform the linear TOF data to the m/z domain.
Signal filtering & background subtraction
Since spectra contain noise from electronic and chemical
sources, signal filtering is often employed before peak detec-
tion. An essential property of a suitable signal filter for mass
spectra is that the filter must not shift the time location of fea-
tures in the spectra. There are many suitable digital filters that
meet this requirement, and moving average or Savitsky–Golay
filters are commonly employed.
Background signals in MALDI and SELDI-TOF can cause a
relatively large background at low mass in MALDI and SELDI
spectra. One method of background correction includes a
standardization of the data to a constant noise scale, and a
849
Fung, Weinberger, Gavin & Zhang
denoising method that utilizes hard thresholding [21]. The
threshold cut-off is determined by a multiple of the standard
deviation. Wavelets can also be applied to TOF spectra for
denoising and compression. Qu and coworkers found that, with
a discrete wavelet, the spectra data could achieve compression
without significant loss of the information when the data were
converted back to the time domain [22]. Malyrenko and coworkers
removed the background from SELDI-TOF data collected on
a ProteinChip Reader® model PBSII (Ciphergen Biosystems,
Inc.) by modeling the response of the detector to overload and
charge accumulation, and applying appropriate digital filters
to correct for the effect [23]. It remains to be seen if the same
techniques can be generally employed, since the effects and the
corrections may be specific to this instrument model.
Peak detection
Peak detection in mass spectra serves to both reduce the
dimensionality of the spectra and allow quantification of the
protein or peptide that gave rise to the peak. Since mass spectra
often contain considerable amounts of noise, typical peak
detection algorithms include user-adjustable parameters to
filter peaks based on height, signal-to-noise or other properties
such as width. Peak detection algorithms are often criticized
because they typically require user adjustment to parameters in
order to optimize results on a given spectra or data set, and this
can introduce a considerable human bias to the process of
biomarker discovery.
In order to determine the location of the peak, many algo-
rithms find a centroid using the top portion of a peak, typically
10% of the total peak height. Since the centroid is the peak
location at which one half of the area is to each side, it provides
a more robust measure of peak location than the apex. The cen-
troid is typically calculated on the top portion of the peak
because peak shapes are often skewed at the base of the peak.
Kempka and coworkers created a peak detection algorithm that
uses the entire peak to find the location in MALDI spectra by
fitting two deconvoluted Gaussian distributions to the peak [24].
They reported that, when compared with other commercial
peak detection methods, this method improved the mass accu-
racy, especially on low signal-to-noise peaks. Carlson and
coworkers have created a method they call simultaneous spectrum
analysis to combine multiple spectra into a single spectra to use
for peak detection [25]. The advantage of such a scheme is that
the average of groups of spectra will have a higher signal-to-
noise ratio than the individual spectra, thus making the detec-
tion of peaks more sensitive and robust. An area-under-the-
curve filter is used to determine peak locations. Alternatively, an
F-test can be used to test that the same peak occurs in replicate
spectra more often (as predicted by chance) than a filter against
peaks that occurred as a result of noise. Coombes and coworkers
combined peak detection and background subtraction by first
finding a set of preliminary peak locations, and then subtracting
the peaks from a spectrum and fitting a baseline to the resulting
spectrum [26]. This baseline is then subtracted from the original
spectrum before performing another pass of peak detection.
850
Spectrum alignment
Since there is always a measurement error associated with the
reported mass of a peak, downstream methods must include
methods to cluster peaks together for comparison. In order to
improve the accuracy of the clustering, an optional spectrum
alignment step may be included in the workflow. Jeffries
describes an algorithm for aligning spectra, utilizing peaks that
are common across the spectra and minimizing the error term
using a Nelder–Mead multivariate optimization procedure [27].
Data mining
Once data have been prepared, they are submitted to data mining
algorithms. Generally, data mining approaches fall into two
categories: unsupervised and supervised. The former refers to
approaches that do not take into account class labels, while the
latter refers to approaches that do. Unsupervised learning
approaches are analogous to clustering, while supervised learn-
ing approaches are analogous to classification. Examples of
unsupervised learning approaches include k-means clustering,
principal component analysis (PCA) and hierarchal clustering.
The gamut of supervised learning techniques includes classifi-
cation and regression trees, neural networks, genetic algorithms
and support vector machines. With the vast array of possibili-
ties, it may be difficult to decide which is the ideal algorithm to
use. Unfortunately, there is no simple answer to this question
and it has been proposed that there is no ideal single method
(no free lunch theorem) [28]. Each algorithm has inherent
strengths and weaknesses, which must be matched to the spe-
cific statistical problem to be addressed. The descriptions below
are intended to describe various algorithms and provide insight
into some of their strengths and weaknesses.
Prior to the introduction of data into these mining algorithms,
a process of feature selection must be undertaken. The ‘curse
of dimensionality’ refers to the asymmetry between the
number of input features (e.g., peaks) and the number of
exemplary features (e.g., samples). Overcoming this problem is
challenging but necessary; any classification algorithm given
too many features will be able to find a solution to the pre-
sented problem. The curse of dimensionality results from the
magnitude of the input data, which are signal amplitudes
recorded at a regular interval (set by the digitizer rate of the
TOF mass spectrometer). A given spectrum can have tens to
hundreds of thousands of such data points; a given sample can
have dozens of spectra depending on the laboratory protocols.
One method to perform feature reduction is to select m/z values
that represent peaks; the process of peak detection is discussed
earlier in this review and is not explored further in this section.
Other approaches to perform feature selection, which can be
performed on the raw data or on the detected peaks, are to use
t-tests, unsupervised learning approaches or supervised learn-
ing approaches that incorporate feature selection. These are
discussed in greater detail below.
Once the features are selected, and prior to implementation
of either unsupervised or supervised learning techniques, data
transformation is often performed. This reduces the impact
Expert Rev. Proteomics 2(6), (2005)

that a high variance in a given input variable might have by
altering the distribution of the original data. For example, the
measurement of a specific analyte may be highly variable, either
for analytic reasons or for clinical reasons. This high variability
in some data analysis approaches may artificially improve its
chances of being selected as a classifying feature. Data trans-
formation reduces the impact of this variability by constraining
the values within a more defined range. Typical approaches to
data transformation include log transformation, square-root
transformation, and linear and logarithmic scaling. Not all data
analysis approaches require transformation; for example, classifi-
cation trees ignore variance and locate cut points that can opti-
mally segregate samples (see below). Therefore, transformation
is not necessary.
Unsupervised learning techniques
A good initial assessment of data quality can be obtained by
using unsupervised approaches to visualize the distribution of
the data [26,29]. This permits the identification of outlier sam-
ples, which may result from sample misclassification, from a
previously unrealized clinical subgroup or from variances in
laboratory sampling. In addition, using unsupervised learning
techniques followed by superimposition of sample labels can
also provide a qualitative assessment of the separation power of
the data. Finally, unsupervised learning techniques can be used
as a basis for feature selection. Each of the uses of unsupervised
learning techniques as well as a general description of these
approaches are summarized below.
One commonly used unsupervised learning technique is
PCA [30]. PCA maps high-dimensional data into a more
manageable set of dimensions by creating eigenvalues (linear
combinations) of the input variables. Each linear combination
(or principal component) is a weighted sum of the amplitude at
each m/z value (or peak if peak detection provides the input
variables). The individual weights assigned to each of the input
variables are derived by calculating the covariance structure of
the input variables. The principal components are then ordered
by the proportion of the overall variance for which each princi-
pal component accounts. The feature extraction capabilities of
PCA are embodied in the fact that the top (usually less than
ten, and often the top two or three) principal components will
be adequate to separate the samples into relatively homogene-
ous clusters; that is, these top principal components account for
the majority of the variance in the data structure. This can be
visualized in 2D or 3D plots in which the calculated values for
each of the top principal components serve as the x, y and
z axes. Moreover, for each principal component, the input vari-
ables that have the largest absolute values for their coefficients
have the greatest weight and, therefore, have the greatest dis-
criminating power. Generally, for PCA to be used in this
capacity, some sort of transformation (e.g., rescaling) needs to
be performed so that peaks with the largest variance are not
unfairly weighted. For a more detailed, mathematical discus-
sion of PCA, the reader is referred to [28]. Lundquist and
coworkers used PCA to analyze data intended to distinguish
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Bioinformatics approaches in clinical proteomics
subspecies of Francisella tularensis by SELDI-TOF-MS [31].
Notably, the first principal component mainly separated the
novicida group from the rest of the strains, and the second prin-
cipal component described differences between the holarctica
and tularensis groups. Finally, the third principal component
described the differences in protein profiles between the media-
siatica group and the rest of the strains. These three principal
components described 72% of the total variance. Lancashire
and coworkers similarly used PCA to cluster species of Neisseria
based on SELDI-TOF-MS data [32]. An interesting approach to
using PCA is to rank peak intensities within each spectrum,
rather than compare peaks directly against each other [33]. Such
an approach has been applied to 2D gel analysis of various
histologic types of lung [34], cervical [35] and borderline ovarian
cancer [36].
Hierarchical clustering is another method to visualize the
distribution of data [37]. Briefly, hierarchical clustering begins
by assigning each sample to its own cluster. It then calculates
similarity scores or distance metrics between samples, and
places samples that are close to each other together. Specific
hierarchical clustering algorithms can differ in their methodol-
ogy for calculating distance metrics. This is done iteratively
until all samples are ordered. Typically, the representation of
the data using hierarchical clustering is in the form of dendro-
grams. Hierarchical clustering can be performed simultane-
ously in two dimensions: typically, samples and peaks. Using
red and green color coding to represent the relative up- and
downregulation of peak intensity generates the familiar heat
map presented in many publications [38,39].
Supervised learning techniques
By definition, supervised learning techniques require class
assignments such that training (learning) can occur on the data
obtained from a subset of the provided samples. The two types
of variables in this exercise are therefore the predictor variables
(intensity at m/z values or peak intensity) and response vari-
ables (disease classes). The most straightforward approach to
identifying differences between groups using a supervised
approach is the t-test. There are numerous ways to calculate the
t-statistic and to implement it. The various t-tests make
assumptions about the sample size of the respective groups, the
distribution (e.g., Gaussian) of values within each group, and
whether the variance is equal or nonequal. The Welch t-test
assumes two independent, small, normally distributed groups
with unequal variance [40]. A more conservative approach is to
assume that the groups are not normally distributed. This is
embodied by the Mann–Whitney test (or Kruskal–Wallis test,
when more than two groups are being analyzed). The
Mann–Whitney assumes equal variances between the two
groups. The t-test, however, suffers from several shortcomings,
some of which can be overcome but others cannot. First, by
calculating the t-statistic for each peak (or m/z value), multiple
hypothesis testing can lead to an artificial inflation of the
number of variables deemed to be significantly different
between the comparison groups. Second, these calculations
851
Fung, Weinberger, Gavin & Zhang
assume that the measurements are independent of each other,
which ignores the fact that proteins are often coregulated.
Additionally, if raw data are used rather than peaks, each m/z
value is related to its adjacent m/z value. Binning is often per-
formed to overcome this problem. Third, these calculations
ignore the possibility that subsets of clinical groups exist for
which a given input variable may be useful. The Bonferroni
correction is one method that attempts to reduce the impact of
multiple hypothesis testing [41]. Permutation tests and assess-
ment of the false-discovery rate are other approaches [42–51]. Sig-
nificance analysis of microarrays also attempts to minimize the
impact of multiple hypothesis testing, and can be applied to pro-
teomics data [52,53]. Permutation tests are discussed in greater
detail below.
t-tests are often used for feature selection to provide the input
into more sophisticated classification algorithms. Alternatively,
classification algorithms themselves can be used for feature
selection as well as for classification. These types of algorithms
can be described as heuristic or exact [54]. Heuristic classifica-
tion algorithms rely on performing multiple iterations to con-
verge on a locally optimal solution, with no guarantee that the
solution is globally optimal. Additionally, repeated application
of such algorithms on a specific data set usually results in vary-
ing classifying outputs (i.e., discriminants) from each run.
Examples of such algorithms include genetic algorithms, deci-
sion trees and neural networks. In contrast, exact algorithms
use closed-form equations and thus are deterministic.
Genetic algorithms attempt to mimic the process of natural
selection found in nature by creating chromosomes of concate-
nated input variables (m/z values) and iteratively recombining
chromosomes and mutating genes [55]. Input variables that
satisfy a fitness function are kept, while those that do not are
eliminated through a process of computational evolution,
achieved by recombination and mutation. This process is con-
tinued until preset criteria have been met, usually relating to
overall classification accuracy and number of fit input variables.
Genetic algorithms have been applied to several MS-derived
data sets [56–60]. A proprietary algorithm combining genetic
algorithms and self-organizing maps, was used to develop mod-
els that could classify ovarian cancer samples with high accuracy
[61], and this algorithm has subsequently been applied to data
sets for prostate cancer [62], Wegener’s granulotomatosis [63],
pheochromocytoma [64] and cystitis [57]. Jeffries provides a
description of the concepts behind the genetic algorithm, as
well as providing some insight into the weaknesses of this algo-
rithm [56]. Notably, the solutions identified by genetic algo-
rithms are dependent on the initial ordering of input variables
on chromosomes as well as parameters set for the fitness func-
tions. In addition, given their heuristic nature, genetic algo-
rithms are susceptible to finding locally optimal solutions, and
will often find different solutions for the same data set when
run repeatedly. This makes selecting a single solution for further
validation difficult. Certain implementations of genetic algo-
rithms may safeguard against these weaknesses. For example,
setting limits on the number of clusters and selected variables, as
852
well as employing cross-validation to define the fitness function,
may help reduce the variability. Alternatively, exact methods,
such as boosting and discriminant analysis (discussed later), do
not suffer from this weakness.
Classification and regression trees utilize recursive portion-
ing to achieve classification [65]. Decision trees begin with the
entire sample set and create a decision rule that partitions the
entire sample set into two more homogeneous groups. The
decision rule examines one or more input features and uses a
‘less than’ function; that is, if the intensity of peak a is less than
x, then a sample is partitioned to the left branch; if not, then it
is partitioned to the right branch. Each branch is then exam-
ined for homogeneity and can be subdivided using a new rule.
Each final, terminal node is then designated as a given class
using a majority rule based on the training samples. New sam-
ples can then be classified by determining how they satisfy each
of the rules in the decision tree and which terminal node they
finish in, and are classified according to the terminal nodes’
designation. An advantage of decision trees is that they create
easily interpretable and applicable decision rules for classifying
samples, and therefore have been used extensively in the analysis
of MS data [40,58,66–86].
A weakness of decision trees is that support diminishes as the
complexity of the tree increases; there are fewer and fewer sam-
ples within each subsequent branch of the tree. Some modifica-
tions, such as bagging and boosting approaches, may help to
stabilize decision tree structures. Bagging produces different
trees from different subsets of the training set, and then aggre-
gates the results on a set of test instances. This helps to reduce
the instability and variance in the decision tree learning process
by combining multiple models. Bagging, in its simplest form, is
performed by bootstrapping. Boosting combines weak individ-
ual base classifiers (c1, c2, c3 and so on) into a more powerful
classifier by performing a weighted stagewise selection of a base
classifier, given all the previously selected base classifiers. At
each stage, higher weights are given to samples that are incor-
rectly classified by the summary classifier; therefore, at each
stage, the added base classifier will be selected due to its ability
to correctly classify previously misclassified examples [87,88]. In
the context of decision trees, boosting generates a sequence of
decision trees (usually small trees with only one decision node,
also called stumps) from a data set in which the misclassifica-
tion rate is used to adjust weights to each sample [87,89]. Each
subsequent tree focuses on the misclassified (more heavily
weighted) samples. After a defined number of iterations, voting
occurs via a committee of experts approach, where a decision is
made by a majority vote. Since each expert is tuned to the
error rate of the prior tree, the committee is able to classify
more accurately than any given member. The goal is to maxi-
mize the margin, which is the difference in the number of votes
between the correctly voting members and the incorrectly
voting members. The larger the margin, the greater the confidence
in the classification. Boosting has been applied to MS data to
improve performance of decision tree analysis [73,80]. A forest
(decision forest or random forest) is a collection of decision
Expert Rev. Proteomics 2(6), (2005)

trees that, in contrast to boosting, are created in parallel (rather
than in series), and no comparisons are made until all the trees
are made [90]. Decision tree forests have two randomizing ele-
ments: the selection of cases used as input for each tree, and the
set of input variables that can be used as splitters for each node.
These randomizing elements, along with the committee deci-
sion, provide the basis for improving classification accuracy. The
drawback of the forest approach is that, rather than a single
easily interpreted decision rule, the output model is complex
and more of a black box. The random forest approach has been
used to analyze data for prostate cancer [75], ovarian cancer [80]
and bacterial proteomics [21].
Artificial neural networks (ANNs) attempt to mimic human
learning computationally [91–93]. Therefore, the jargon sur-
rounding ANNs is taken from our understanding of learning in
the nervous system. Neurons integrate information obtained
from inputs, which could be the outside world (i.e., primary
data) or previously integrated data (i.e., other neurons). There-
fore, there are multiple layers of neuronal integration. Like real
neurons, these computational neurons require that the input
function exceeds a certain activation threshold. Most neural
networks are feed-forward, that is, information flow (and deci-
sion making) proceeds only in one direction, starting with the
input layer, flowing through n layers of neurons to the output
layer. The parameters that can be varied and determine the
learning process include the weights given to the input func-
tions, activation thresholds for each neuron and computation
function performed by each neuron. Training the neural net-
work involves decreasing the error rate by adjusting these
parameters. The two major criticisms of ANNs are that they are
prone to overtraining and that they result in black box, difficult-
to-assay solutions. Despite these drawbacks, their ability to gen-
erate solutions with low error rates has made them attractive
algorithms for the analysis of proteomics data [32,39,58,94–107].
Partial least squares (PLS) is one form of exact supervised
learning [108]. Conceptually, it is quite similar to PCA, except
that knowledge of the class assignments allows it to perform
classification in addition to feature selection. The distinction
between PCA and PLS is that in deriving the weights for each
input variable in each component, PCA uses only the covari-
ance structure of the input variables, while PLS utilizes the
covariance structure of the input variables with the response
variables (i.e., class assignment).
Discriminant analysis (DA) attempts to maximize the ratio
of the difference in class mean to the within-class variance
[109–111]. After separation into n-dimensional space using PCA
or PLS, a linear discriminant is derived using a hyperplane
that maximizes between-class variance, while minimizing
within-class variance. A test spectrum is dimensionally
reduced by projection onto the principal components and
then onto the calculated hyperplane. The distances between
the respective clusters (healthy vs. disease) determine its classi-
fication, and the confidence in the classification is based on a
Gaussian distribution emanating from the center of the cluster.
Linear discriminant analysis examines the two-class problem,
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Bioinformatics approaches in clinical proteomics
while multiple discriminant analysis looks at multiple classes.
Logistic regression is now sometimes used in place of DA as
it usually involves fewer violations of assumptions (namely,
normal distribution and equal within-group variances), is
robust, handles categorical as well as continuous variables
and has coefficients that many workers in the field find easier
to interpret [112,113]. Logistic regression is preferred when
data are not normal in distribution or group sizes are very
unequal. k-nearest neighbors (k-NNs) is a form of nonpara-
metric discriminant analysis that examines the k-nearest clus-
ters samples and takes a majority voting approach among
these samples. Majority voting can be thought of conceptu-
ally as being equivalent to calculating the prior probability
based on the proportion of a given class of the k examples [80].
k-NN is nonparametric and, therefore, makes no assump-
tions regarding the distribution of samples. Li and coworkers
used a genetic algorithm for feature selection, and then
k-NN for classification [114]. Lundquist and coworkers dem-
onstrated how PCA, PLS and DA can be used together [31].
After performing PCA to determine the optimal linear com-
binations, PLS-DA was used to determine the most impor-
tant contributors to the most important principal compo-
nents, thus identifying the specific peaks that underlie the
differences in bacterial subspecies. PLS-DA has also been
used to discriminate species of wheat [103] and to assess wheat
quality [115].
A form of machine learning that has been applied to pro-
teomics data is the use of support vector machines (SVMs),
which were invented by Vapnik [116]. SVMs operate first by dis-
tributing samples in n-dimensional space and then by finding a
hypersurface that attempts to split the cases from the controls.
The split will be chosen to have the largest distance (margin)
from the hypersurface to the nearest of the case and control
examples. A detailed explanation of SVMs can be found in
[117]. Several studies have used SVMs for analysis of proteomics
data [69,80,118–123]. In this example, support vector machines
could be adequately substituted for linear discriminant analysis
and combined with PLS regression [54].
Examining a single data set using multiple algorithms can be
instructive. Perhaps the most scrutinized data set is the ovarian
cancer data set generated by Petricoin and coworkers [61]. Levner
comprehensively describes various approaches used to analyze
this data set as well as prostate cancer data sets generated by
Adam and coworkers [66,124]. However, potential flaws in the
design of these studies preclude any real conclusions regarding
the relative performance of various statistical approaches to
those data [3]. Levner points out that the availability of high-
quality MS data sets will be required before direct comparisons
of various algorithms can be made. However, several smaller
studies have benefited from the application of multiple algo-
rithms. Sorensen has applied both neural networks and PLSs to
data obtained from analysis of wheat proteins, and has shown
that, while both approaches could provide comparable classifi-
cation capabilities, PLS was able to provide additional data
regarding wheat quality [103].
853
Fung, Weinberger, Gavin & Zhang
Implementation of techniques
The authors prefer to utilize several means of feature reduction
as well as classification. The actual implementation of these
approaches is as follows. The data are divided into a training set
and a validation set. Preprocessing (e.g. baseline subtraction and
peak detection) is performed on the full complement of data.
Once this is performed, the data from the validation set are
ignored until the time for validation. If the size of the training
data set is adequate (e.g., n > 60 in each class), the training data
set can be further divided into a training and a test set. Feature
selection and model training are performed on the training set
and their performance is assessed in the test set. This can be per-
formed repeatedly to assess multiple sets of features and multi-
ple algorithms. If the training data set is too small (almost
always the case), typically, cross-validation is performed. Note
that cross-validation can still be performed in the training set
even if it is a separate set. In effect, cross-validation is a method
by which the sample size may be artificially increased. v-fold
cross-validation refers to subdividing the training data set into
equal v-sized subsets. Typically, v is equal to five or ten. When
v is equal to n, it is termed ‘leave-one-out’ cross-validation. Dur-
ing cross-validation, v-1 subsets are used to train the model, and
then test the model on the remaining subset. This is done itera-
tively until each subset has been used for testing once. One
additional note of caution involves selection bias [75,125]. When
cross-validation is performed on algorithms generated on a
preselected set of variables, rather than selecting the variables
with each iteration, it may overestimate the suitability for gen-
eralization of an algorithm. An alternative approach to mini-
mizing selection bias is to perform cross-validation during the
feature selection process.
Regardless of the specific feature selection and classification
approaches used to identify the most important classifying
peaks, in the diagnostic setting, the simplest, most transparent
algorithm is desired. Therefore, a short decision tree may be a
good diagnostic tool due to its interpretability and robustness
to outliers. Another approach to a final diagnostic algorithm is
to combine the optimal classifiers into a logistic regression
model. Finally, discriminant analysis may generate attractive
diagnostic models. These models can be linear, quadratic or
nonparametric (k-NN).
For any given model, there are two measures that need to be
assessed. The most obvious is the overall error rate in the
cross-validated sample set. The lower the error rate, the more
accurate the model. However, examining the error rate alone
is inadequate, since most analysis algorithms can be driven to
find a local optimum solution, that is, a solution particularly
fit (trained) to the specific input data, but unsuitable for gen-
eralization. Therefore, it is important to determine how stable
the model is, and whether it truly differs from a random solu-
tion. One method by which this can be assessed is a permuta-
tion test. Class assignments are randomized in an iterative
fashion, and the model is applied to these random class
assignments. The average error rate (and standard deviation)
can be calculated; the error rate when the model is applied to
854
the true assignments should be statistically significant from
when it is applied to the random, permuted class assignments.
Lilien and coworkers note that, in their experiments, they
could achieve 100% classification accuracy whenever they
used more than 50% of the spectra for training, when they
analyzed the National Cancer Institute clinical proteomics
data [54]. This emphasizes the need to identify multiple
train/test splits and to report the performance and variance in
performance of the models.
Once a model has been chosen due to the low error rate and
robustness, it can be applied to the independent validation
sample set. Typically, the independent validation sample set is a
subset of the original sample set (as described above). More
rigorous independent validation includes samples taken from
other medical institutions, as well as those processed at a differ-
ent time and in a different laboratory from the original study.
These steps in independent validation are required to deter-
mine whether the findings can be generalized across parameters
such as patient demographics, clinical subtypes and laboratory
handling techniques [126].
From data mining to clinical statistics
The most common goal of a data mining program is to minimize
the error rate. However, in clinical testing, there are different
types of errors, and not all errors are created equally. The classic
two-by-two table reflects the two types of errors associated with
clinical testing (FIGURE 2). Put simply, these misdiagnose a
healthy person as sick, and a sick person as healthy. One can
substitute responder and nonresponder, or prone to adverse
reaction and not prone to adverse reaction and so on for the
labels healthy and sick. Misdiagnosing a healthy person as hav-
ing a disease (termed a false positive) results in a loss of specifi-
city. From a patient management viewpoint, it leads to unnec-
essary testing and therapeutic intervention, leading to
discomfort (or worse) for the individual as well as increased
healthcare cost. The opposite error, missing a person with dis-
ease (termed a false negative), results in loss of sensitivity.
Although the ramifications of missing this diagnosis are appar-
ently obvious, the natural history of the disease is also impor-
tant. Frequent testing can compensate for decreased sensitivity,
particularly if the disease is slowly progressive.
Sensitivity and specificity almost always trend in opposite
directions. An ideal test has bimodal distribution with no over-
lap. In practice, most biomarkers have some degree of overlap;
this overlap is called the gray zone. Identifying the right cut-off
value for distinguishing healthy from disease within that gray
zone means finding the appropriate compromise between sensi-
tivity and specificity. In FIGURE 3A, if a cut-off at point A was
chosen and an individual with levels of biomarker above that
cut-off were diagnosed as having a disease, then the biomarker
would never miss a single case of the disease. At this cut-off, the
test would have 100% sensitivity, but many individuals without
disease would be inaccurately diagnosed (poor specificity).
Alternatively, if a cut-off at point B were chosen, no healthy
person would be misdiagnosed as having disease, but many sick
Expert Rev. Proteomics 2(6), (2005)
 Bioinformatics approaches in clinical proteomics

A Actual positive Actual negative B Actual positive Actual negative

Positive test result True positive False positive Positive test result 90 20

Negative test result False negative True negative Negative test result 10 80

Sensitivity Specificity Sensitivity = 90% Specificity = 80%

= TP/(TP + FN) = TN/(FP + TN)
Positive predictive Negative predictive Positive predictive Negative predictive
value = TP/(TP + FP) value = TN/(TN + FN) value = 81.8% value = 88.9%

C Actual positive Actual negative D Actual positive Actual negative

Positive test result 90 180 Positive test result 90 90

Negative test result 10 720 Negative test result 10 810

Sensitivity = 90% Specificity = 80% Sensitivity = 90% Specificity = 90%

Positive predictive Negative predictive Positive predictive Negative predictive
value = 33.3% value = 98.6% value = 50% value = 98.8%

Expert Review of Proteomics

Figure 2. Two-by-two diagnostic tables. (A) A candidate test (marker) is compared against a gold standard (actual). Instances in which the test matches the
actual are called true positives or negatives, and discordances are termed false positives or negatives. Based on the error rate and types of errors, clinical
parameters for sensitivity, specificity and predictive value can be derived. (B) Most clinical proteomics studies examine an equal number of cases and controls.
This artificially increases the calculated positive predictive value. (C) Most diseases have a prevalence far lower than the distribution used in clinical proteomics
studies, and therefore the actual positive predictive value would be lower. (D) Increased specificity will result in improved positive predictive value. Conversely,
increased sensitivity will result in improved negative predictive value.
FN: False negative; FP: False positive; TN: True negative; TP: True positive.

people would be missed. The trade-off between sensitivity and
specificity can be graphically visualized by a receiver operator
characteristic (ROC) curve, which plots the sensitivity on the
y axis and (1-specificity) on the x axis (FIGURE 3B) [127]. The ideal
test would have 100% sensitivity and specificity, and the ROC
plot would show a single point at the upper left corner. In prac-
tice, no test has 100% accuracy, and the ROC plot shows the
relationship between sensitivity and specificity by choosing dif-
ferent cut-offs in the gray zone. The overall accuracy of the test
can be assessed by calculating the area under the ROC curve,
and the aim is to maximize this. More recently, there has been
an interest in examining only the portion of the ROC plot of
clinical relevance (i.e., at high sensitivity or specificity) and cal-
culating the area under the curve in these regions [128–131].
These partial ROC plots, which have been used extensively in
radiology [132,133], are important when assessing particularly low
prevalence diseases (see later).
Sensitivity and specificity do not reside in a vacuum. The
frequency of disease impacts the relative importance of sensitivity
versus specificity. The frequency of disease is generally described
as prevalence and measured as a ratio of individuals with disease
at a given time point to the number of individuals at risk for
that disease at that time point. Incidence refers to the ratio of
the number of new cases in a given time period to the number
of individuals at risk for that disease during that time period.
The theoretical two-by-two tables, with varying prevalence of 50
and 10%, with fixed sensitivity and specificity (false-negative
and -positive) rate, demonstrate this principle. Most clinical
proteomics studies have a prevalence of 50%; that is, there are
generally an equal number of individuals with the disease as
there are healthy (or other relevant disease) controls (FIGURE 2B).
However, the disease may have an actual prevalence of 10%.
Note the dramatic decrease in positive predictive value; that is,
the proportion of tested positives who are actually positive.
Those who tested positive and that do not have the disease will
go on to have unnecessary additional procedures and therapies.
Of course, the negative predictive value increases dramatically
because, in this example, there are many more healthy individ-
uals. Diagnostic clinical tests generally place a greater demand
on positive predictive value and, therefore, maximize specificity.
Tests designed to rule out a disease or a therapy generally place
a greater demand on negative predictive value and, therefore,
maximize sensitivity. FIGURE 2D shows the improvement in posi-
tive predictive value by improving the specificity. While sensi-
tivity, specificity and ROC analysis are not prevalence depend-
ent, they are less useful measures of the quality of a biomarker
than predictive value.
Biomarker identification
Once biomarkers have been confidently discovered and validated,
the next task is to purify and identify them. The purification
of biomarkers is a biochemical exercise and beyond the scope
of this review. However, actual identification requires the
searching of databases and therefore deserves comment in

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Fung, Weinberger, Gavin & Zhang
this review. Currently, protein identification and characteriza-
tion are almost universally achieved using single or MS/MS in
combination with computational algorithms.
Peptide mass fingerprinting
With the continued growth of protein sequence databases, as
well as with the emergence of complementary (c)DNA databases,
it has become possible to derive peptide sequence and protein
identity by correlating MS measurements with theoretical pep-
tide fragments of known sequences. Henzel and coworkers intro-
duced a computer algorithm, later coined Fragfit, for the auto-
mated identity determination of proteins separated by 2D gels
[134,135]. Peptides were generated by reduction, alkylation and
tryptic digestion and then analyzed via MALDI-TOF. Fragfit
functioned by searching an existing protein sequence database for
multiple peptides of individual proteins that match the measured
masses. To ensure that the most recent database updates were
included, a theoretical digest of the entire database was generated
each time the program was executed. In a parallel effort, Mann
A created. A parameterized multilevel scoring algorithm was com-
Percentage of population
Healthy Disease
X Y
Biomarker concentration
B 1
Sensitivity
0 1
1-Specificity Expert Review of Proteomics
Figure 3. Distribution of values in the population and receiver
operator characteristic (ROC) plot. (A) Almost all analytes have an
overlapping bimodal distribution in concentration between healthy and
diseased individuals. This plot shows an equal frequency of cases and
controls, which are present in most clinical proteomics studies, but does
not accurately represent the general population. The cut-off level for
designating the presence of disease at a level between X and Y affects the
number of people misidentified as being healthy or sick. (B) The ROC curve
plots the relationship between sensitivity (true-positive rate) and
specificity (true-negative rate). A dashed line indicates a random test.
Curves trend towards the upper left corner as they become increasingly
more accurate.
856
and coworkers created software routines to correlate MS results
to protein identities found within protein databases [136]. Initial
search routines focused upon protein molecular weight. While
sufficient at times, this approach failed as the databases increased
in complexity and mass determination error was excessive. Ulti-
mately, search routines based upon partial mass spectrometric
peptide maps of target proteins were created. In general, approx-
imately four to six proteolytic peptides, measured with a mass
accuracy between 0.1 and 0.01%, allowed for useful search
activities in the Protein Identification Resource database.
Today, the process of identifying proteins based upon single
MS measurements of specific proteolytic fragments searched
against protein or cDNA databases is generically referred to as
peptide mass fingerprinting (PMF). High-throughput PMF
analysis is frequently performed in hyphenated 2D gel MALDI-
MS analysis. Alternatively, several in-gel digestions are queued
for automated analysis using LC/ESI-MS. High-throughput
PMF studies generate a tremendous amount of data, creating
corresponding challenges in ensuring quality protein identifica-
tions. Accordingly, computer algorithms directed towards
improving protein identification from PMF studies have been
bined with an optimized peak detection scheme to improve
identifications derived from 2D gel MALDI-TOF analysis [137].
Proteolytic specificity, species origin, protein isoelectric point,
determined molecular weight, chemical modifications and
potential number of missed cleavages are differentially weighted
and considered to create a hierarchical report of potential protein
identities. In another effort, a new PMF algorithm was devel-
oped that provided improved m/z calibration and peak rejection
as well as the use of a meta-search approach that employed vari-
ous PMF search engines [138]. The program successfully
improved routine PMF identification rate from 6 to 44% when
examining 1891 PMF spectra. More recently, a modular, script-
able automated analysis tool suite for high-throughput PMF
studies has been introduced [139]. The tool suite consists of auto-
matic peak extraction, peak filtering and protein database
matching modules that communicate via extensible markup lan-
guage (XML). This modular approach affords flexibility and
each module can be easily replaced with other software if desired.
Sequencing & protein identification via tandem
mass spectrometry
While PMF can often provide initial protein identification, in
cases in which insufficient protein purification is achieved, or
in studies with limited peptide coverage, substantial irregular
peptide cleavage and/or PTMs, PMF algorithms generally fail
to provide a confident and complete list of all proteins found
in the original sample. Consequently, MS/MS analysis is relied
upon as the gold standard of establishing peptide primary
sequence, post-translational modifications and protein identi-
fication. One of the earliest MS/MS studies of peptides
involved high-energy collision-induced dissociation (CID) of
peptide ions generated by fast atom bombardment in a dou-
ble-focusing MS/MS analyzer [140]. A year later, the same
Expert Rev. Proteomics 2(6), (2005)
 Bioinformatics approaches in clinical proteomics

group created a computer program for derivation of primary
sequence from CID peptide fragmentation known as SEQPEP
[141]. The only required input was a list of product ion masses
and relative abundances, the mass of the precursor ion and the
mass of any C-terminal modification. The program was capa-
ble of processing approximately 100 product ions in no more
than 5 min.
In 1990, pioneering work for today’s modern peptide MSn
analysis was performed by Cooks and Stafford using quadru-
pole ion trap (qIT) MS [142]. A number of small peptides were
ionized using Cs+ surface ionization, injected into the trap,
mass selected and then activated by low-energy CID, resulting
in dissociation. Product ions were mass-selectively ejected and
then analyzed to determine primary sequence. MS/MS data
on subfemtomole levels of gramicidin S were demonstrated.
In the same year, Van Berkel and others combined ESI with
qIT single and multiple MS analysis to demonstrate low
energy CID fragmentation and peptide sequencing [143].
A year later, online capillary reverse-phase (RP) LC was com-
bined with ESI-qIT-MS analysis [144]. Online HPLC/MS
molecular weight determinations for cytochrome c, human
serum albumin and myoglobin were shown, as well as
LC/MS/MS and LC/MS/MS/MS analysis of selected tryptic
peptides in a protein tryptic digest. In addition to ESI,
MALDI-generated ions were also analyzed using qIT-MS
[145]. Today, the majority of MS/MS experiments are per-
formed using online capillary RP-HPLC with ESI ion trap
devices. Sequences are automatically processed and protein
identification conferred using various algorithms such as
SEQUEST. Other MS/MS schemes currently used for pep-
tide sequence determination include the ESI tandem quadru-
pole TOF-MS analyzers [146], ESI-FTICR-MS [147], MALDI
post-source decay analysis [148], MALDI quadrupole TOF
analysis [149], MALDI-TOF/TOF-MS/MS analysis [150–152]
and MALDI-qIT-TOF-MS analysis [153,154].
Expert commentary & five-year view
Many approaches exist to mine high-dimensional data, and
novel ones are continually being developed. No specific algo-
rithm can be termed an ideal approach; consequently, the
authors believe that the best approach to mining data is to utilize
a number of feature selection algorithms with appropriate
cross-validation, and to utilize the optimal features to generate
a final diagnostic algorithm. The authors recommend utilizing
various approaches, both in parallel and in sequence, to gener-
ate these final algorithms. In the future, as computational
power increases, the parallel approaches to data mining will
become easier and more high throughput. Moreover, as appro-
priate data sets become publicly available, more rigorous com-
parisons of data preprocessing and mining techniques will be
possible. Proteomics researchers are urged to work in inter-
disciplinary groups that include physicians, laboratorians and
statisticians. Ultimately, the only true validation of a conclu-
sion is to apply the fixed algorithm to a newly collected set of
samples. Therefore, multiple rounds of validation must be
undertaken. While the end point of a highly accurate set of
biomarkers is often the focus, there can be no substitute for
appropriate study design, taking into account the relevant pre-
analytic and analytic variables that can confound data mining.
A major limitation to the successful application of clinical pro-
teomics programs thus far has been throughput, both clinical
and analytic. On the clinical side, there is a tremendous need
for well-documented clinical samples with great care in collec-
tion, aliquotting and storage. Most studies performed to date
have examined a limited number of samples obtained from a
single institution, with the result that promising biomarkers
fail to pass validation. The failure to be validated may be due
to specific sample acquisition parameters, approaches to sam-
ple analysis or demographic and clinical parameters of the
small population being examined. On the analytic side, auto-
mation will become increasingly important so that more
powerful fractionation technologies can be used to detect low-
abundance proteins as well as to analyze the larger number of
samples required for more adequate biomarker discovery and
validation. As proteomics and sample preparation technolo-
gies become more high-throughput and reproducible, higher
powered studies will become feasible, accelerating the discov-
ery and validation of biomarkers that can be translated into
clinical practice.
Acknowledgement
The authors thank Leslie Roth for assistance in making the figures.

Key issues

• Data preprocessing steps such as background correction and spectrum alignment are critical before data mining.
• No single mathematical approach is ideal or applicable to all study designs.
• High-dimensional data need to be reduced to fewer variables via a process of feature selection.
• While the temptation is to drive classification algorithms to the lowest error rate achievable, this approach is likely to result in
overfitting. Stable, globally optimal solutions are preferred.
• The statistics and proteomics community need more sample data to be made publicly available so that direct comparisons of
statistical approaches and, therefore, development of better tools, can be made.
• Identification of biomarker candidates needs to address protein identity as well as all salient post-translational modifications.

www.future-drugs.com 857
Fung, Weinberger, Gavin & Zhang
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Affiliations
• Eric T Fung, MD, PhD
Vice President of Clinical Affairs, Ciphergen
Biosystems, Inc., 6611 Dumbarton Circle,
Fremont, CA 94555, USA
Tel.: +1 510 505 2242
Fax: +1 510 505 2101
efung@ciphergen.com
• Scot R Weinberger, BSc
President & Founder, GenNext Technologies,
PO Box 370645, Montara,
CA 94037-0645, USA
Tel.: +1 650 563 9577
Fax: +1 650 563 9577
sweinberger@ix.netcom.com
• Ed Gavin, BSc
Director of Software Development, Ciphergen
Biosystems, Inc., 6611 Dumbarton Circle,
Fremont, CA 94555, USA
Tel.: +1 510 505 2244
Fax: +1 510 505 2101
egavin@ciphergen.com
• Fujun Zhang, PhD
Staff Statistician, Ciphergen Biosystems, Inc.,
6611 Dumbarton Circle, Fremont,
CA 94555, USA
Tel.: +1 510 505 2332
Fax: +1 510 505 2101
fzhang@ciphergen.com
Expert Rev. Proteomics 2(6), (2005)