Proteomics and applications within the drug
development pipeline
René Houtman and Ian Humphery-Smith
Introduction
Rough estimates at the end of the last century pre-
dicted the existence of approximately 100,000
human genes. However, with the completion of the
draft of the human genome in early 2001 came what
many had already suspected: the actual count
reduced this figure by 75%. A shocking conclusion:
humans have only twice as many genes as a fruit fly
or roundworm, and the gene count is comparable to
that of a mouse! The big question that arises is: How
in theory do we manage to operate at a higher level
of complexity? The answer: It is proteins, and not
genes, that are responsible for an organism’s overall
complexity.The interaction of proteins in a complex
network adds up to how an organism functions.This
is further complicated by lipids,sugars,the dynamics
of molecular shape changes, and the intra-cellular
compartmentalization of specific reagents and their
respective concentrations.
Indeed, proteins are referred to as the ‘molecular
workhorses’ that determine a phenotype which
makes the PROTEOME,the protein complement encod-
ed by a genome, an important object for scientific
research, i.e., PROTEOMICS. Post-transcriptional mecha-
nisms that control the rate of synthesis and half-life
of proteins [1] are responsible for a poor correlation
between mRNA expression levels and protein abun-
dance [2, 3]. Moreover, the importance of CO- and
POST-TRANSLATIONAL MODIFICATIONS (PTMs) for the reg-
ulation of protein function demands that an
improved understanding of molecular systems in
health and disease will depend upon access to tech-
niques that provide both quantitative and qualitative
information derived from complex protein mixtures.
The complexity and number of samples to be
analyzed makes PROTEOMICS a technology-driven sci-
B5
ence and current approaches can be classified as
either traditional or array-based PROTEOMICS. These
approaches will be discussed in this chapter.
Traditional proteomics
Traditional PROTEOMICS is based on the separation sci-
ence and involves sample preparation by extraction
of proteins from the tissue or cells of interest followed
by quantitative (expression level) and qualitative
(identification) analysis. Technologies employed for
separation of the proteins include capillary electro-
phoresis, liquid chromatography, one- and two-dimen-
sional gel electrophoresis (2DE), continuous flow
electrophoresis, and variations of mass spectrometry
(MS), namely single or tandem MS. The latter can be
expanded to MSn and is based upon repeated ion
selection and increasing more refined screening of
the breakdown products of a particular polypeptide.
2-Dimensional electrophoresis
Although 2DE was already introduced in the mid
1970s [4], various modifications over the years
helped in improving this technique into the powerful
tool currently employed in PROTEOMICS research. To
date,2DE has been the mainstay of PROTEOMICS,but is
increasingly being complemented by a variety of
techniques.
Protein samples are first separated by ISOELECTRIC
FOCUSING (IEF, 1st dimension) as illustrated in Figure
1A.This is referred to as charge-driven separation. At
a pH above their ISOELECTRIC POINT (pI), amino acids
(α) behave as proton donors and hence become
negatively charged.When an electric field is applied,
these negatively charged residues migrate to the
214 Proteomics and applications within the drug development pipeline

FIGURE 1. SEPARATION OF PROTEINS BY TWO-DIMENSIONAL ELECTROPHORESIS

A. Proteins are separated in the 1st dimension based on their isoelectric point (pI), i.e.: isoelectric focusing. Subse-
quently, B. these proteins are separated in the 2nd dimension based on their molecular mass (Mr) by SDS-PAGE. Sep-
aration based on the combination of two protein characteristics enables analysis of complex protein mixtures.

(positive) anode until they reach their pI. At this
point the residue’s charge is neutralized by proton
uptake from its environment resulting in an arrest of
migration.Alternatively, at a pH above below their pI,
amino acids (β) behave as proton acceptors result-
ing in a positive charge. Accordingly, in an electric
field they move to the cathode until their pI is
reached. The pI of a protein is determined by the
average of the pIs of the residues of which it consists.
Proteins with different amino acid compositions
therefore have different pIs. Originally, IEF of proteins
was performed in rod-shaped polyacrylamide gels
with mixed-in synthetic ampholytes, pH gradient-
forming chemicals. However a major improvement
of the technique was accomplished with the intro-
duction of immobilized pH gradients (IPG) in the
late 1980s. The latter, formed by ampholytes cross-
linked to a plastic carrier strip by acryl amide, result-
ed in enhanced resolution,improved reproducibility,
higher loading capacity for preparative gels and pos-
sibility of separation of basic proteins under equilib-
rium conditions [5], but is usually practiced in the
presence of ampholytes and thus referred to as
‘mixed-bed’ electrophoresis.
Following IEF, proteins are separated with respect
to mass within a polyacrylamide sieving matrix,

TABLE 1. CLEAVAGE SITE OF SEVERAL PROTEASES
COMMONLY USED IN PROTEIN IDENTIFICATION
Enzyme Cleavage site
Asp-N X-D1
Arg-C X-R
Glu-C(i) E-X
Glu-C(ii) E-X
D-X
Lys-C K-X
Trypsin R-X
K-X
For amino-acid coding see Table 3; X, any amino acid
namely SDS-PAGE in the 2nd dimension. An anionic
detergent sodium dodecyl sulphate (SDS) within the
electrophoresis buffer is employed to form charged
moieties encompassing the polypeptides needing to
be separated.Hence proteins previously separated in
an IEF gradient become saturated with a negative
charge. Application of an electric field induces
migration of these proteins towards the anode from
the IPG strip into the PAGE gel where they are sepa-
rated by means of their molecular mass (Mr), small
proteins migrating faster through the maze of acryl
polymers then large proteins.
Once separated, proteins can be visualized by
various fluorescent and non-fluorescent staining
methods.
The power of separation by 2DE,based on a com-
bination of different protein parameters, is illustrated
in Figure 1B. Proteins of identical Mr (I and III) can
be distinguished with respect to pI due to different
amino acid composition, while proteins inseparable
by IEF (II and IV) are separated by PAGE due to dif-
ference in Mr. In this manner, mixtures containing
thousands of proteins can be resolved.
For identification, protein spots are excised from
the 2DE gels and subjected to in-gel digestion by an
ENDOPEPTIDASE with known specificity as listed in
Table 1. PROTEOLYTIC FRAGMENTS diffuse out of the gel
and are analyzed by mass spectrometry (MS).
Traditional proteomics 215
One of the inherent qualities of 2DE is its ability
to separate and quantitatively visualize PTMs of pro-
teins, because accompanying changes in mass (see
also Table 2) and, most importantly, accompanying
charge, result in altered migration and a visible shift
on the gel.
Procedures for protein characterization following
protein isolation have been reviewed by Humphery-
Smith and Ward [6].
Protein identification by mass spectrometry
One of the major breakthroughs for PROTEOMICS
research has been the advent of soft-ionization MS-
based techniques to facilitate analysis of macromol-
ecules (polypeptides) by mass spectrometry. The
instruments used, which enable protein identifica-
tion from only a few femtomoles of material, can be
grouped into two categories: single-stage mass spec-
trometers producing peptide mass fingerprints
(PMF) and tandem MS- (MS/MS) based systems.
Basically, MS is performed by three consecutive
events. First, the components of the ANALYTE require
ionization/ desorption; second, the ions generated
are separated based on their size and charge; and
third,the separated ions are detected.Internal resolu-
tion of peptides can be obtained by enhancing frag-
mentation by post-source decay, collision-induced
dissociation or electron-coupled dissociation of a
parent ion of interest.
Ionization
There are several ways by which the PROTEOLYTIC
FRAGMENTS can be ionized, of which matrix-assisted
laser desorption/ionization (MALDI) and electro-
spray ionization (ESI) are most commonly used, illus-
trated in Figure 2A. In MALDI, the PROTEOLYTIC FRAG-
MENTS are mixed with a crystalline matrix, spotted
onto a solid surface and dried. Next, crystals are tar-
geted by a laser. The energy of the laser is absorbed
by the crystal,causing it to vaporize and leading to an
‘explosion’ and subsequent desorption/ionization of
peptides, which then enter the analyzer in gas phase.
Using electrospray ionization (ESI), ionization is
achieved by a process referred to as COLOMBATION.
216 Proteomics and applications within the drug development pipeline

per droplet size to a critical value, at which time the

TABLE 2. MASSES OF SEVERAL COMMON POST- droplet explodes into naked ions carrying charge.
TRANSLATIONAL MODIFICATIONS
Separation of ions
Modification Mass
Ionized peptides are separated with respect to their
Acetylation 42 mass according to the principle of time-of-flight
Amidation –1 (TOF). As illustrated in Figure 2B, in an electric field
Biotinylation 226 (V) the time (tD) it takes for ions to travel the dis-
Carbamylation 41 tance (d) between the ion source (t0) and the detec-
Carboxylation 44 tor is inversely correlated with their mass-over-charge
Deamidation 1 ratio (m/z). Of two equally charged peptides the one
Deoxyhexose 146 with the highest mass travels more slowly and arrives
Formylation 28 at the detector later.A typical TOF mass spectrograph
O-GlcNac 203 is shown in Figure 3 in which each peak represents a
Glucosylation 162 parent ion of particular m/z ratio, calculated from tD.
Hexosamines 161
Hydroxylation 16
Methylation 14 Peptide mass fingerprinting by MALDI-TOF
Myristoylation 210
N-acetyl hexosamines 203 Protein identification can be achieved through pep-
Oxidation 16 tide mass fingerprinting (PMF),usually performed by
Palmytoylation 238 a single-stage MS instrument using MALDI linked to
Pentose 132 TOF (MALDI-TOF).The resulting spectrum resolves a
Phosphorylation 80 peak list of masses corresponding to PROTEOLYTIC
Sialytation 291 FRAGMENTS of the excised protein of interest and auto-
Sodium 22 proteolytic peptides from the protease that was used
Sulphation 80 for the digestion.The characteristic masses of the lat-
ter,indicated with * in Figure 2B,are used for internal
calibration of the raw mass spectrum, enabling the
generation of a high-accuracy list of molecular mass-
Here, the peptide solution is sprayed under vacuum es, or peptide mass fingerprint (PMF). The PMF is
from a gold-coated glass capillary directly into the then compared with those derived from databases of
analyzer.Before the analyzer is reached,fast evapora- which all proteins have been digested in silico with
tion of the solvent induces a rapid increase of charge the same protease that was used for digestion of the

FIGURE 2 (following page)

Protein identification via single-stage mass spectrometry is performed in three subsequent events. A: First, proteolytic
fragments are ionized. This is usually achieved by matrix-assisted laser desorption/ionization (MALDI) or electrospray
ionization (ESI) B: Second, ions are separated based on their molecular mass by the principle of Time-Of-Flight (TOF)
and, finally, detected. This results in a mass-over-charge spectrum (M/Z) spectrum C: A quadrupole can be used as an
ion gate so select an ion with a particular M/Z. Tandem-mass spectrometry (MS/MS) is performed for peptide sequenc-
ing. The selected ion is fragmented by gas collision. In the resulting M/Z spectrum of all fragments the mass difference
between adjacent peaks represents the mass of a terminal amino acid that was lost due to fragmentation. Hence
MS/MS is performed for peptide sequencing.
Traditional proteomics 217
218 Proteomics and applications within the drug development pipeline
FIGURE 3
Schematic representation of a quadrupole ion trap, which
is used for ion gating. The total mix of ionized peptides is
‘trapped’ within the space between the ring and end-cap
electrodes. A combination of a constant trapping poten-
tial (VAC) and alteration of the radio frequency (Vr.f.)
amplitude enables selective elution of ions for analysis by
TOF.
excised protein spot. A probability score for protein
identity is calculated by an algorithm that takes the
percentage of overall peptide sequence coverage
taken across the undigested protein and the extent
observed: expected mass errors. The success rate of
this technique can be improved by the use of more
than one protease so that results from more than one
PMF can be used to increase the statistical confi-
dence extracted from the search results obtained.
[7]. As a general rule in PROTEOMICS, whenever
orthogonal techniques and/or orthogonal algo-
rithms are employed on the same sample or experi-
ment, one can enhance the associated statistical
confidence in the conclusions through concordance
of research findings.
De novo protein sequencing by QTOF
PROTEOLYTIC FRAGMENTS can also be analyzed by tan-
dem MS instruments (MS/MS or MSn), such as the
quadrupole coupled to TOF (QTOF),as illustrated in
Figure 2C, by which information pertaining to the
amino acid sequence and composition of peptides
can be extracted.A quadrupole consists of four par-
allel rods or poles and serves as a gate for ions, pep-
tides, of interest. The poles have a fixed direct cur-
rent, DC, while alternating radio frequency voltages
are applied to them. Depending on the electric field
produced, only ions of a particular m/z will be
focused on the detector (resonant ions), while all
the others are deflected into the rods (nonresonant
ions). Using QTOF, peptides are usually ionized by
ESI before entering the instrument,which during the
analysis switches back and forth between scanning
and MS/MS-(sequencing) mode. During scanning
mode, when the quadrupole is switched of, the ANA-
LYTE is separated by TOF.When an ion is detected that
meets predetermined criteria,e.g.,a proper signal-to-
noise ratio and having a mass that does not belong
to an autoproteolytic fragment of the applied pro-
tease, the instrument switches to scanning mode.
When the quadrupole is switched on, it creates an
electric field that selects the m/z range for the ion of
interest, i.e., parent ion gating. The gated ions are
then led through an argon-filled chamber posi-
tioned inline with the quadrupole, the collision cell,
and are fragmented by collision-induced dissocia-
tion prior to entering the TOF. In the resulting spec-
trograph, as shown in Figure 4, each peak represents
a fragment of the parent ion and the mass difference
between two adjacent peaks may correspond to that
of an amino acid, the masses of which are listed in
Table 3.These peaks are derived from the parent ion
on the right (Fig. 2C), each having lost varying por-
tions of the intact peptide.The sequence of m/z dif-
ferences between each consecutive peak in the
spectrum can be deconvoluted so as to determine
the actual sequence of the selected peptide. After
fragmentation of the parent ion and detection of the
ion fragments, the instrument switches to scanning
mode until a new ion meets the selection criteria.
The amino acid sequences obtained by this process
can then be employed for use in protein identifica-
tion by database searching. In addition, MS/MS spec-
tra can be used to study the location and nature of
PTMs by focusing on mass differences between
adjacent peaks that can only be explained by a net
 Traditional proteomics 219

FIGURE 4
Quantitative mass spectrometry-based analysis of protein expression by stable isotope labeling. Comparison of pro-
tein abundance between two samples is performed by labeling the proteins of the samples with different mass tags.
This labeling is mainly done A. metabolically, B. chemically, e.g., isotope-coded affinity tagging (ICAT, see also D.) or
C. proteolytically. Subsequently, labeled samples are mixed, digested and subjected to mass spectrometry. In the result-
ing spectrum, identical peptides of the different samples are separated by the mass difference of the two isotopes and
the ratio of their relative abundance in the spectrum reflects the ratio of protein expression between the two samples.

mass of an amino acid and a PTM as listed in
Table 2.
2DE drawbacks
Although 2DE is unsurpassed in its resolving power
for separation of complex protein mixtures, it is not
without difficulties and shortcomings. Low-abun-
dance proteins are probably the most interesting
ones from the point of view of understanding cellu-
lar and regulatory proteins; however, only highly
expressed proteins are detected and identified with
current 2DE protocols [2, 8, 9].The tendency of 2DE
to detect only highly expressed proteins is caused by
the intrinsic limitations in dynamic range of various
stages of the technique on one hand and the enor-
mous dynamic range of protein expression level
across a PROTEOME on the other.
The dynamic range of detection by classical
methods such as Coomassie Blue (low sensitivity) or
silver staining (non-linear at higher protein concen-
220 Proteomics and applications within the drug development pipeline

TABLE 3 CHARACTERISTICS OF AMINO ACIDS

Residue 1-letter code Residue mass Mass of most abundant immonium ion

Alanine A 71 44
Arginine R 156 129
Asparagine N 114 87
Aspartic Acid D 115 88
Cysteine C 103 76
Glutamic Acid E 129 102
Glutamine G 128 101
Glycine Q 57 30
Histidine G 137 110
Isoleucine H 113 86
Leucine I 113 86
Lysine L 128 101
Methionine M 131 104
Phenylalanine F 147 120
Proline P 97 70
Serine S 87 60
Threonine T 101 74
Tryptophan W 186 159
Tyrosine Y 163 136
Valine V 99 72

trations) is too narrow and therefore fluorescent
staining methods with new probes with enhanced
SENSITIVITY and dynamic range have been developed
and are becoming increasingly popular. In addition,
the number of proteins that can be separated practi-
cally by 2DE is limited by the physical dimensions of
the gel itself. One solution to enhance resolution is
separation of a protein sample on multiple 2DE gels
of consecutive partially overlapping narrow-range
IPGs. Subsequently, the images of generated gels are
combined in silico to construct so-called ‘ZOOM GELS’
[5, 10].
The high variety in protein expression levels is
illustrated by the fact that 90% of the PROTEOME of a
typical cell is made up of only 10% of the
10,000–20,000 different protein species. An addition-
al factor that adds to this complexity is the large vari-
ety of biochemical characteristics (hydrophobicity,
charge, pI, etc.) of proteins, which makes solubiliza-
tion of all proteins during sample preparation virtu-
ally impossible. To reduce complexity, samples can
be pre-fractionated for specific subsets of proteins by
affinity enrichment and application of specific
extraction buffers and methods. Still, hydrophobic
proteins such as membrane receptors often precipi-
tate during IEF, which prevents transfer into the SDS-
PAGE gel and are thus lost during separation.
2DE also suffers from high variation that is intro-
duced during sample preparation, protein loading
and the complex nature of the staining procedures
employed. In order to obtain statistically significant
quantitative data from which valid conclusions
regarding biology can be drawn, it is necessary to
produce a number of replicate gels from the same
sample [11–13]. Furthermore, qualitative and quanti-
tative analysis of 2DE gels performed by dedicated
software is still difficult to automate and requires a
high degree of human intervention.The latter step is

therefore time consuming and currently the rate-lim-
iting factor in 2DE-based PROTEOME analysis. These
shortcomings have been ostensibly overcome and
fully automated image analysis is becoming a long-
awaited reality in the discipline.As with any process,
when one bottleneck has been overcome, the goal-
posts are transposed elsewhere. Independent of
method employed, the major challenge for PRO-
TEOMICS is that of total proteomic coverage, which
remains low, except in microbes.
Qualitative MS-based proteomics
Because of the limitations of 2DE, other techniques
have been developed for qualitative PROTEOME analy-
sis based on liquid separations coupled to on-line
mass spectrometry. In general, complex mixtures of
proteins,or peptides,are separated by microcapillary
format liquid chromatography, capillary elec-
trophoresis or hybrids thereof prior to ESI and subse-
quent mass analysis by MS/MS. This approach using
one-dimensional separation is sufficient for the
analysis of low-complexity samples; however,its reso-
lution is by far inferior to that of 2DE.To date,no stud-
ies have been conducted on multiple analyses of the
same sample set, but initial indications are that vari-
ance due to technology and biology will mean that
large numbers of replicates will be necessary to
allow valid conclusions due to significant popula-
tion variance in both test and control groups.
One approach to obtain a higher order of resolu-
tion of separation prior to MS is termed multidimen-
sional protein identification technology (MudPIT)
[14]. This orthogonal combination of cation
exchange and reverse-phase chromatography con-
sists of sequential step-elutions (~15) from global
styptic peptides from a strong cation exchange resin
with slow ethanol gradients inserted between each
salt elution step. After ESI, eluted peptides are ana-
lyzed using a quadrupole ion trap (QIT).This instru-
ment consists of two hemispherical end-cap elec-
trodes,spatially separated by ring electrodes,illustrat-
ed in Figure 3. The trapping potential, AC voltage,
applied to the end-cap electrodes, is used to confine
ion species with respect to their respective m/z ratio
for subsequent analysis.A mass spectrum is obtained
Traditional proteomics 221
by linearly altering the amplitude of the radio fre-
quency applied to the ring electrode so as to elute a
particular ion series from the trap, after which they
are analyzed by TOF.
The MudPIT method was shown to be effective in
identification of proteins with a wide range of bio-
chemical properties and expression levels, including
low-abundance proteins [14,15].Although MudPIT is
easy to automate and harbors the appropriate reso-
lution and dynamic detection range needed to man-
age the complexity of global PROTEOME analysis, it
will only generate qualitative data.This is a restriction
of all MS-based techniques because the relative
abundance of a peptide in the spectrum depends on
its biochemical make-up and subsequent ability to
become ionized and is not necessarily correlated
with its concentration in the sample, i.e., relative ion
intensity at the detector may be significantly differ-
ent from that entering the mass spectrometer.
Quantitative MS-based proteomics
Several alternative methods have been proposed for
relative quantification of protein expression between
paired samples, of which one has been subjected to
stable isotope labeling, which can be achieved by
several methods (Fig. 4).
Metabolic labeling
In this approach (Fig.4A),two populations of cells or
organisms are grown in parallel, one on a normal
source of nutrition and the other on a source deplet-
ed or enriched for an isotope of N, C or H [16–18].
This process is referred to as stable isotope labeling
with amino acids during culture,or SILAC [19].Next,
protein samples from both populations are mixed,
digested and analyzed in parallel by MS. The incor-
porated isotope induces a mass change reflected by
a characteristic shift in the resultant MS spectrum.
The mean ratio in relative ion intensity of correspon-
ding peaks is employed as an indication of that of
the relative difference in protein expression
between the two populations (Fig. 4E). Unfortunate-
ly, peptides from the same protein show much varia-
tion with respect to the estimates of protein abun-
222 Proteomics and applications within the drug development pipeline
dance derived from ion intensity measures, due to
differences in a peptide’s overall charge and associ-
ated willingness to enter the gas phase and ‘fly’ in
the MS.
Isotope-coded affinity tagging
Another method is isotope-coded affinity tagging
(ICAT) [20] (Fig. 4B) or variants thereof based upon
isotope tagging of sulphydryl groups, amino groups,
active sites for serine & cysteine hydrolases, phos-
phate ester groups, and N-linked carbohydrates. The
ICAT reagents (Fig. 4D), consist of a protein-(cys-
teine) reactive group,a linker region and a biotin tag.
The linkers are composed of either 8 deuterium (d8,
heavy reagent) or 8 hydrogen (d0, light reagent)
atoms, resulting in an 8Da mass difference between
corresponding peaks in the MS spectrum.A reduced
protein sample from one population is derivatized,
on cysteine,with the isotopically heavy version of the
ICAT reagents, while the other is derivatized with the
isotopically light variant. Derivatized samples are
mixed and subjected to digestion, after which the
mix is enriched for cysteine-containing peptides by
affinity purification using the biotin tag of the ICAT
linker. This results in an approximate tenfold reduc-
tion in sample complexity. One of the weaknesses of
this method is the fact that 1 out of 7 proteins does
not contain any cysteine and thus does not react
with the ICAT reagents.These proteins are lost during
affinity purification using biotin and therefore
missed in the analysis. Furthermore, the ICAT
reagents are relatively large molecules compared
with the peptide to which they are attached and
thereby influence chromatographic separation prior
to ionization and peptide ionization itself, which
complicates data interpretation. A detailed compari-
son of 2DE and ICAT technologies has shown
healthy complementarity of these techniques,where-
by together enhanced proteomic coverage was
achieved with the latter demonstrating a bias for
high-Mr proteins and quantification based on the
sum of the protein species, while the former showed
improved resolution of low-Mr proteins, post-transla-
tionally modified polypeptides, processed polypep-
tides and identified cysteine-free proteins, otherwise
invisible to ICAT [21].
Mass coded abundance tagging
To avoid problems associated with poor quantifica-
tion derived from estimates based on ion intensity
measures, mass coded abundance tagging (MCAT)
has been introduced [22].The methodology is based
on guanidination of C-terminal lysine residues on
tryptic peptides, corresponding elution times, direct
peptide identification by MS and a predictable mass
change of 21 m/z units. Here, however, ACCURATE pro-
tein quantification can be achieved from a recon-
structed ion chromatogram for comparison of ‘areas
under the curve’ for differential screening. The latter
is somewhat limited by the resolution afforded by
the liquid chromatography system. Another variant
of MCAT is absolute quantification (AQUA) internal
standard peptides [23].
Proteolytic labeling
A third labeling method is performed during protein
digestion (Fig.4C).Two samples are separately digest-
ed in either H216O or H218O.The oxygen atom derived
from the aqueous solvent is incorporated into the
newly formed C-terminus of each peptide, providing
an EPITOPE tag for relative quantification [23]. Differ-
ential resolution can be achieved in a similar man-
ner by incorporation of 15N [24].
Array-based proteomics
MICROARRAYS provide the potential for determining
thousands of different binding events in a single
experiment in a massively parallel fashion. The
genomic revolution of the last decade which has
delivered information on whole organisms at the
level of DNA sequence and transcriptome analysis
has been based notably on parallelization, miniatur-
ization and automation – all of which become possi-
ble in a protein biochip environment. Usually, a num-
ber of different molecules with distinct binding char-
acteristics, CAPTURE MOLECULES or ligands, are
immobilized in a grid-like fashion on a solid SUB-
STRATE, the array.The latter is incubated with an ANA-
LYTE solution containing the molecules of interest,
TARGETS.Detection of binding is usually performed by

FIGURE 5. BIOCHEMICAL DIVERSITY
OF AMINO ACIDS
fluorescent staining methods either before or after
TARGET binding.
The microarray format was originally designed
for studying DNA dot blots and later mRNA expres-
sion levels and is currently being extrapolated for
applications to study protein expression and func-
tion.The origins and applications of array-based PRO-
TEOMICS have been reviewed by Humphery-Smith
[25].A number of practical problems are associated
with the setup of high throughput parallel binding
assays involving proteins. Proteins are far more
diverse with respect to their biochemical properties
than their nucleic acid counterparts, whereby each
amino acid combines to rapidly produce an incred-
ibly large potential variety as to properties of the
final polypeptide, i.e., the number of amino acid in a
polypeptide string to the power of 20 (Fig. 5).
Miniaturization of systems allows for less ANALYTE
consumption. But perhaps more importantly,
reduced spot size associated with the area devoted
to CAPTURE MOLECULES on the array will concomitant-
ly allow for MICROSPOTS with high overall density of
binding sites. This results in enhanced SENSITIVITY
compared to the use of larger spot areas, i.e.,
MACROSPOTS. This phenomenon is explained by the
theory of ‘AMBIENT ANALYTE CONCENTRATION’ [26, 27]
and is explained below.
Array-based proteomics 223
Microspot
CAPTURE MOLECULES are immobilized at high density
on the solid support and localized within a very
small surface area. During the assay, the TARGETS are
captured by the MICROSPOT in which the absolute
number of capture-TARGET complexes is low owing to
its small area. As a result, the capture process does
not significantly change the TARGET concentration in
the ANALYTE solution, even for TARGETS in low concen-
tration and/or for binding reactions that occur with
high affinity. This remains true whenever <0.1/K of
the CAPTURE MOLECULES are captured, where K is the
initial concentration of potential TARGETS in solution.
Under these ambient ANALYTE conditions,the amount
of the TARGET captured from solution directly reflects
its concentration in the assay system. Such a system
is independent of the actual ANALYTE volume and is
capable of high-sensitivity concentration measure-
ments with low sample consumption.
The SENSITIVITY that can be obtained is high for
two reasons. First, the binding activity occurs at the
highest possible TARGET concentration. Second, the
CAPTURE MOLECULE-target complex is found only in a
small area of the MICROSPOT, resulting in high local
signal (Fig. 6).As demonstrated in this diagram, CAP-
TURE MOLECULES are immobilized in a constant sur-
224 Proteomics and applications within the drug development pipeline

FIGURE 6. SIGNAL AND SIGNAL DENSITY IN MICROSPOTS (adapted from [28]).

Capture molecules are immobilized in a constant surface density in all spots. The largest spot harbors the highest total
amount of capture molecules and overall signal. Here signal density is lowest because of the limiting amount of tar-
get, evenly distributed on the area without saturating it. With decreasing spot size the overall signal will decrease but
the signal density will increase for smaller spots. Below a certain spot size, the signal density approaches an optimum
(ambient analyte conditions, unlimited amount of target) and will stay approximately constant with any further
decrease of spot size.

face density onto spots that have increased spot
size. With increasing spot size, the total amount of
CAPTURE MOLECULES in the assay increases,as does the
sum of the obtained signal in the spot. The signal
density, however, starts to decrease with increasing
spot size because the amount of TARGET starts to
become a limiting factor.The capture process leads
to a significant reduction of TARGET concentration in
solution and at the same time the probe-TARGET com-
plexes are distributed over a larger area. As a result
the maximal signal that can be obtained from any
spot in the spot is decreased. Decreasing the spot
size will decrease the overall signal per MICROSPOT
but the signal density will increase for smaller spots.
Below a certain spot size, the signal density
approaches an optimum (ambient ANALYTE condi-
tions, unlimited amount of TARGET) and will stay
approximately constant with any further decrease of
spot size.Therefore the highest signal intensities and
optimal signal-to-noise ratios can be achieved in
small spots. Small spot size also allows for dramatic
parallelization on a low-cost biochip format.
Furthermore, when conducted in conjunction with
grating-coupled surface plasmon resonance, real-
time association and dissociation constants can be
acquired in parallel so as to provide still greater

FIGURE 7. A WIDE VARIETY OF ASSAY TYPES IN ARRAY-BASED PROTEOMICS
insight into the dynamics of biomolecular interac-
tions.
To date, protein biochips have been employed to
examine a wide variety of assay types, see Figure 7.
These include detection of interactions between
antibody-antigen; protein-protein; protein-nucleic
acid, protein-small molecule, membrane-bound
receptors and TARGET,domain screening,and analysis
of enzymatic function. In addition, a parallelized for-
mat has been adopted for a variety of other applica-
tions in PROTEOMICS.These applications include tissue
arrays, EPITOPE mapping via peptide arrays, reverse
arrays for the examination of naturally occurring pro-
tein isoforms found in cellular extracts as eluted
from liquid chromatography,cellular arrays for bioas-
says, and immobilization of various chromatography
affinity capture reagents. The latter are now being
used extensively to seek out biomarkers associated
Summary 225
with the progression, prognosis and early detection
of disease and are also being applied to patient
cohorting during drug trials, i.e., pharmacopro-
teomics. Protein arrays hold equally great potential
to improved lead development and optimization as
a means of verifying TARGET SPECIFICITY in the presence
of numerous potential recombinant binders and
thereby working towards reducing adverse drug
effects as a direct spin-off from increased knowledge
of the human genome.
Summary
Here,we have set out to demonstrate that PROTEOMICS
in its various forms has demonstrated its utility
across the entire drug development pipeline from
initial TARGET discovery and validation through to
226 Proteomics and applications within the drug development pipeline
monitoring drug responses during clinical trials. In
recent times, proteomic techniques are also increas-
ingly making their impact felt in the very last step in
this pipeline, namely process validation during the
production phase.This more generalized impact has
only become possible as a result of improved repro-
ducibility,sensitivity,user-friendliness and throughput
of the techniques employed. The next phase of
genomics is now generally hailed as focusing on
PROTEOMICS.These techniques are evolving rapidly to
meet the demands of the scientific and pharmaceu-
tical communities. Such specialized approaches as
chemoproteomics are likely to usher in still further
advances in global screening of biomolecule bind-
ing to therapeutic agents and high-throughput char-
acterization on line via mass spectrometry of these
binders.The results of such analyses can then be fed
to in silico analysis so as to better understand the
shared structural motifs and domains that have given
rise to drug binding across a wide spectrum of cellu-
lar components. In such a manner, PROTEOMICS data
are likely to be seen increasingly to actually drive
many aspects of future drug development and
improved safety of therapeutic agents, be they tradi-
tional small molecules or biomolecules.
Selected Readings
Aebersold R,Mann M (2003) Mass spectrometry-based pro-
teomics. Nature 422: 198–207
Phizicky E, Bastiaens PI, Zhu H, Snyder M, Fields S (2003)
Protein analysis on a proteomic scale. Nature 422:
208–215
Recommended Websites
Web resources for protein scientists: http://www.proteinso-
ciety.org/docs/WWWResources.html (Accessed De-
cember 2004)
IonSource: http://ionsource.com/ (Accessed December
2004)
ExPASy Proteomics Server: http://www.expasy.org/ (Ac-
cessed December 2004)
Proteomics Interest Group (ProtIG): http://proteome.
nih.gov/links.html (Accessed December 2004)
An Introduction to Mass Spectrometry: http://www.ast-
bury.leeds.ac.uk/Facil/MStut/mstutorial.htm (Acces-
sed December 2004)
Swiss Proteomics Society (SPS): http://www.swisspro-
teomicsociety.org/links.html (Accessed December
2004)
Proteomics – Spectroscopic Applications in Proteomics:
http://www.spectroscopynow.com/Spy/basehtml/Sp
yH/1,1181,10-4-0-0-0-directories_new-0-0,00.html
(Accessed December 2004)
North Carolina Genomics & Bioinformatics Consortium,
LLC: http://www.ncgbc.org/resources/links/proteo-
mics-links.cfm (Accessed December 2004)
http://www.hupo.org/ (Accessed December 2004)
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