Malaysian Journal of Biochemistry and Molecular Biology (2007) 15, 1-7 1

Review Article
Overview of Proteomics

Onn Haji Hashim

Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur.

Abstract
The era of genomics has brought tremendous advancement especially in the fields of medicine and life sciences. Despite the
overwhelming growth of information generated from genomics research, a huge gap specifically in relation to genome expression
persists, and became increasingly noticeable. This has sparked interest in studies of proteins expression and eventually added
limelight to the field of proteomics. Aside from the need to fill the gap, the emergence of proteomics is also deemed to have
occurred due to the advancement in capabilities of research techniques, particularly in the separation and identification of proteins.
Proteomics has since progressed and is slowly extending into other research as well as applied subspecialties. This brief overview
was written to provide a basic and simplified understanding of proteomics in view of its growing interest from newcomers to the
area.

Keyword: proteomics

Defining Proteomics the processes that uphold the normal physiological

Proteomics is broadly defined as the systematic analysis functions of the human body, is increasingly in demand.
of the protein expression of a proteome. The term
‘proteome’ was introduced to describe an entire protein Although the synthesis of proteins is directed by the
complement expressed by a genome, or by a particular genome, the expression of proteins within a cell cannot
cell or specific tissue type [1,2]. In many cases, the be directly predicted from genomic information. It is not
proteome is a virtual reality that arises from accumulation always a direct complement of the genome. For example,
of data obtained from separate analyses of subproteomes, the proopiomelanocortin gene when expressed in the
which were done at different times and under different corticotrophic cells of the anterior pituitary generates
experimental settings and conditions. It is therefore an adrenocorticotropic hormone and β-lipotropin, but
annotation of all normal/wild type proteins that may be expression of the same gene in the cells of the
expressed by a genome with complete disregard of the intermediary pituitary gland produces a different set of
differences of time, conditions or regulatory parameters. hormones comprising α-melanocyte-stimulating hormone,
Thus, all studies annotating or analyzing the genomic corticotrophin-like intermediary peptide, γ-lipotropin and
expression in the form of proteins being expressed within β-endorphin [3]. This phenomenon which is attributed
a defined proteome or subproteome are termed proteomics. to differential peptide processing, together with other
mechanisms like alternative splicing, mRNA editing,
polyadenylation and post-translational modifications, by
Why is Proteomics Necessary? and large, results in different forms of protein products
Aside from water, protein is the main constituent of that all arise from the same genome (Fig. 1). Further
the human body. The body’s function is also orchestrated additions to this complexity are the facts that different
by proteins in the form of its structures, biocatalysts, cells or tissues express different genes and many of these
modulator and transporter molecules, metabolites as well genes are not constitutively expressed but rely on the
as an energy source at crucial times of requirement. presence of specific modulators. All proteins are also
Proteins not only form the human body but operate it. It continuously degraded, sorted and may be modified upon
is also the main site of action of most drugs that are used interaction with other proteins. Thus, the dynamics of
to manipulate and assert necessary changes to the protein expression and interaction within a defined
physiological conditions of the human body as and when proteome at various stages and conditions, more often
required. Perhaps the greatest expectation from than not, could only be unraveled at its level and not
proteomics comes from research carried out to identify possibly deduced from mere genomic information. This
new protein targets in transformed cell lines and diseased has become even clearer when analysis of mRNA
tissues for the actions of specific drugs. Added knowledge
on proteins interaction may also drive new initiatives to
Author for correspondence: Prof. Dr. Onn Hashim Department
design better drugs. In order to gain further insight into of Molecular Medicine, Faculty of Medicine, University of
many diseased conditions, in-depth knowledge and Malaya, 50603, Kuala Lumpur. Phone: 603-79674906
understanding of protein expression and interaction, i.e., Fax: 603-79674957 E-mail: [email protected]
Overview of proteomics 2

maturation translation
RNA mRNA PROTEINS

transcriptional alternative splicing translational proteolysis,

regulation mRNA editing regulation post-translational
polyadenylation modification,
transcription & sorting

DNA MULTIPLE PROTEIN

PRODUCTS
Figure 1: Genomic expression. Due to multiple processing and regulatory mechanisms at different stages of the expression
of a gene, multiple protein products may be generated.

expression using methods like serial analysis of gene expression between samples that differ by some variables
expression and DNA microarray technology have now using a combination of separation and identification
shown poor correlation between mRNA and protein techniques like 2-DE and mass spectrometry. In this
expression levels [4-6]. case, variables would include comparison between
different cells or tissues, stages of a disease, the states of
At tandem with the need to perform its studies, the era being induced or inhibited, normal or diseased, treated or
of proteomics has also evolved from the advancement of non-treated and many others. The data obtained from
techniques used in the identification and separation of such comparison studies will certainly enhance
proteins. A tremendous leap has generally taken place as understanding in the specific areas of medicine or life
proteins can now be identified using mass spectrometry sciences. Differences in the expression of the proteins
and the separation of proteins by two-dimensional gel that are detected in the analyses of the proteome or
electrophoresis (2-DE) is modified and improved. An subproteome may also form a basis for further
attribute to the genomic work, compilation of cDNA investigations to seek improvements in techniques and
sequences of proteins are directly translated into putative methodologies that are currently being applied in diagnosis
amino acid sequences. By identifying specific positions and treatment of patients.
of trypsin action, amino acid sequences of polypeptides
within the cleavage points of the enzyme are identified. Our studies comparing the serum 2-DE protein profiles
The masses of the putative tryptic peptides of proteins of patients with breast and nasopharyngeal cancers with
are deciphered from databases, which are then used as that of matching normal healthy controls are examples of
references, in the form of peptide mass fingerprints (PMF), expression proteomics. By comparing the serum protein
for identification of proteins. In the process of identifying profiles of normal individuals with that of newly detected
a protein, masses of its tryptic peptides, obtained by and untreated patients with breast cancer, we were able
mass spectrometry analysis, are generally used to search to demonstrate significant differences in the expression
for matching PMFs in the database. Identification is of several acute-phase proteins [12]. Identities of the
generally achieved when considerable matches are made. serum proteins were verified by mass spectrometry. The
aberrant expression of some of the proteins was validated
As for the separation of proteins, although the use of by ELISA as well as by immunohistochemical staining
2-DE has been reported as early as 1975 by O’Farrell [7] of the respective lesions of the breast. Unlike that of
and Scheele [8], its popularity was only seen patients with breast cancer, the sera of patients with
approximately twenty years later when the technique was nasopharyngeal carcinoma (NPC) demonstrated 2-DE
proven to be reproducible and more convenient [9]. This protein profiles similar to that of the controls, with
was made possible mainly when the problem of gel exception of the ceruloplasmin spots, which appeared
gradient drift [10] was remedied with the use of up-regulated [13]. The enhanced ceruloplasmin
immobilised pH gradient in replacement of carrier expression, which was also validated by ELISA studies,
ampholytes [11]. was normalized when the NPC patients were treated.
Immunohistochemical analysis of nasopharyngeal lesions
of NPC patients demonstrated positive staining for
Applications of Proteomics ceruloplasmin only at the malignant areas. These
At present, proteomics may be categorized into three expression proteomics studies may form a basis for further
different types, i.e., expression proteomics, structural research to investigate whether the differentially expressed
proteomics and functional proteomics, depending on its proteins may be used as additional biomarkers to aid
application. Expression proteomics, which is also called diagnoses of both the cancers or to monitor the progress
classical proteomics, is the quantitative study of protein of their treatment towards patient’s recovery.
Overview of proteomics 3

Structural proteomics are studies whose goal is to Proteomics Technology

map out all free proteins or protein complexes that are The technology of proteomics is generally divisible
present in a specific cellular organelle. This involves into three different stages involving separation,
attempts to identify all proteins within an organelle or a identification and quantification of proteins.
multiprotein complex, determine where they are located,
and characterize all protein-protein interactions.
Establishment of subproteomes by isolating specific Separation of Proteins
subcellular organelles or protein complexes by purification Proteomics analysis by mass spectrometry may be
can greatly simplify analysis of structural proteomics carried out on proteins that are separated by one-
[14]. Identifying the proteins and activities localized to dimensional gel electrophoresis (1-DE). The technique
specific cellular compartments provides insight on the is simple to perform, reproducible and can generally
structural organization of the cell. Analysis of the nuclear separate proteins that differ by a few kDa. Nevertheless,
pore complex of yeast is a good example of this subtype the resolving power of 1-DE is rather limited and thus,
of proteomics [15]. The data obtained has helped piece the technique is usually carried out on protein mixtures
together the overall architecture of yeast cells and explain after some stages of purification. Separation of proteins
how expression of certain proteins contributes to the by 1-DE occurs on the basis of differences of molecular
unique characteristics of a cell. mass.

Functional proteomics is a broad term generally used For more complex mixtures of proteins such as a
to define many specific, directed proteomic approaches, crude cell lysate, high-resolution 2-DE is a better choice.
usually under non-denaturing conditions and smaller 2-DE separates proteins on the basis of their net charge
subset of proteins. In some studies, specific subproteomes in the first dimension and molecular mass in the second
are isolated by affinity chromatography and analyzed for dimension. Despite being labor-intensive and time
their protein expression. This could include the isolation consuming, the technique has become a popular choice
of protein complexes or the use of protein ligands to among researchers mainly due to its high resolving power
isolate specific types of proteins or glycoproteins. This and reproducibility. The technique has the ability to
approach, which seems to have attracted more interest in resolve proteins that have undergone some form of post-
recent years, allows a selected group of proteins to be translational modification like phosphorylation [18] or
analyzed and characterized in their native forms, and can glycosylation [19]. The primary application of 2-DE is
provide important information about disease mechanisms, in protein expression profiling. The appearance and
protein signaling or protein-drug interactions. disappearance of spots, usually detected by subjecting
the gels to silver staining, provide information on
More recently, the use of protein microarrays as an differential protein expression while the intensity of the
approach in functional proteomics has been explored [16]. spots provides quantitative information on protein
Microarrays, generated by spotting biomolecules on a expression levels.
solid surface at high spatial density, offer miniaturized,
robust platforms for high-throughput study of proteins. Although 2-DE is generally performed using a standard
Two different forms of protein microarrays have been protocol, modifications may be required depending on
described. The abundance-based microarrays measures samples to be analyzed. Analysis by 2-DE generally
the abundance of specific biomolecules using analyte- requires samples that are denatured, free of contaminants
specific reagents such as antibodies, while the function- (mainly salt) and at optimal loading concentration.
based microarrays examines protein functions by printing Denaturation of proteins is important so as to prevent
a collection of target proteins on the array surface and oxidation, aggregation and protein-protein interaction.
assessing their interactions and biochemical activities. Under native conditions, proteins exists in different
conformations, may aggregate and interact with other
A good example of functional proteomics is the use of proteins and therefore cannot be analyzed by 2-DE. Too
protein microarrays in the form of chip-based antibodies much salt in the sample disturbs isoelectric focusing and
to screen labeled sera from patients with prostate cancer leads to streaky patterns. For comparative purposes, all
for potential biomarkers [17]. A capture microarray samples are to be kept and treated similarly as proteins
containing 184 antibodies targeting serum proteins, are continuously degraded and the rate of degradation of
proteins previously detected in sera of cancer patients protein varies at different conditions.
and intracellular proteins were used. From this, five
proteins were demonstrated to be able to discriminate Despite many improvements, 2-DE is still limited in
prostate cancer serum from control. Four of these serum its ability to resolve proteins that are large, hydrophobic
proteins were those that had been previously associated or those of extreme acidity or basicity [20]. A eukaryotic
with prostate cancer. cell lysate protein mixture may be too complexed to be
Overview of proteomics 4

completely resolved in a single 2-D gel [21]. Similarly, restrictive and cannot be applied on proteins that are N-
it is also impossible to resolve all proteins of samples terminally blocked. Nevertheless, N-terminal sequencing
with broad dynamic range, like plasma [22], in a single is still a viable alternative to mass spectrometry despite
2-DE gel. Due to these limitations of 2-DE, several its waning application. In addition, an alternative protocol
alternative approaches have been used. One approach is for the sequencing of proteins, in the form of mixed
to subject the entire protein sample mixture to trypsin peptide sequencing, has been developed for protein
digestion and then purify the peptides before subjecting identification purposes [26].
them to analysis by mass spectrometry. Several methods
have been used to purify the tryptic peptides including Before analysis using a mass spectrometer, proteins
liquid chromatography [23], capillary electrophoresis [24] are initially subjected to peptide cleavage, usually with
and a combination of ion-exchange chromatography and trypsin (Fig. 2). For proteins that are separated using gel
reverse-phase chromatography [25]. However, these electrophoresis, trypsin digestion is usually performed
methods require considerable effort, time and computing “in-gel” [27] as it is more efficient for sample recovery
power for data analysis. than other methods like electroblotting or electroelution
[28]. Before being analyzed by mass spectrometry, the
Identification of Proteins peptide fragments recovered following in-gel digestion
need to be concentrated and purified to remove
Progress in peptide analysis using mass spectrometry
contaminants such as salts, buffers and detergents, which
has advanced very rapidly in recent years. It is currently
interfere with mass spectrometric analysis [29]. This is
the state of the art technique used in the identification of
usually performed using miniaturized chromatography
proteins in proteomics analysis. Prior to the use of mass
columns in the form of ZipTips, which purify proteins on
spectrometry, proteins were identified by Western blotting
the basis of reverse-phase, ion exchange or affinity
and binding recognition using specific antibodies or on
chromatography principles. Alternatively the samples
the basis of their N-terminal sequences or Edman
can also be subjected to high performance liquid
sequencing. While the earlier technique is dependent
chromatography.
on availability of antibodies, the latter technique is

protein

trypsin digestion

(b) ESI-MS/MS
peptide fragments

(a) MALDI-TOF

m/z
b-ions 116 229 342 429 528 643 806 907 1022

D I L S V D Y T D I
Database Search 12781184 1071 984 885 770 607 506 391 y-ions

Figure 2: Mass spectrometric identification of proteins. Analyses of tryptic peptide fragments by (a) MALDI-TOF and (b)
ESI-MS/MS generate data in the form of peptide mass fingerprints and sequence of amino acids, respectively.
Proteins may be identified by search of the peptide mass fingerprints or amino acid sequence in protein
databases (Table 1).
Overview of proteomics 5

Analysis of the concentrated peptide fragments by collection and analysis can be automated. As such,
mass spectrometry generates data in the forms of peptide MALDI is more suitable for ionization of peptide samples
mass fingerprints or amino acid sequences. In peptide in high throughput studies. Lately, the use of surface-
mass fingerprinting, a mass fingerprint or map of peptides enhanced laser desorbtion/ionization (SELDI) in
is derived from mass spectrometric analysis, e.g., by proteomics analysis is also becoming popular [32]. Unlike
using MALDI-TOF, of the trypsin-digested peptides (Fig. MALDI or ESI, SELDI is usually used to analyze complex
2a). By searching protein databases, the molecular masses protein mixtures directly without the need of sample
of this set of peptides are then measured and compared preparation and purification steps.
against the theoretical molecular masses of known proteins
cleaved with the same protease in the database. Subsequent to their conversion to molecular ions,
Identification is achieved when many of the molecular peptides are then subjected to mass analysis using the
masses match. It is often not to find matches for all the mass analyzers in mass spectrometers. Analysis is usually
peptides as some may have been covalently modified accomplished on the basis of the mass-to-charge (m/z)
(e.g., glycopeptide) or resisted cleavage. ratio of a peptide ion in vacuum. There are currently
three main types of mass analyzers. The time-of-flight
In the second approach, a tandem mass spectrometer (TOF) instrument is among the simplest mass analyzers,
(MS/MS), which combines two mass analyzers or two which measures the m/z ratio of an ion by measuring the
mass analysis steps, is indirectly used to determine the time required for it to traverse the length of a flight tube
amino acid sequence of a peptide. In this approach, e.g., [29]. The quadrupole analyzer consists of four parallel
by using ESI-MS/MS, a peptide ion is further activated metal rods through which the gas phase ions have to
using a gas-phase collision to break peptide bonds creating achieve a stable trajectory. The analyzer is operated by
a ladder of fragment ions (Fig. 2b). Peptide fragmentation the application of a voltage to create an electric field that
which maintains the charge on the N-terminus is acts to transmit all ions or as mass filter to allow the
designated b-ion, whereas fragmentation which maintains transmission of a certain m/z ratio [33]. If multiple
the charge on the C-terminus is designated a y-ion. The quadrupoles are combined, they can be used as a tandem
fragment ions produced in the dissociation of peptides mass spectrometer to obtain information on the amino
reflect the sequence of amino acids and thus can be used acid sequence of a peptide. Quadrupoles can also be
to search theoretical sequence databases. Table 1 combined with the TOF mass analyzers to generate the
demonstrates some of the common databases available in hybrid type quadrupole-TOF tandem mass spectrometer
the internet that can be searched for peptide mass [34]. However, even more sensitive and accurate are the
fingerprints or sequencing. ion trap mass analyzers (e.g., Fourier transform ion
cyclotron resonance mass spectrometer), which have the
The two main stages involved in mass spectrometry ability to store ions and then selectively eject them from
are sample ionization and mass analysis. Mass the ion trap unlike the quadrupole mass analyzer in which
spectrometry requires protein samples to be charged and ions are discarded before analysis begins [35,36]. These
in dry form. For this purpose, the highly concentrated, mass analyzers also have the unique ability to perform
purified and digested protein samples are initially multiple stages of mass spectrometry.
converted to desolvated ions either by matrix-assisted
laser desorbtion/ionization (MALDI) or electrospray
ionization (ESI). In MALDI, the sample is incorporated Quantification of Proteins
into matrix molecules and then subjected to irradiation In 2-DE, in order to compare the expression of proteins
by a laser [30], while in ESI, the formation of desolvated between different samples, gels are often scanned and
ions occur when a liquid sample evaporates as it flows subjected to image analysis. The evaluation and
through a microcapillary tube that is induced with a comparison of the complex 2-DE profiles with the eye is
potential difference [31]. The biggest advantage of impossible. For a proper evaluation, it is important to
MALDI is that sample application can be performed acquire the image as a grey-scale TIFF file with adequate
using a robot so that the entire process including data resolution. Gels with visible spots have to be scanned in

Table 1: Common databases for peptide search in the internet.

Site name URL Information

MASCOT www.matrixscience.com Peptide mass fingerprinting

PeptIdent www.expasy.ch/tools/peptident &
BLAST www.ncbi.nlm.nih-gov/BLAST Sequencing
PepFrag www.proteometrics.com
FindMod www.expasy.ch/tools/findmod Post-translational modification
Overview of proteomics 6

transmission mode while blot membranes are scanned in these advancements, proteomics technology is still
reflectance mode. Dedicated scanners are required to restrictive in several aspects. Unlike genomics, proteomics
scan proteins that are labeled with fluorescent dyes or lacks a “PCR-like” technique, which could be useful for
radioisotopes. studies on proteins of low abundance. Many important
proteins that may be targets for drugs such as transcription
Quantification of individual protein spots can be factors, regulatory proteins and protein kinases are low-
performed by using computerized image analysis. Many copy proteins. Proteomics analysis is generally dependent
image analysis softwares are currently available. on theoretical databases that are derived mainly from
PDQuest, ImageMaster, MELANIE and Progenesis are genomics studies and these are not always available for
among the common softwares widely used by researchers. certain organisms and plants. The existing reference
All of these softwares have the capability to quantify the proteome maps are also not complete. For example,
expression of peptide spots in the form of volume (i.e., quite a number of proteins that are detected in the human
optical density x area in square mm of the region) plasma, which are provided with primary accession
subsequent to a background intensity correction. Some numbers, are yet to be identified and validated.
are sophisticated enough to match gels, correct gel Proteomics is also faced with problems like post-
distortion and perform automated quantification of translational modifications of proteins and the analysis of
annotated proteins. Also currently available is software proteomes with a broad dynamic range of proteins (e.g.
solely meant for the quantitative image analysis of 2-DE plasma proteins has an estimated concentration range of
gels that simultaneously separate two distinct protein ten orders of magnitude [22]) although substantial progress
samples that are labeled with different spectrally has been made to tackle the problems. Despite these
resolvable fluorescent dyes [37]. Because samples are shortcomings and within the boundaries of its limitations,
distinctively labeled, differential expression of proteins proteomics remains a major impact to scientists at the
can be detected directly using this method. Besides turn of this century mainly because of its role in
computerized image analysis, quantification can also be enlightening the basis of life sciences in terms of proteins
performed using mass spectrometers [38]. However, this expression and interaction, and this cannot be obtained
requires the creation of internal standards, which must be from the study of individual or groups of proteins.
chemically similar to the molecule that is to be measured
by mass spectrometry, for each molecule to be measured.
Acknowledgement
The author would like to thank Ms Puteri Shafinaz
Challenges for Proteomics Abdul Rahman from the Department of Molecular
The last decade has seen an explosion of data derived Medicine, Faculty of Medicine, University of Malaya,
from proteomics research as well as tremendous for helpful discussion during preparation of the
improvements in the technology of proteomics. Despite manuscript.

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