Proteomics: Principles, Techniques and

Applications
Article Published: December 9, 2020 | Natasha Beeton-Kempen, PhD

Credit: Technology Networks

Contents

What is the proteome?

What is proteomics?

What are the key questions that proteomics can answer?

Proteomics: Techniques
 - Low-throughput methods:

• 1. Antibody-based methods
• 2. Gel-based methods
• 3. Chromatography-based methods

- High-throughput methods:

• 1. Analytical, functional and reverse-phase microarrays

• 2. Mass spectrometry-based proteomics

Mass spectrometry workflow

What is tandem MS?

Top-down proteomics vs. bottom-up proteomics

Data analysis in proteomics

Applications of proteomics

The future of proteomics

References

What is the proteome?

Proteins are biological molecules made up of building blocks called amino acids.
Proteins are essential to life, with structural, metabolic, transport, immune,
signaling and regulatory functions among many other roles.1

The term “proteome” was coined by an Australian PhD student, Marc Wilkins, in a
1994 symposium held in Siena, Italy.2 It is a blanket term that refers to all of the
proteins that an organism can express. Each species has its own, unique proteome.
Unlike the genome (the complete set of genes within each organism), the
composition of the proteome is in a constant state of flux over time and
throughout the organism.3 Therefore, when scientists refer to the proteome, they
are also sometimes referring to the proteome at a given point in time (such as the
embryo versus the mature organism), or to the proteome of a particular cell type
or tissue within the organism.

Figure 1: There are approximately 20,000 genes in the human genome, ~100,000
transcripts in the human transcriptome and over >1000000 proteoforms in the
human proteome. Credit: Technology Networks

What is proteomics?

Proteomics is the study of the proteome – investigating how different proteins

interact with each other and the roles they play within the organism.4
Although protein expression can be inferred by studying the expression of mRNA,
which is the middle man between genes and proteins, mRNA expression levels do
not always correlate well with protein expression levels.1,3 Furthermore, the study
of mRNA does not consider protein posttranslational modifications, cleavage,
complex formation and localization, or the many variant mRNA transcripts that can
be produced; all of which are key to protein function.

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The first experiments that fit the label of “proteomic” studies were performed in
1975 with the development of 2D protein electrophoresis.5

However, truly high-throughput identification of multiple proteins per sample only

became possible with the development of mass spectrometry (MS) technology
over 20 years later.6

Since then, the sensitivity and accuracy of MS have advanced to the point where
proteins can be reliably detected down to the attomolar range (1 target protein
molecule per 1018 molecules),7 and various other proteomic techniques have been
developed and optimized.

What are the key questions that proteomics can answer?

Broadly speaking, proteomic research provides a global view of the processes

underlying healthy and diseased cellular processes at the protein level.3,4 To do
this, each proteomic study typically focuses on one or more of the following
aspects of a target organism’s proteome at a time to slowly build on existing
knowledge:

Protein
Which proteins are normally expressed in a particular cell type, tissue or
identificatio
organism as a whole, or which proteins are differentially expressed?
n

Protein Measures total (“steady-state”) protein abundance, as well as investigating

quantificati the rate of protein turnover (i.e., how quickly proteins cycle between being
on produced and undergoing degradation).

Where a protein is expressed and/or accumulates is just as crucial to

Protein protein function as the timing of expression, as cellular localization controls
which molecular interaction partners and targets are available.
localization

Post-
translationa Post-translational modifications can affect protein activation, localization,
l stability, interactions and signal transduction among other protein
modificatio characteristics, thereby adding a significant layer of biological complexity.
ns

This area of proteomics is focused on identifying the biological functions of

Functional
specific individual proteins, classes of proteins (e.g., kinases) or whole
proteomics
protein interaction networks.

Structural Structural studies yield important insights into protein function, the
proteomics “druggability” of protein targets for drug discovery, and drug design.

Protein-
Investigates how proteins interact with each other, which proteins interact,
protein
and when and where they interact.
interactions

Proteomics: Techniques

Low-throughput methods:
1. Antibody-based methods

Techniques such as ELISA (enzyme-linked immunosorbent assay) and western

blotting rely on the availability of antibodies targeted toward specific proteins or
epitopes to identify proteins and quantify their expression levels.

2. Gel-based methods

Two-dimensional gel electrophoresis (2DE or 2D-PAGE), the first proteomic

technique developed, uses an electric current to separate proteins in a gel based
on their charge (1st dimension) and mass (2nd dimension). Differential gel
electrophoresis (DIGE) is a modified form of 2DE that uses different fluorescent
dyes to allow the simultaneous comparison of two to three protein samples on the
same gel. These gel-based methods are used to separate proteins before further
analysis by e.g., mass spectrometry (MS), as well as for relative expression
profiling.

3. Chromatography-based methods

Chromatography-based methods can be used to separate and purify proteins from

complex biological mixtures such as cell lysates. For example, ion-exchange
chromatography separates proteins based on charge, size exclusion
chromatography separates proteins based on their molecular size, and affinity
chromatography employs reversible interactions between specific affinity ligands
and their target proteins (e.g., the use of lectins for purifying IgM and IgA
molecules). These methods can be used to purify and identify proteins of interest,
as well as to prepare proteins for further analysis by e.g., downstream MS. 8
High-throughput methods:

1. Analytical, functional and reverse-phase microarrays

Protein microarrays apply small amounts of sample to a “chip” for analysis (this is
sometimes in the form of a glass slide with a chemically modified surface). Specific
antibodies can be immobilized to the chip surface and used to capture target
proteins in a complex sample. This is termed an analytical protein microarray, and
these types of microarray are used to measure the expression levels and binding
affinities of proteins in a sample. Functional protein microarrays are used to
characterize protein functions such as protein–RNA interactions and enzyme-
substrate turnover. In a reverse-phase protein microarray, proteins from e.g.,
healthy vs. diseased tissues or untreated vs. treated cells are bound to the chip,
and the chip is then probed with antibodies against the target proteins.

Figure 2: The differences between forward phase and reverse phase protein
microarrays. Credit: Technology Networks.
2. Mass spectrometry-based proteomics

There are several “gel-free” methods for separating proteins, including isotope-
coded affinity tag (ICAT), stable isotope labeling with amino acids in cell culture
(SILAC) and isobaric tags for relative and absolute quantitation (iTRAQ). These
approaches allow for both quantitation and comparative/differential proteomics.
There are also other, less quantitative techniques such as multidimensional
protein identification technology (MudPIT), which offer the advantages of being
faster and simpler. Other gel-free, chromatographic techniques for protein
separation include gas chromatography (GC) and liquid chromatography (LC). 8,9

Mass spectrometry workflow

Regardless of how the protein sample is separated, the downstream MS workflow

comprises three main steps:

1. The proteins/peptides are ionized by the ion source of the mass spectrometer.

2. The resulting ions are separated according to their mass to charge ratio by the
mass analyze.

3. The ions are detected.

When using gel-free techniques upstream of MS such as iTRAQ or SILAC, the

samples are used directly for input into the mass spectrometer. When using gel-
based techniques, the protein spots are first cut out of the gel and digested before
being either separated by LC or directly analyzed by MS.

There are two main ionization sources, namely:

• Matrix-assisted laser desorption/ionization (MALDI)

 • Electrospray ionization (ESI)

Figure 3: The two main ionization sources in MS-based proteomics. Credit:

Technology Networks.

Other, less common sources include chemical ionization, electron impact and glow
discharge ionization.

There are four main mass analyzers:

• Time-of-flight (TOF)
• Ion trap
• QuadrupoleFourier-transform ion cyclotron (FTIC)
• Electrostatic sector and magnetic sector are two other, less commonly
adopted mass analyzers.
What is tandem MS?

Peptides can be subjected to multiple rounds of fragmentation and mass analysis –

a process termed tandem-MS, MS/MS or MSn. By combining the same or different
mass analyzers in tandem, such as quadrupole-TOF (Q-TOF) or triple-quadrupole
(QQQ) MS, the strengths of different mass analyzers can be leveraged to further
improve the capacity for proteome-wide analysis. Simple MS setups such as
MALDI-TOF are only used for peptide mass measurements, whereas tandem mass
spectrometers are used to determine peptide sequences.

Top-down proteomics vs. bottom-up proteomics

In top-down proteomics, the proteins in a sample of interest are first separated

before being individually characterized.1,10

With bottom-up proteomics – also termed “shotgun” proteomics – all the proteins
in the sample are first digested into a complex mixture of peptides, and these
peptides are then analyzed to identify which proteins were present in the
sample.1,10

Appro
Description Method
ach
 Protein separation is performed based on mass and charge
with e.g., 2DE, DIGE or MS. When using 2D electrophoresis
Proteins in a
techniques, the proteins are first resolved on the gel and
Top- sample of interest
then individually digested into peptides that are analyzed by
down are first separated
a mass spectrometer. When using MS directly, the
proteo before being
undigested sample containing the whole proteins is injected
mics individually
into the mass spectrometer, the proteins are separated, and
characterized.
individual proteins are then selected for digestion and a
further round of MS for analysis of the peptides.

Proteins are first digested, and the digested peptide mixture

All the proteins in
Botto is fractionated and subjected to MS, frequently in an LC-
the sample are first
m-up MS/MS configuration. The resulting peptide sequences are
digested into a
proteo compared to existing databases using automated search
complex mixture of
mics, algorithms. These search engines match the experimentally
peptides, and these
or obtained peptide spectra to the predicted spectra of
peptides are then
"shotg proteins produced by in silico digestion (this is called
analyzed to identify
un “peptide-spectrum matching”). There are several
which proteins
proteo different bottom-up workflows possible, including data-
were present in the
mics" dependent and data-independent methods, as well as
sample.
hybrids of these.

Both the top-down and bottom-up approaches have their own set of advantages
and disadvantages and applications to which each is more suited.10,11 For example,
top-down MS is more appropriate for research on different PTMs and protein
isoforms. However, it is limited by difficulties inherent in separating complex
mixtures of proteins and the decreasing sensitivity of MS toward larger proteins
(particularly > 50 to 70 kDa).1

In contrast, while the peptides used in bottom-up MS (~5 to 20 amino acids in

length) are much easier to fractionate, ionize and fragment, this approach provides
an indirect measure of the proteins originally present in samples and relies heavily
on inference.1 A hybrid “middle-down” approach has been developed, which
employs larger peptide fragments than conventional bottom-up proteomics,
thereby potentially allowing more unique peptide matches.
Figure 4: The differences between top-down and bottom-up proteomics. Credit:
Technology Networks.

Data analysis in proteomics

Proteomic studies, particularly those employing high-throughput technologies, can

generate huge amounts of data.12 In addition to the sheer quantity of data
produced, proteomic data analysis can also be relatively complex for certain
techniques such as shotgun MS.13 Adding to this complexity is the range
of bioinformatics tools available for proteomic analyses.14-17
Listicle

Data-dependent vs. Data-independent Proteomic Analysis

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Proteomic researchers are faced with many hurdles when attempting to optimize
how they warehouse and analyze their proteomic data.12

When planning proteomic experiments, scientists need to factor in not only the
costs of the reagents and laboratory equipment but also that of data storage and
analysis, and they have to appraise the level of bioinformatics skills and
computational resources required.

Proteomic studies often require multiple data processing and analysis steps that
need to be performed in a specific sequence.12 To address this need, researchers
are increasingly assembling the needed scripts, tools and software into
customized proteomic analysis pipelines suited to their particular research
questions.
Applications of proteomics

The applications of proteomics are incredibly numerous and varied. The table
below lists just some of these applications and provides links to examples of
studies using these approaches.

Proteomi
cs
Description and examples
applicati
on

Tailoring disease treatment to each patient based on their genetic and

Personali epigenetic makeup, so as to improve efficacy and reduce adverse effects.
zed While genomics and transcriptomics have been the main focus of such studies
medicine to date, proteomics data will likely add a further dimension for patient-specific
management.

Biomark Identification of protein markers e.g., for the diagnosis and prognosis
er of glioblastoma, and evaluating patients’ response to therapeutic interventions
discovery such as stem cell transplantation.

Drug
discovery Identifying potential drug targets, examining the druggability of selected
and protein targets, and developing drugs aimed at candidate therapeutic protein
develop targets (e.g., for hepatocellular carcinoma).
ment

System-wide investigations of disease pathways and host–pathogen

Systems interactions to identify potential biomarkers and therapeutic targets; system-
wide investigations of drug action, toxicity, resistance and efficacy.
biology

Agricultu Investigations of plant–pathogen interactions, crop engineering for increased

re resilience to e.g., flooding, drought and other environmental stresses.

Food Food safety and quality control, allergen detection and improving the
science nutritional value of foods.

Paleopro The study of ancient proteins to further our understanding

teomics of evolution and archeology.

Investigations of how mammals’ immune systems may respond to exo-

Astrobiol
microbes found in space and studies of the prebiotic organic matter found on
ogy
meteorites.

The future of proteomics

Currently, proteomic workflows rely heavily on MS.1 As powerful as this technology

has proven, researchers are now looking ahead to a future for proteomics that lies
“beyond MS.” Despite the attomolar sensitivity of MS, millions of the target
molecule still need to be present in the sample for it to be detected. This implies
that low-concentration target molecules (e.g., serum biomarkers) can be
undetectable in complex milieu such as human serum unless first enriched for.

Scientists are still searching for the holy grail of high-throughput proteomic
techniques that 1) has excellent sensitivity across the dynamic range of the target
proteome (e.g., 107 for the human proteome), 2) can directly read entire protein
sequences and identify their PTMs, and, therefore, 3) does not need to draw
inferences from databases of theoretical protein matches.1

There are several promising technologies that, while currently hampered by

limitations in sensitivity, throughput or cost, may yet come to dominate the
proteomic field.1 These include nascent fluorescent fingerprinting methods and
yet-to-be-developed subnanopore arrays for the high-throughput single-molecule
sequencing of proteins.

Along with the expected advances in proteomic techniques, approaches to

proteomic data analysis are expected to evolve just as rapidly. For example, there
is a strong impetus towards developing data technologies such as cloud
computing, software containers and workflow systems, which will “democratize”
access to top-notch computing resources for proteomic data analysis regardless of
researchers’ location, IT infrastructure or computational expertise.12,18,19

References
(Click to expand)

References:

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