Proteomics is the study of the entire set of proteins produced by a cell type, examining how proteins interact with cellular processes and the environment. It aims to highlight differences in proteomes under various conditions, with applications in cancer research for early detection and individualized treatment plans. Key techniques in proteomics include mass spectrometry, x-ray crystallography, nuclear magnetic resonance, and protein microarrays to analyze protein structure and functionality.

1. Proteomics
• Proteomics is the study of the entire set of proteins
produced by a cell type in order to understand its
structure and function.
Key Points
• Proteomics investigates how proteins affect and are
affected by cell processes or the external environment.
• Within an individual organism, the genome is constant,
but the proteome varies and is dynamic.
• Every cell in an individual organism has the same set of
genes, but the set of proteins produced in different
tissues differ from one another and are dependent on gene
expression. Dr. Shiny C Thomas, Department of Biosciences, ADBU

2. Proteomics: the branch of molecular biology that studies
the set of proteins expressed by the genome of an
organism
Proteome: the complete set of proteins encoded by a
particular genome
Genomics: the study of the complete genome of an
organism

4. • Studying proteins generates insight into how they affect
cell processes.
• This study also investigates how proteins themselves are
affected by cell processes or the external environment.
• Proteins provide intricate control of cellular machinery;
they are, in many cases, components of that same
machinery.
• They serve a variety of functions within the cell; there are
thousands of distinct proteins and peptides in almost
every organism.

5. • The goal of proteomics is to analyze the varying
proteomes of an organism at different times in order to
highlight differences between them.
• Put more simply, proteomics analyzes the structure and
function of biological systems. For example, the protein
content of a cancerous cell is often different from that of
a healthy cell.
• Certain proteins in the cancerous cell may not be present
in the healthy cell, making these unique proteins good
targets for anti-cancer drugs.

6. • The realization of this goal is difficult; both purification
and identification of proteins in any organism can be
hindered by a multitude of biological and environmental
factors.
• The study of the function of proteomes is called
proteomics.
• A proteome is the entire set of proteins produced by a
cell type.
• Genomics led to proteomics (via transcriptomics) as a
logical step. Proteomes can be studied using the
knowledge of genomes because genes code for mRNAs
and the mRNAs encode proteins.
• Although mRNA analysis is a step in the right direction,
not all mRNAs are translated into proteins.

7. • Proteomics complements genomics and is useful when
scientists want to test their hypotheses that were based on
genes.
• Even though all cells of a multicellular organism have
the same set of genes, the set of proteins produced in
different tissues is different and dependent on gene
expression.
• Thus, the genome is constant, but the proteome varies
and is dynamic within an organism.
• In addition, RNAs can be alternately spliced (cut and
pasted to create novel combinations and novel proteins)
and many proteins are modified after translation by
processes such as proteolytic cleavage, phosphorylation,
glycosylation, and ubiquitination.

8. • There are also protein-protein interactions, which
complicate the study of proteomes.
• Although the genome provides a blueprint, the final
architecture depends on several factors that can change
the progression of events that generate the proteome.

9. The context of proteomics is systems biology, rather than structural
biology. In other words, the point of proteomics is to characterize the
behaviour of the system rather than the behaviour of any single
component.

10. Basic Techniques in Protein Analysis
• The basic techniques used to analyze proteins are mass
spectrometry, x-ray crystallography, NMR, and protein
microarrays.
Key Points
• Mass Spectrometry is a technique that is useful for
determining the size of a protein or protein complex.
• X-ray crystallography and NMR are techniques useful for
determining the 3-D structure of a protein or protein
complex.
• Protein microarrays are useful for determining protein-
protein interactions.

11. Basic Techniques in Proteome Analysis
• The ultimate goal of proteomics is to identify or
compare the proteins expressed in a given genome
under specific conditions, study the interactions
between the proteins, and use the information to
predict cell behaviour or develop drug targets.
• Just as the genome is analyzed using the basic technique
of DNA sequencing, proteomics requires techniques
for protein analysis.
• The basic technique for protein analysis, analogous to
DNA sequencing, is mass spectrometry.

12. Mass Spectrometry
• Mass spectrometry is used to identify and determine the
characteristics of a molecule.
• It is a technique in which gas phase molecules are
ionized and their mass-to-charge ratio is measured by
observing acceleration differences of ions when an
electric field is applied.
• Lighter ions will accelerate faster and be detected first.
• If the mass is measured with precision, then the
composition of the molecule can be identified.
In the case of proteins, the sequence can be identified.
•

13. • The challenge of techniques used for proteomic analyses
is the difficulty in detecting small quantities of proteins,
but advances in spectrometry have allowed researchers
to analyze very small samples of protein.
• Variations in protein expression in diseased states,
however, can be difficult to discern.
• Proteins are naturally-unstable molecules, which makes
proteomic analysis much more difficult than genomic
analysis.

14. X-ray crystallography and Nuclear Magnetic Resonance
• X-ray crystallography enables scientists to determine the
three-dimensional structure of a protein crystal at
atomic resolution.
• Crystallographers aim high-powered X-rays at a tiny
crystal containing trillions of identical molecules.
• The crystal scatters the X-rays onto an electronic
detector that is the same type used to capture images in
a digital camera.

15. • After each blast of X-rays, lasting from a few seconds to
several hours, the researchers precisely rotate the
crystal by entering its desired orientation into the
computer that controls the X-ray apparatus.
• This enables the scientists to capture in three
dimensions how the crystal scatters, or diffracts, X-rays.
• The intensity of each diffracted ray is fed into a
computer, which uses a mathematical equation to
calculate the position of every atom in the crystallized
molecule.
• The result is a three-dimensional digital image of the
molecule.

16. NMR
• Another protein imaging technique, nuclear magnetic
resonance (NMR), uses the magnetic properties of atoms to
determine the three-dimensional structure of proteins.
• MR spectroscopy is unique in being able to reveal the
atomic structure of macromolecules in solution,
provided that highly concentrated solution can be
obtained.
• This technique depends on the fact that certain atomic
nuclei are intrinsically magnetic.

17. • The chemical shift of nuclei depends on their local
environment.
• The spins of neighboring nuclei interact with
each other in ways that provide definitive structural
information that can be used to determine complete three
dimensional structures of proteins.

18. Protein Microarrays and Two- Hybrid Screening
• Protein microarrays have also been used to study
interactions between proteins.
• These are large-scale adaptations of the basic two-
hybrid screen.
• The principle behind the two-hybrid screen is that most
eukaryotic transcription factors have modular activating
and binding domains that can still activate transcription
even when split into two separate fragments, as long as
the fragments are brought within close proximity to
each other.

19. • Generally, the transcription factor is split into a
DNA-binding domain (BD) and an activation domain (AD).
• One protein of interest is genetically fused to the BD
and another protein is fused to the AD.
• If the two proteins of interest bind each other, then the
BD and AD will also come together and activate a
reporter gene that signals interaction of the two hybrid
proteins.

20. The yeast genome on a chip

21. Western Blot
• The western blot, or protein immunoblot, is a technique
that combines protein electrophoresis and antibodies to
detect proteins in a sample.
• A western blot is fairly quick and simple compared to
the above techniques and, thus, can serve as
an assay to validate results from other experiments.
• The protein sample is first separated by gel
electrophoresis, then transferred to a nitrocellulose or
other type of membrane, and finally stained with a
primary antibody that specifically binds the protein of
interest.

22. • A fluorescent or radioactive-labeled secondary antibody
binds to the primary antibody and provides a means of
detection via either photography or x-ray film,
respectively.
Cancer Proteomics
Proteomics, the analysis of proteins, plays a prominent role
in the study and treatment of cancer.

23. Cancer Proteomics
• Genomes and proteomes of patients suffering from
specific diseases are being studied to understand the
genetic basis of diseases.
• The most prominent set of diseases being studied with
proteomic approaches is cancer.
• Proteomic approaches are being used to improve
screening and early detection of cancer, which is
achieved by identifying proteins whose expression is
affected by the disease process.

24. • An individual protein that indicates disease is called a
biomarker, whereas a set of proteins with altered
expression levels is called a protein signature.
• For a biomarker or protein signature to be useful as a
candidate for early screening and detection of a cancer, it
must be secreted in body fluids (e.g. sweat, blood, or
urine) such that large-scale screenings can be performed
in a non-invasive fashion.
• The current problem with using biomarkers for the early
detection of cancer is the high rate of false-negative
results.

25. • A false-negative is an incorrect test result that should
have been positive.
• In other words, many cases of cancer go undetected,
which makes biomarkers unreliable.
• Some examples of protein biomarkers used in cancer
detection are CA-125 for ovarian cancer and PSA for
prostate cancer.
• Protein signatures may be more reliable than biomarkers
to detect cancer cells.

26. Key Points
• Identifying those proteins whose expression is affected
by disease processes can be used to improve screening
and early detection of cancer.
• Different biomarkers and protein signatures are being
used to analyze each type of cancer. (biomarker: a
substance used as an indicator of a biological state, most
commonly disease)
• A future goal of cancer proteomics is to have a
personalized treatment plan for each individual.
Explain the ways in which cancer proteomics may lead to better
treatments

27. • Proteomics is also being used to develop individualized
treatment plans, which involves the prediction of
whether or not an individual will respond to specific
drugs and the side effects that the individual may
experience.
• In addition, proteomics can be used to predict the
possibility of disease recurrence.
• The National Cancer Institute has developed programs
to improve the detection and treatment of cancer.
• The Clinical Proteomic Technologies for Cancer and the
Early Detection Research Network are efforts to identify
protein signatures specific to different types of cancers.

28. • The Biomedical Proteomics Program is designed to
identify protein signatures and design effective
therapies for cancer patients.

29. Tools of Proteomics
• The first tool is the database.
• Protein, EST, and complete genome sequence
databases collectively provide a complete catalog of all
proteins expressed in organisms for which the databases
are available.
• Based on analyses of all the coding sequences for
Drosophila, for example, we know that there are 110
Drosophila genes that code for proteins with EGF-like
domains and 87 genes that code for proteins with tyrosine
kinase catalytic domains.

30. • Accordingly, when doing proteomics in Drosophila, we
are searching a large, but known index of possible
proteins.
• When searched with limited sequence information or
even raw mass spectral data (see below), we can identify
a protein component from a match with a database
entry.

31. The second tool is mass spectrometry (MS).
• MS instrumentation can offer three types of analyses
• First, MS can provide accurate molecular mass
measurements of intact proteins as large as 100 kDa or
more.
• migration on sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) is the best way to
estimate protein masses
MS also can provide accurate mass measurements of
peptides from proteolytic digests.
The data from these peptide mass measurements
can be searched directly against databases, frequently to
obtain definitive identification of the target proteins.

32. • Finally, MS analyses can provide sequence analysis
of peptides obtained from proteolytic digests.
• MS sequence data provide the most powerful and
unambiguous approach to protein identification.

33. The third essential tool for proteomics is an emerging
collection of software that can match MS data with
specific protein sequences in databases.
• it is possible to determine the sequence of a peptide
from MS data.
• However, this de novo sequence interpretation is a
relatively laborious task, particularly when one has to
interpret hundreds or thousands of spectra.
•
• These software tools take uninterpreted MS data and
match it to sequences in protein, EST, and genome-
sequence databases with the aid of specialized
algorithms.

34. The fourth essential tool in proteomics is analytical
protein-separation technology.
• Protein separations serve two purposes in proteomics.
• First, they simplify complex protein mixtures by
resolving them into individual proteins or small
groups of proteins.
• Second, because they also permit apparent
differences in protein levels to be compared between
two samples, protein analytical separations allow
investigators to target specific proteins for analysis.

35. • Two-dimensional SDS-PAGE (2D-SDS-PAGE) is most
widely associated with proteomics.
• Two-dimensional gels represent perhaps the best single
technique for resolving proteins in a complex sample.
• However, other protein separation techniques, including
1D-SDS-PAGE, high-performance liquid chromatography
(HPLC), capillary electrophoresis (CE), isoelectric
focusing (IEF), and affinity chromatography all can be useful
tools in analytical proteomics.
• Perhaps most powerful is the integration of different
protein and peptide separations as multidimensional
techniques.

36. • For example, ion-exchange liquid chromatography (LC) in
tandem with reverse-phase (RP)-HPLC is a powerful tool for
resolving complex peptide mixtures.
• It is the integration of these four tools that provides the
current technology of proteomics.

37. Analytical Protein
and Peptide Separations
• First convert proteins to peptides. This is generally done
with proteolytic enzymes.
• Second, we must separate very complex mixtures of
proteins or peptides into somewhat less complex
mixtures.
• This gives MS instruments a better opportunity
to obtain useful data on the components of the mixture.
• The MS instruments used to obtain data on peptides are
capable of extracting a great deal of information from
relatively complex mixtures.

39. How many different proteins and peptides
we may be dealing with in a typical proteomic analysis?
• Based on the number of known human genes, a
typical human cell may contain about 20,000 different
expressed proteins.
• If we assume that they average about 50 kDa and
contain average numbers of lysine and arginine
residues, then each would yield about 30 tryptic
peptides.
• Thus, one cell’s proteins would yield about 6,000,000
tryptic peptides.

40. Extracting Proteins from Biological Samples
• In any real study, we start with a biological sample: a
piece of tissue, a plate of cultured cells, a flask of
bacteria, a leaf, and so on.
• The sample then is usually pulverized, homogenized,
sonicated, or otherwise disrupted to yield a soup that
contains cells, subcellular components, and other
biological debris in an aqueous buffer or suspension.
• Proteins are extracted from this soup by a number of
techniques.

41. • For proteomic analysis, the objective here is to recover
as much of the protein as possible with as little
contamination by other biomaterials (e.g., lipids,
cellulose, nucleic acid, etc.) as possible.
• This is generally done with the aid of:
• Detergents (e.g., SDS, 3-([3-cholamidopropyl] dimethyl
ammonio)-1- propane sulfonate (CHAPS), cholate, Tween),
which help to solubilize membrane proteins and aid their
separation from lipids
• Reductants (e.g., dithiothreitol [DTT], mercaptoethanol,
thiourea), which reduce disulfide bonds or prevent protein
oxidation

42. • Denaturing agents (e.g., urea and acids), which disrupt
protein protein interactions, secondary and tertiary
structures by altering solution ionic strength and pH
• Enzymes (e.g., DNAse, RNAse), which digest
contaminating nucleic acids, carbohydrates, and lipids.

43. Protein Separations Before Digestion
The three principal separation approaches used with intact
proteins are
• 1D- and 2D-SDS-PAGE and preparative isoelectric
focusing (IEF).
• Although these are most widely used,
• there are alternatives, particularly HPLC (reverse
phase (RP), size exclusion, ion exchange, or affinity
chromatography).
• Regardless of the method used, the idea behind
separating intact proteins is to take advantage of
their diversity in physical properties, especially
isoelectric point and molecular weight.

44. • The mixture may be separated into a relatively small
number of fractions (as in 1D-SDS-PAGE and preparative
IEF) or into many fractions (as in the many spots in
2D-SDS-PAGE).
• The fractions then are taken for proteolytic digestion
followed either by further separation of the peptide
fragments or direct MS analysis of the peptides.

45. One-Dimensional SDS-PAGE
• In 1D-SDSPAGE, the protein sample is dissolved in a
loading buffer that usually contains a thiol reductant
(mercaptoethanol or DTT) and SDS (Fig. 2).
• The separation method is based on the binding of SDS to
the protein, which imparts negative charge (from the SDS
sulfate group) to the protein in roughly constant
proportion to molecular weight.
When the gel is subjected to high voltage, the protein-SDS
complexes migrate through the cross-linked polyacrylamide
gel at rates based on their ability to penetrate the pore
matrix of the gel.
• The proteins thus are resolved into bands in order of
molecular weight.

47. • The 1D-SDS-PAGE analysis will often give a single, clean-
looking band, whereas 2D-SDS-PAGE of the same sample
will resolve the sample into multiple spots along the
same molecular-weight band, but with different
isoelectric points.
• This can reflect multiple post-translational modifications
that do not significantly affect SDS binding or migration
through the polyacrylamide gel.

48. Two-Dimensional SDS-PAGE
• This separation method has become synonymous with
proteomics and remains the single best method for
resolving highly complex protein mixtures.
• Two-dimensional SDS-PAGE is actually a combination
of two different types of separations.
• In the first, the proteins are resolved on the basis of
isoelectric point by IEF.
• In the second, focused proteins then are further resolved
by electrophoresis on a polyacrylamide gel (Fig. 3).
• Thus 2D-SDS-PAGE resolves proteins in the first
dimension by isoelectric point and in the second
dimension by molecular weight.

50. • Two-dimensional separation (2D) involves first separating
proteins based on their isoelectric point (pI) using
isoelectric focusing (IEF).
• The isoelectric point is the pH at which there is no net
electric charge on a protein.
• IEF is an electrophoretic technique whereby proteins are
separated in a pH gradient.
• An electric field is applied to the gradient and proteins
migrate to the position in the pH gradient equivalent to the
pI (Fig. 2.1).
• Because the pI of a protein is based on its amino acid
sequence, this technique has good resolving power.
• The resolution can be adjusted further by changing the
range of the pH gradient.

51. • The use of immobilized pH gradient (IPG) strips has
enabled reproducible micropreparative fractionation of
protein samples
• The second step in 2D electrophoresis is to separate
proteins based on molecular weight using SDS-PAGE.
• Individual proteins are then visualized by Coomassie or
silver staining techniques or by autoradiography.
• Because 2D gel electrophoresis separate proteins based
on independent physical characteristics, it is a powerful
means to resolve complex mixtures proteins (Fig. 2.1).
• Modem large-gel formats are reproducible and are the
most common method for protein separation in proteomic

52. Figure 2.1. Schematic
illustration of two-dimensional
gel electrophoresis.
• Proteins are extracted from
the organism of interest and
solubilized.
• The first dimension
separates proteins based on
isoelectric point.
• The pI strip is reduced and
alkylated and applied to an
SDS-PAGE gel for separation
by molecular weight.
• Proteins can be
visualized using a number of
staining techniques.

53. Limitations of two-dimensional gel electrophoresis
(i) Poor reproducibility,
(ii) limited dynamic range, and importantly,
(iii) the fact that certain proteins stain poorly or not at all.
Reducing complexity: Protein fractionation prior to
electrophoresis
• Because of the difficulties above mentioned
• fractionation steps are often performed on protein
mixtures prior to 2D gel separation to reduce the
complexity of the mixtures.
• Pre-fractionation of proteins can be achieved by (i)
isolation of cell compartments such as the plasma
membrane or organelles such as mitochondria or nuclei,

54. (ii) By sequential extraction procedures with alternative
solubilization capacities such as aqueous buffers versus
detergents, or
(iii) by fractionation methods such as free flow
electrophoresis or chromatography.

55. Figure 2.2. Fractionation of
protein extracts before 2D
gel electrophoresis.
• Crude lysates can be
fractionated by affinity
purification or by a
number of
chromatographic
techniques.
• In addition, organelles or
other cellular structures
can be purified and
lysates from these
organelles can be
fractionated or separated
directly on 2D gels.
• By repeating this
procedure using a number
of conditions it may be
possible to visualize a
large fraction of a cell's
proteome.

56. Protein identification by mass spectrometry
Basics of mass spectrometry analysis
• One of the major advances in proteomics has been the
development of mass spectrometry as a reliable, high-
throughput method of protein identification.
• Mass spectrometry provides extremely sensitive
measurements of the mass of molecules and this data can
be used to search protein and nucleotide databases directly
to identify a protein.
• Mass spectrometry relies on the digestion of gel
separated proteins into peptides by a sequence specific
protease such as trypsin.
• Peptides are used rather than proteins because the
molecular weight of an entire protein is not sufficiently
discriminating for database identification.

58. Figure 2.3. A. Mass spectrometer consisting of an ionization
source, a mass analyzer and an ion detector.
• The mass analyzer shown is a time-of -flight (TOF) mass
spectrometer.
• Mass-to-charge (m/z) ratios are determined by
measuring the amount of time it takes an ion to reach
the detector.
• B. Tandem mass spectrometer consisting of an ion
source, a first mass analyzer, a collision cell, a second
mass analyzer and a detector.
• The first mass analyzer is used to choose a particular
peptide ion to send to the collision cell where the
peptide is fragmented.
• The mass of the spectrum of fragments is determined in
the second mass analyzer and is diagnostic of the amino

59. • Mass spectrometers measure the mass-to-charge ratio
(m/z) of ions.
• They consist of an ionization source that converts
molecules into gas-phase ions and a mass analyzer
coupled to an ion detector to determine the m/z
ratio of the ion (Yates III, 2000).
• A mass analyzer uses a physical property such as time-of-
flight (TOF) to separate ions of a particular m/z value that
then strike the detector (Fig. 2.3).

60. • The magnitude of the current that is produced at the
detector as a function of time is used to determine the
m/z value of the ion.
• While mass spectrometers have been used for many years
for chemistry applications, it was the development of
reproducible techniques to create ions of large molecules
that made the method appropriate for proteomics.

61. Ionization of biological macromolecules
Matrix-assisted laser desorption ionization (MALDI) creates
ions from the energy of a laser with the help of an energy
absorbing matrix.
• The molecules to be ionized are desiccated in a
crystalline matrix and the laser causes excitation of the
matrix and the ejection of ions into the gas-phase.
• This method of ionization is often used in conjunction
with time-of-flight detection (MALDI-TOF) to identify
proteins by peptide mass fingerprinting.

62. • The masses of peptides derived from an in-gel
proteolytic digestion of protein spots from a 2D gel are
measured and searched against a computer generated
list of peptides created by a simulated digestion of a
protein database using a specific protease such as
trypsin.
• The accuracy of the mass measurement is often
sufficient to identify proteins from completely sequenced
genomes, such as the bacterium Haemophilus influenzae

63. • This is possible because the masses of all of the tryptic
peptides from the predicted open reading frames can be
precisely calculated for comparison to the mass data.
• The power of this approach has increased due to
advances in automation such that hundreds of proteins
can be visualized, excised, digested enzymatically, and
their mass determined and automatically searched
against databases

64. • Figure 2.4. Peptide fingerprinting by MALDI-TOF mass Spectrometry.
Proteins are extracted and separated on by 2D gel electrophoresis. A
spot of interest is excised from the gel, digested with trypsin, and
ionized by MALDI.
• The precise mass of proteolytic fragments is determined by time-of-
flight mass spectrometry.
• The identity of the protein is determined by comparing the peptide
masses with a list of peptide masses generated by a simulated
digestion of all of the open reading frames of the organism.

65. • Electrospray ionization (ESI) creates ions by holding a
liquid at a high potential difference.
• This results in a local separation of charges.
• The repulsion of these charges overcomes the surface
tension of the liquid and gives rise to a spray of charged
droplets of solvent containing analyte.
• Cycles of evaporation remove the solvent and result in
the formation of ions.
• The ions enter a mass analyzer such as TOF and give rise
to an m/z spectrum.

66. • A variation of the method is nanoelectrospray
ionization, which is commonly used in proteomic studies.
• This method involves the use of a miniaturized
electrospray source consisting of a metal-coated glass
capillary with an inner diameter of 1 μM.
• The spray droplets that are approximately 100 times
smaller in volume than those produced by conventional
electrospray sources.
• An advantage of nano-electrospray ionization is that little
of the sample is lost in large droplets from which
biomolecules cannot be ionized.
In addition, very small amounts of a sample can be subjected
to mass spectrometric analysis over a long period of time.

78. Peptide mass fingerprinting
• Peptide mass fingerprinting (PMF) (also known
as protein fingerprinting) is an analytical technique
for protein identification in which the unknown protein
of interest is first cleaved into smaller peptides, whose
absolute masses can be accurately measured with
a mass spectrometer such as MALDI-TOF or ESI-TOF

79. • The peptide masses are compared to either a database
containing known protein sequences or even the
genome.
• This is achieved by using computer programs that
translate the known genome of the organism into
proteins, then theoretically cut the proteins into
peptides, and calculate the absolute masses of the
peptides from each protein.
• They then compare the masses of the peptides of the
unknown protein to the theoretical peptide masses of
each protein encoded in the genome.
• The results are statistically analyzed to find the best
match.

80. • The advantage of this method is that only the masses of
the peptides have to be known.
• Time-consuming de novo peptide sequencing is then
unnecessary.
• A disadvantage is that the protein sequence has to be
present in the database of interest.
• Additionally most PMF algorithms (Probabilistic Matrix
Factorization and Collaborative Filtering) assume that the
peptides come from a single protein.
• The presence of a mixture can significantly complicate the
analysis and potentially compromise the results.
• Typical for the PMF based protein identification is the

81. • Therefore, the typical PMF samples are isolated proteins
from two-dimensional gel electrophoresis (2D gels) or
isolated SDS-PAGE bands.
• Additional analyses by MS/MS can either be direct, e.g.,
MALDI-TOF/TOF analysis or downstream nanoLC-ESI-
MS/MS analysis of gel spot eluates.

83. • Due to the long, tedious process of analyzing proteins,
peptide mass fingerprinting was developed.
• Edman degradation was used in protein analysis, and it
required almost an hour to analyze one amino acid
residue.
• SDS-PAGE was also used to separate proteins in very
complex mixtures,

84. Sample preparation
• Protein samples can be derived from SDS-
PAGE or reversed phase HPLC, and are then subject to
some chemical modifications.
• Disulfide bridges in proteins are reduced and cysteine
amino acids are carbamidomethylated chemically or
acrylamidated during the gel electrophoresis.
• Then the proteins are cut into several fragments using
proteolytic enzymes such as trypsin, chymotrypsin. A
typical sample:protease ratio is 50:1.
• The proteolysis is typically carried out overnight and the
resulting peptides are extracted with acetonitrile and
dried under vacuum.
• The peptides are then dissolved in a small amount of
distilled water or further concentrated and purified and
are ready for mass spectrometric analysis

85. Mass spectrometric analysis
• The digested protein can be analyzed with different types
of mass spectrometers such as ESI-TOF or MALDI-TOF.
• MALDI-TOF is often the preferred instrument because it
allows a high sample throughput and several proteins can
be analyzed in a single experiment, if complemented
by MS/MS analysis.
• LC/ESI-MS and CE/ESI-MS are also great techniques for
peptide mass fingerprinting.

86. • A small fraction of the peptide (usually 1 microliter or
less) is pipetted onto a MALDI target and a chemical
called a matrix is added to the peptide mix.
• Common matrices are Sinapinic acid, Alpha-Cyano-4-
hydroxycinnamic acid, and 2,3-Dihydroxybenzoic acid.
• The matrix molecules are required for the desorption of
the peptide molecules.
• Matrix and peptide molecules co-crystallize on the MALDI
target and are ready to be analyzed.
• There is one predominantly MALDI-MS sample
preparation technique, namely dried droplet technique.

87. • The target is inserted into the vacuum chamber of the
mass spectrometer and the desorption and ionisation of
the polypeptide fragments is initiated by a pulsed laser
beam which transfers high amounts of energy into the
matrix molecules.
• The energy transfer is sufficient to promote the ionisation
and transition of matrix molecules and peptides from the
solid phase into the gas phase.
• The ions are accelerated in the electric field of the mass
spectrometer and fly towards an ion detector where their
arrival is detected as an electric signal.
• Their mass-to-charge ratio is proportional to their time of
flight (TOF) in the drift tube and can be calculated
accordingly.

88. • Coupling ESI with capillary LC can separate peptides from
protein digests, while obtaining their molecular masses
at the same time.
• Capillary electrophoresis coupled with ESI-MS is another
technique; however, it works best when analyzing small
amounts of proteins.

89. Computational analysis
• The mass spectrometric analysis produces a list of
molecular weights of the fragments which is often called
a peak list.
• The peptide masses are compared to protein databases
such as Swissprot, which contain protein sequence
information.
• Software performs in silico digests on proteins in the
database with the same enzyme (e.g. trypsin) used in the
chemical cleavage reaction.
• The mass of these peptide fragments is then calculated
and compared to the peak list of measured peptide
masses.
• The results are statistically analyzed and possible matches
are returned in a results table.

90. Applications of Proteomics
In current practice, proteomics encompasses four principal
applications.
These are: 1) mining, 2) protein-expression profiling, 3)
protein-network mapping, and 4) mapping of protein
modifications.

91. Mining is simply the exercise of identifying all (or as many
as possible) of the proteins in a sample.
• The point of mining is to catalog the proteome directly,
rather than to infer the composition of the proteome
from expression data for genes (e.g., by microarrays).
• Mining is the ultimate brute-force exercise in
proteomics:
• one simply resolves proteins to the greatest extent
possible and then uses MS and associated database
and software tools to identify what is found.
• There are several approaches to mining and each
offers advantages. What these approaches
collectively offer is the ability to confirm by direct
analysis what could only be inferred from gene-

92. • Protein-expression profiling is the identification of
proteins in a particular sample as a function of a
particular state of the organism or cell (e.g.,
differentiation, developmental state, or disease state) or
as a function of exposure to a drug, chemical, or physical
stimulus.
Expression profiling is actually a specialized form of mining.
It is most commonly practiced as a differential analysis, in
which two states of a particular system are compared.
• For example, normal and diseased cells or tissues can be
compared to determine which proteins are expressed
differently in one state compared to the other.
• This information has tremendous appeal as a means of
detecting potential targets for drug therapy in disease.

93. Protein-network mapping
• It is the proteomics approach to determining how
proteins interact with each other in living systems.
• Most proteins carry out their functions in close
association with other proteins.
• It is these interactions that determine the functions of
protein functional networks, such as signal-transduction
cascades and complex biosynthetic or degradation
pathways.
• Much has been learned about protein-protein
interactions through in vitro studies with individual,
purified proteins and with the yeast two-hybrid system.

94. • However, proteomics approaches offer the opportunity to
characterize more complex networks through the creative
pairing of affinity-capture techniques coupled with
analytical proteomics methods.
• Proteomics approaches have been used to identify
components of multiprotein complexes.
• Multiple complexes are involved in point-to-point signal-
transduction pathways in cells.
• Protein-network profiling would offer the ability to assess
at once the status of all the participants in the pathway.
• As such, protein-network profiling represents one of the most
ambitious and potentially powerful future applications of
proteomics.

95. Mapping of protein modifications
• It is the task of identifying how and where proteins are
modified.
• Many common posttranslational modifications govern
the targeting, structure, function, and turnover of
proteins.
• In addition, many environmental chemicals, drugs, and
endogenous chemicals give rise to reactive electrophiles
that modify proteins.
• A variety of analytical tools have been developed to
identify modified proteins and the nature of the
modifications.

96. • Modified proteins can be detected with antibodies (e.g.,
for specific phosphorylated amino acid residues), but
the precise sequence sites of a specific modification
often are not known.
• Proteomics approaches offer the best means of
establishing both the nature and sequence specificity of
posttranslational modifications.
• The extension of this approach to simultaneous
characterization of the modification status of regulated
proteins in a network again represents a powerful
extension of proteomics technology.
• These approaches will provide fresh avenues of
approach to questions of how chemical modification
of the proteome affects living systems.