Techniques in proteomics

1. Techniques used in proteomics

2. Proteomics
 A biotechnology branch concerned
with applying the techniques of
molecular biology, biochemistry, and
genetics to analyzing the structure,
function, and interactions of
the proteins produced by the genes
of a particular cell, tissue, or
organism, with organizing the
information in databases.
 Real functional molecules of the
cell.
 Protein-protein interactions of an
organism.

3. Proteome = protein
compliment of the
genome
Proteomics = study of
the proteome
Protein world = study
of less abundant
proteins
Transcriptomics –
often insufficient to
study functional aspects
of genomics

4. Proteomics
 Organisms have
one genome
 But multiple
proteomes
 Proteomics is
the study of the
full complement
of proteins at a
given time.

5. Single gene produce multiple proteins:
 Alternative sequencing
 Introns (non coding regions)
 Exons (Coding regions)
 Series of steps
 Mature mRNA
 Different proteins

7. Proteomics and Genomics
 Proteomics is the analysis of the protein
complement to the genome.
 Genomics is the study of all gene, the
study of all proteins is proteomics. It is
the study of all the proteins in a cell,
tissue, organ, leaf.
 analysis and identification of proteins.

8. Proteomics and Genomics
 The proteome is much more complex than either the
genome or the transcriptome (see transcriptomics).
 Each protein can be chemically modified in different
ways after synthesis.
 Many proteins have carbohydrate groups added to
them. Others are phosphorylated or acetylated or
methylated.

9. Proteomics is multidisciplinary
Proteomics
Molecular
Biology
Biology
Analytical
Chemistry
Protein
Biochemistry
Bioinformatics

10. Proteomics Research
• Basic research:
To understand the molecular mechanisms
underlying life.
• Applied research:
Clinical testing for proteins associated with
pathological states (e.g. cancer).

11. Steps in Proteomic Analysis
 Purification of proteins:
Extraction of protein samples from whole cell,
tissue or sub cellular organelles
 Separation of proteins:
gel electrophoresis, Spots are detected using
fluorescent dyes or radioactive probes.
 Identification of proteins:
separated protein spots on gel, mass
spectrometry.

12. Proteomic tools to study proteins
 Protein isolation
 Protein separation
 Protein identification

13. Proteomics is important
 Proteomics, the study of the proteome, is important
because proteins represent the actual functional
molecules in the cell.
 When mutations occur in the DNA, it is the proteins
that are ultimately affected.
 Drugs, when they have beneficial effects, do so by
interacting with proteins.

14. Types of proteomics
Expression proteomics
The large-scale analysis of protein
expression.
identify the main proteins in sample and
proteins differentially expressed in related
samples, such as diseased v.s healthy tissue.
Represent a useful drug target or diagnostic
marker.
Proteins with similar expression profiles may
also be functionally related.
Technologies such as 2D-PAGE and mass
spectrometry are used here.

15. Structural proteomics
The large-scale analysis of protein
Protein structure comparisons can help to identify the
functions of newly discovered genes.
Structural analysis can also show where drugs bind to
proteins and where proteins interact with each other.
X-ray crystallography and mass spectroscopy.
Identification by matel ion, drug, toxins.

16. Interaction proteomics
The large-scale analysis of
protein interactions.
protein-protein interactions
helps to determine protein
functions .
how proteins assemble in
larger complexes.
Affinity purification, mass
spectrometry and the yeast
two-hybrid system are
particularly useful.

17. Techniques used in
proteomics
Mass spectroscopy.
Microarray technology.
ICAT(isotope coded affinity tag)
SILAC(stable isotope labeling with amino acids
inside culture)
SELDI(surface enhanced laser desorption
ionization)
Bioinformatics
Blotting

18. PROTEIN RESOLUTION
TECHNIQUE:

19. 2D-GEL ELECTROPHORESIS
 Electrophoresis is the migration of charged molecules, particles or ion in a liquid
or solid medium under the influence of an electric field.
 1969 - introduction of denaturing agents especially SDS separation of protein
subunit (Weber and Osborn).
 This technique combines the technique IEF (first dimension), which separates
proteins in a mixture according to charge, with the size separation technique of
SDS-PAGE second dimension).
 The combination of these two technique to give 2-D PAGE provides a highly
sophisticated analytical method for analyzing protein mixtures.

20. Some media for
Electrophoresis
Medium Conditions Principal Uses
Paper Filter paper
moistened
With Buffer, placed
Between electrods
Small molecules
Amino acid, nucleotides
Polyacrylamide
gel
Cast in tubes or
slabs;
Proteins and nucleic
acids
Agarose gel As polyacrylamide, Very large proteins,
Nucleic acid and
Nucleoprotiens etc

21. 2DE
 Several forms of PAGE exist and can provide different types
of information about the protein(s).
 SDS-PAGE, the most widely used electrophoresis technique,
separates proteins primarily by mass.
 Non denaturing PAGE, also called native PAGE, separates
proteins according to their mass: charge ratio.
 Two-dimensional PAGE (2D-PAGE) separates proteins by
isoelectric point in the first dimension and by mass in the
second dimension.

22. What is a gel….?
Gel is a cross linked polymer whose composition and
porosity is chosen based on the specific weight and
porosity of the target molecules.
Types of Gel:
Agarose gel
Polyacrylamide gel

23. AGAROSE GEL…
A highly purified uncharged polysaccharide derived
from agar.
Used to separate macromolecules such as nucleic
acids, large proteins and protein complexes.
 It is prepared by dissolving 0.5% agarose in boiling
water and allowing it to cool to 40°C.
It is fragile because of the formation of weak hydrogen
bonds and hydrophobic bonds.

25. Polyacrylamide Gels:
Polyacrylamide gels are tougher than
agarose gels.
Acrylamide monomers polymerize into
long chains that are covalently linked by
a cross linker.
Polyacrylamide is chemically complex,
as is the production and use of the gel.

26. General Procedure
General operations performed in conventional
electrophoresis include:
 separation
 staining
 detection
 Quantification

27. Principle:
Proteins move in the electric
field.
Their relative speed depends on
the charge, size, and shape of
the protein.

29. Factors Affecting Migration Rate:
The sample: Charge, Size,
Shape
The electric field: Current,
Voltage, Resistance, Heat
The Buffer: Composition,
Concentration, PH
Supporting Media

30. Types of 2DE:
Isoelectric Point:
There is a pH at which there is no net charge on a
protein; this is the isoelectric point (pI).
Above its isoelectric point, a protein has a net negative
charge and migrates toward the anode in an electrical
field.
Below its isoelectric point, the protein is positive and
migrates toward the cathode.

32. ISOELECTRIC FOCUSING:
Electrophoretic method that separates proteins
according to the iso-electric points
Is ideal for separation of amphoteric substances.
Separation is achieved by applying a potential
difference across a gel that contain a pH gradient.
Isoelectric focusing requires solid support such as
agarose gel and polyacrylamide gel

33. ISOELECTRIC FOCUSING:

34. Applications:
 Using this method one can routinely resolve between
1000 and 3000 proteins from a cell or tissue extract and
in some cases workers have reported the separation of
between 5000 and 10000 proteins.
The result of this is a gel with proteins spread out on its
surface. These proteins can then be detected by a
variety of means, but the most commonly used stains
are silver and coomasie staining.

35. MICROARRAY

36. MICROARRAY TECHNOLOGY
 Microarray technology evolved from Southern
blotting. The concept of microarrays was first
proposed in the late 1980s by Augenlicht and his
colleagues.

37. MICROARRAY
 Microarray analysis involves breaking and opening a
cell, isolating its genetic contents, identifying all the
genes that are turned on in that particular cell, and
generating a list of those genes.

38. STEPS INVOLVED IN MICCROARRAY
Sample
preparation
and labeling
Hybridization Washing
Image
acquisition
and Data
analysis
There are four major steps in performing a
typical microarray experiment.

39. SAMPLE
PREPARATION AND LABELING
• Isolate a total RNA
containing mRNA
• Preparation of cDNA from
mRNA using a reverse
transcriptase enzyme.
• Short primer is required to
initiate cDNA synthesis.
• Each cDNA (Sample and
Control) is labeled with
fluorescent cyanine dyes

40. ARRAY HYBRIDIZATION
• Here, the labelled
cDNA (Sample and
Control) are mixed
together.
• Purified
• After purification, the
mixed labelled cDNA is
competitively
hybridised against
denatured PCR
product or cDNA
molecules spotted on a
glass slide.

41. IMAGE ACQUISION AND DATA ANALYSIS
 slide is dried and scanned to
determine how much labeled
cDNA (probe) is bound to each
target spot. Hybridized target
produces emissions.
 Microarray software often uses
 Green spots on the microarray
to represent upregulated genes.
 Red to represent those genes
that are downregulated
 Yellow to present in equal
abundance

42. TYPES OF MICROARRAY
 Protein microarrays
 DNA microarrays
 Transfection microarrays
 Antibody microarray
 Tissue microarray
 Chemical compound microarray

43. PROTEIN MICROARRAY
 A protein microarray (protein chip)is a high
throughput method used to track the interactions &
activities of proteins,& to determine their functions,&
determining function on a large scale.
 Protein arrays can be screened for their ability to
bind other proteins in a complex , receptors,
antibodies, lipids, enzymes, pepyides, harmones,
specific DNA sequence

44. PREPERATION OF PROTEIN
MICROARRAY
 Protein microarrays are typically prepared by
immobilizing proteins onto a microscope slide using a
standard contact spotter or non contact microarray.
DIFFERENT METHODS OF
ARRAING THE PROTEINS
 Robotic spotting method
 Ink jetting method
 Piezoelectric spotting
 In these methods, robotic is contact microarray method
while the other two are non contact microarray
methods.

45. TYPES OF PROTEIN MICROARRAYS
There are three types of protein microarrays that
are currently used to study the biochemical activities
of proteins.
 Analytical protein microarrays
 Functional protein microarrays
 Reverse phase protein microarrays

46. 1.ANALYTICAL MICROARRAYS
Analytical microarrays(or antibody microarrays) have
antibodies arrays on solid surface, and are used to
detect proteins in biological samples.

47. 2.FUNCTIONAL PROTEIN MICROARRAYS
 Also known as target protein array. With functional
protein microarrays purified recombinant protein are
immobilized onto the solid phase.
 These can be used to identify enzyme substrates.
 These can also be used to detect antibodies in a
biological specimen to profile an immune response.

48. 3.REVERSE PHASE PROTEIN
MICROARRAY
 Involves complex samples, such as tissue lysates.
 cells are isolated from various tissues of interest and
lysed.
 The lysate is arranged onto the microarray & probed
with antibodies against the target protein of interest.
 These antibodies are typically detected with
chemiluminescent,fluoresent or colorimetric assays.

49. APPLICATIONS
 There are five major areas where protein arrays are
being applied:diagnostics,proteomics,protein
functional analysis,antibody characterisation &
treatment.
 Diagnostics involves the detection of antigens &
antibodies in blood samples; to discover new
disease biomarkers
 Proteomics pertains to protein expression profilling.

50. APPLICATIONS
 Protein functional analysis is the identification of
protein-protein interactions, protein- phospholipid
interactions, small molecule targets, enzymatic
substrates.
 Antibody characterization is characterizing cross
reactivity,specificity & mapping epitopes.
 Treatment development involves the development of
antigen-specific therapies for autoimunity,cancer

51. Mass spectrometry in
proteomics

52. Mass spectrometry
Mass spectrometry is an instrumental technique in
which sample is converted to rapidly moving
positive ions by electron bombardment and charged
particles are separated according to their masses.
Mass spectrum
Mass spectrum is a plot of relative abundance
against the ratio of mass/charges (m/e).

53. Mass spectrometer based proteomics
• This area is most commonly associated with
proteomics.
• A method to determine which proteins are
expressed and the amounts of those proteins.

54. MASS SPECTROMETER BASED
PROTEOMICS
Principle
• Ions of differing charge and mass will move
differently in a magnetic field.
• Proteins are first separated and then analyzed
with a mass spectrometer.

55. • Protein separation can be performed using gel
electrophoresis,
`usually separates proteins first by isoelectric
point and then by molecular weight.
• Once proteins are separated and quantified,
they are identified.

57. • Individual spots are cut out of the gel and
cleaved into peptides with proteolytic enzymes.
• These peptides can then be identified by mass
spectrometry.
• Specifically: matrix-assisted laser desorption-
ionization time-of-flight (MALDI-TOF) mass
spectrometry.
• In this procedure, a peptide is placed on a matrix,
which causes the peptide to form crystals.

58. • Then the peptide on the matrix is ionized
with a laser beam.
• An increase in voltage at the matrix is used.
• The higher the mass, the longer the time of
flight of the ion.

59. • In a MALDI-TOF mass spectrometer, the
ions can also be deflected with an
electrostatic reflector that also focuses the ion
beam.
• Masses of the ions reaching the second
detector can be determined with high
precision.
• These masses can reveal the exact chemical
compositions of the peptides, and therefore
their identities!

60. • Protein mixtures can also be analyzed without
prior separation.
• These procedures begin with proteolytic
digestion of the proteins in a complex
mixture.
• The resulting peptides are often injected onto
a high pressure liquid chromatography
column (HPLC).
• HPLC can be coupled directly to a time-of-
flight mass spectrometer using electrospray
ionization

61. Electrospray ionization: A technique used in mass
spectrometry to produce ions.
• It is especially useful in producing ions from
macromolecules
• because it overcomes the propensity of these
molecules to fragment when ionized.

63. • Peptides eluting from the column can be
identified by tandem mass spectrometry
(MS/MS).
• The first stage of tandem MS/MS isolates
individual peptide ions.
• Secondly, breaks the peptides into
fragments.
• Labeling with isotope tags.

64. Finally, use databases.
• Computer compares sequences to other
sequences stored in an internationally
accessible database.
• Determines the identity of the isolated
protein.

65. • As the entire human genome is known,
computers are able to determine nearly
every potential protein.
• New proteins are “discovered” when they
match sequences predicted by the computer
that have not previously been found.

66. Isotope coded affinity tag

67. 5
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Master Layout (Part 1)
This animation consists of 2 parts:
Part 1: ICAT
Part 2: Application of ICAT
Gygi, S. P. et al., Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotech. 1999,
17:994-999.
LC-MS/MS analysis
Relativeabundance
Retention time
Sample 1
Sample 2
Light (d0)
ICAT
labeled
Heavy (d8)
ICAT labeled
Mixed samples
Affinity
purification
Affinity purified
peptides

68. History:
 Introduced in 1999
 Isotope coded affinity tagging is based on a class of
chemical reagents called 'Isotope coded affinity tags'
(ICAT).
 Light reagent:
 Heavy reagent:
Both depends on the number of deuteriums.

69. ISOTOPE-CODED AFFINITY TAG (ICAT): a
quantitative method
 Label protein samples with heavy and light
reagent
 Reagent contains affinity tag and heavy or
light isotopes
Chemically reactive group: forms a
covalent bond to the protein or peptide
Isotope-labeled linker: heavy or light,
depending on which isotope is used
Affinity tag: enables the protein or
peptide bearing an ICAT to be isolated by
affinity chromatography in a single step

70. Principles
1.The peptide sequence of the protein to
be quantified (between 5-25 Amino acids
long)
 contains sufficient information
2.peptides tagged with the light and
heavy reagents respectively are
chemically identical

71.  The principles of Isotope coded affinity tags as
documented by Aebersold et al. are divided into four
stages:
 Sampling
 Tagging
 Isolation
 Quantification.

72.  ICAT is an in vitro labeling procedure that involves
tagging of protein or peptide samples with the ICAT
reagent specifically at their Cys residues. The ICAT
reagent consists of a biotin tag, a light or heavy
linker chain and a Cys-reactive group. One sample
is tagged with the light ICAT reagent while the other
is tagged with heavy ICAT.

73. Sampling and tagging
 Protein samples
 Side chains having reduced cystein residues
 Through breaking cell structure
 Samples are tagged with light isotope and heavy
isotope
 Light form of the ICAT reagent (containing zero
deuterium)
 Heavy form of ICAT reagent (containing eight
deuterium)
 Combine both into one complex mixture
 Protease act to break

74. Peptide isolation
 Avidin is then introduced to isolate the ICAT-tagged
peptides from the mixture through affinity
chromatography.
 The isolated peptides are then analysed and
separated by micro-capillary high performance liquid
chromatography- mass spectrometry (HPLC-
MS/MS).

75. Protein quantification
 Quantification is achieved by comparing the
integrated peak intensities for simultaneously eluted
pairs of identical, doubly charged peptide ions.
 the chemically identical ICAT-labelled peptide ions
are readily identified because as they co-elute, they
differ in mass-to-charge (m/z) ratio because of an 8
deuterium difference in the mass of the ICAT-
reagents.

76. Preparation for ICAT Reagent
Isotope Coded Affinity Tag
(ICAT)
 Clean the surface of the balance, place a
butter paper and tare the weight.
 Prepare reducing solution buffer of pH 8.5
consisting of 5mM tributyl phosphine, 50mM
TRIS, 6M guanidine HCl dissolved in required
volume of water.
 Set the pH of reducing buffer to pH 8.5 using
NaOH.
 Add NaOH slowly to the buffer solution and
just in case of an increase in pH, balance it
by adding HCl.
 Add labeling solution to the pellet and
dissolve it completely by vortexing.

77.  Add reducing solution to the pellet and vortex it. This
helps for reducing the disulphide bonds in the
protein.
 Place the sample at 37°C for 1hour to allow
reducing reaction to proceed.
 Estimate the protein quantity to get an idea of
proteinsample to be added during the experiment.
 Around 150μg of protein is considered ideal for
experiment.

78.  Add light and heavy ICAT reagent in control and
drug treated samples.
 Cysteinyl groups in each mixture are biotinylated
with five fold molar excess of appropriate ICAT
reagent.
 Place labelled samples in incubator at 37°C for 1 hr
and mix samples in 1:1 ratio.

79. 79
 Isotope Coded Affinity Tags Technology, ICAT

80. 5
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Master Layout (Part 2)
This animation consists of 2 parts:
Part 1: ICAT
Part 2: Application of ICAT
Kang, U. B. et al., Differential profiling of breast cancer plasma proteome by isotope-coded affinity tagging method reveals
biotinidase as a breast cancer biomarker. BMC Cancer 2010, 10:114.
Plasma sample
of normal,
healthy control
Plasma sample
from breast
cancer patient
Immune-affinity
column
chromatograpy
ICAT
155 proteins identified of
which, 33 showed 1.5-fold
abundance changes in plasma
of breast cancer patients
compared to healthy controls
Immunodeplete
d plasma

81. Definitions of the
components:
Part 2- Application of ICAT
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1 1. Normal healthy control: Normal healthy control refers to those who do not have the
disease/condition that is being studied. The authors made use of plasma proteomes
obtained from 6 healthy control samples.
2. Breast cancer patients: Plasma samples from 6 patients with breast cancer were
analyzed using ICAT labeling technique.
3. Immune-affinity column chromatography: Immune-affinity column chromatography is
a process that is carried out in order to remove the high abundance proteins present in
sera, which tend to hamper the process of detection of medium or low abundance protein
markers. Antibodies specific to the high abundance proteins of interest are immobilized on
the column and used to specifically remove them.
4. Immunodepleted serum: The serum from which the high abundance proteins have
been removed, leaving behind only the medium and low abundance proteins thereby
reducing the dynamic range, is known as immunodepleted serum.