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Protein chemistry to proteomics
Technological advancements in protein analysis with
increased sensitivity, resolution and capability to carry
out high throughput studies has led to a transition
from protein chemistry to the new field of proteomics.
Harini Chandra
Affiliations
Master Layout (Part 1)
1
This animation consists of 3 parts:
Part 1 – Mass spectrometric analysis Vs Edman degradation
Part 2 – Immobilized pH gradient (IPG) strips Vs tube gels
Part 3 – Completion of several genome sequence projects
Sample inlet
Charged peptide fragments
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+
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Mass analyzer
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Intact protein
to be analyzed
Ionization source
Mass spectrometry
Detector
Edman degradation
Labeling
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Release
Labeling
Polypeptide chain
Labelled amino acid
Release
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Definitions of the components:
Part 2 – Mass spectrometric analysis Vs Edman
degradation
1. Mass spectrometry: A powerful protein detection and analysis technique that
produces charged molecular species in vacuum, separates them by means of
electric and magnetic fields and measures the mass-to-charge ratios and relative
abundances of the ions thus produced.
2. Protein to be analyzed: Mass spectroscopy is commonly used to identify
proteins by breaking them into smaller, charged peptide fragments and analyzing
their mass-to-charge ratio. Several advancements have been made in MS to
facilitate this process.
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3. Sample inlet: The first point of contact where the sample is introduced within the
mass spectrometer either as liquid nano-droplets or alongside small matrix
molecules.
4. Ionization source: The protein of interest must be ionized with a suitable source
so that the charged molecules can be detected in the mass spectrometer. Biological
samples are most often ionized by electrospray ionization (ESI), wherein liquid
containing the analyte of interest is dispersed into a fine aerosol by means of an
electrospray. The other commonly used technique is matrix assisted laser
desorption/ionization (MALDI), another soft ionization technique that involves the
use of a laser beam (nitrogen) for ionization. The biomolecule of interest is
embedded in a solid matrix that prevents it from being damaged by the laser.
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Definitions of the components:
Part 2 – Mass spectrometric analysis Vs Edman
degradation
5. Mass analyzer: The charged molecules are segregated after ionization based on
their mass-to-charge ratio. The four most common analyzers are Time of Flight
(TOF), quadrupole, ion trap and Fourier Transform Ion Cyclotron Resonance (FTICR), each having its own level of sensitivity and accuracy. The ion motion is
manipulated either by electric or magnetic field and ions are directed to the detector
for analysis.
6. Charged peptide fragments: The peptide fragments generated by the ionization
source carry positive, negative as well as neutral charges. The settings of the mass
analyzer can be adjusted such that only certain ions are taken up for detection. A
cut off range can be specified whereby only ions in that particular range move
ahead for detection. Sensitivity of detection for positive ions is higher than negative
ions while neutral ions cannot be detected by MS.
7. Detector: The final component of the spectrometer is the detector which can
record either the current produced or the charge induced when an ion hits a
surface. Electron multipliers are commonly used as detectors.
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8. Edman degradation: This is a technique for sequencing amino acid residues in
a polypeptide chain starting from the N-terminus without disrupting any other
peptide bonds. The peptide is treated with phenylisothiocyanate and then
hydrolyzed such that only the derivatized amino acid is liberated, leaving the
remaining peptide chain intact for the next round of analysis. Although this is an
extremely useful technique, it is time consuming and cumbersome.
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Koichi Tanaka and John
Bennett Fenn were
awarded the Nobel Prize
in Chemistry in 2002 for
development of these
techniques!
The development of these soft
ionization techniques which
could be used for protein
samples was a major turning
point for proteomic studies.
Sample ionization
Laser
beam
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Electron
beam
Action
Small circles
must be show
to enter tube
and then the
coil must be
zoomed into to
show the
figures below.
ESI
Description of the action
First show some coloured circles
entering the transparent tube on top
and moving towards the grey coil. This
is then zoomed into and the figures
below must be shown with the circles
moving towards the red rectangle at
the end. The text box on right top must
appear followed by the bubble on top
left.
MALDI
Audio Narration
Protein analysis by MS had proved challenging due to complete
degradation of samples with then available hard ionization
techniques. This was overcome by development of soft ionization
techniques, MALDI and ESI. The vapourized sample is ionized by
means of an electron beam in ESI or by a laser beam in MALDI.
This results in charged peptide fragments which get accelerated
towards the mass analyzer. These two techniques greatly impacted
proteomic studies as they facilitated MS analysis of protein samples.
1 Part 1, step 2:
Edman degradation
Release
Release
…...
Polypeptide chain
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Labeling
Labelled amino acid
Labelled amino acid
Multiple rounds of sequencing required to
analyze long polypeptide chains by Edman
degradation. Sequencing by mass
spectrometry is rapid thereby facilitating
analysis of large number of samples.
Mass spectrometry
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Sample inlet
Charged
peptide
fragments
+
+ ++
++
Intact protein
to be analyzed
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Action
(Please
redraw all
figures.) As
shown in
animation.
5
+
+
Mass analyzer
Ionization source
Rapid
sequencing
MS data analysis
Detector
Description of the action
1st
First show the chain with circles on top. The
circle must be attached to a
triangle as shown. This must then be released after which the second circle is
attached to the triangle. Again this is released leaving behind the remaining
circles after which the dots must appear to show that this process continues.
Next the figure below must be shown. The purple ‘protein’ must enter via the
inlet and arrive at the grey circles. Here, it must be broken down into smaller
fragments as shown. The smallest green fragments must travel rapidly to the
‘detector’ followed by the red fragments and finally the violet fragments which
must move slowly. Next, the arrow and the computer must appear followed by
the text box shown.
Audio Narration
Protein sequencing by Edman
degradation is time-consuming and
cumbersome. Several rounds of
sequencing are required for analysis of
long polypeptide chains. Protein
sequencing by MS, however, is much
faster thereby allowing large number of
samples to be analyzed in same
amount of time.
Master Layout (Part 2)
1
This animation consists of 3 parts:
Part 1 – Mass spectrometric analysis Vs Edman degradation
Part 2 – Immobilized pH gradient (IPG) strips Vs tube gels
Part 3 – Completion of several genome sequence projects
2-D Gel electrophoresis
Separation based on pI
2
IPG strip
Tube gel
3
Separation based on
molecular weight
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SDS-polyacrylamide gel
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Source: Biochemistry by A.L.Lehninger, 4th edition (ebook); http://www.gelifesciences.com/
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Definitions of the components:
Part 2 – IPG strips Vs tube gels
1. 2-D gel electrophoresis: This is an advanced electrophoretic separation
technique that carries out separation in two dimensions. Proteins are separated
based on their isoelectric points (pI) in the first dimension followed by SDS-PAGE in
the second dimension, which separates proteins based on their molecular weight.
2. IPG strip: Commercially available immobilized pH gradient (IPG) gel strips have
considerably facilitated the process of isoelectric focusing by eliminating the tedious
steps of gel preparation and pH gradient establishment using ampholyte solutions.
These strips, available across the pH range, contain a preformed pH gradient
immobilized on a precast polyacrylamide gel placed on a plastic support. Narrow
pH ranges can be selected for fine separations while broader pH ranges are also
available for crude separations. These strips only need to be rehydrated with a
suitable buffer before use.
3. Tube gel: Isoelectric focusing using tube gels is a tedious process compared to
the readily available IPG strips. Here, the gels first need to be cast and then run
with a suitable ampholyte solution before sample application, in order to establish
the pH gradient. These pH gradients are not very stable and tend to breakdown on
application of some concentrated samples.
4. SDS-polyacrylamide gel: It is one of the most commonly used gels for
separation of proteins based on their molecular weights. It is prepared by free
radical induced polymerization of acryl-amide and N, N’-methylenebisacrylamide
and also contains the anionic detergent, sodium dodecyl sulhpate (SDS), and
reducing agent, dithiothreitol (DTT). These are responsible for denaturing the
protein and facilitate their separation solely on the basis of size.
1 Part 2, step 1:
2-DE of same protein sample carried out using tube gels
Increasing pH
pH 9
pH 3
2
3
Tube gel
Decreasing
molecular
weight
SDS-PAGE
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Action
Description of the action
As shown
in
animation.
(Please redraw all figures.)
First show the tube on top with the bands followed
by the empty blue slab below. The tube must then
move down and get fixed n the groove of the slab.
The blue spots must then appear which move
down the blue slab in the direction indicated until
they reach the positions shown. This is repeated
for the remaining two slabs as well.
Audio Narration
The pH gradient in tube gels is established by means of
ampholyte solutions which consist of low molecular
weight organic acids and bases that are subjected to an
electric field. These gradients are not always very stable
and tend to break down upon addition of concentrated
samples. Analysis of the same protein mixture by 2-DE
using tube gels often gives a lot of variation in results
across gels.
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Part 2, step 2:
Reproducibility across gels is not obtained
in 2-DE using tube gels. Commercially
manufactured IPG strips ensure minimal
variations in the pH gradient of the strip
thereby giving better reproducible results.
2-DE of same protein sample carried out using
IPG strips
Increasing pH
2
Increasing pH
pH 9
pH 3
pH 3
Increasing pH
pH 9
pH 3
pH 9
IPG strip
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4
Decreasing
molecular
weight
SDS-PAGE
Action
As shown
in
animation.
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Description of the action
(Please redraw all figures.)
First show the strip on top with the bands followed
by the empty blue slab below. The tube must then
move down and get fixed n the groove of the slab.
The blue spots must then appear which move
down the blue slab in the direction indicated until
they reach the positions shown. This is repeated
for the remaining two slabs as well.
Audio Narration
The problem of reproducibility has been overcome to a
large extent by the development of IPG strips which are
commercially manufacture gel strips having a preformed
pH gradient. These strips only need to be rehydrated
before use for 2-DE. Minimal gel-to-gel variation is
observed when the same sample is run by 2-DE using
IPG strips, thereby making them extremely suitable for
large scale proteomic applications.
Master Layout (Part 3)
1
This animation consists of 3 parts:
Part 1 – Immobilized pH gradient (IPG) strips Vs tube gels
Part 2 – Mass spectrometric analysis Vs Edman degradation
Part 3 – Completion of several genome sequence projects
Genomic DNA
Digestion &
insertion into BACs
2
Contigs identified
& mapped
3
Sequencing
4
Sequence overlaps
reveal final sequence
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Source: Biochemistry by A.L.Lehninger, 4th edition (ebook)
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Definitions of the components:
Part 3 – Completion of several genome sequence
projects
1. Genomic DNA: The entire DNA sequence of all chromosomes of an
organism constitutes its genomic DNA. There have been several projects
aimed at deciphering the complete genome sequence of organisms
including humans.
2. BAC: Bacterial Artificial Chromosomes are DNA constructs that are
useful for cloning purposes. These cloning vectors can carry DNA inserts
of around 150-350 kbp and have been extremely useful in the various
genome sequencing projects carried out.
3. Contigs: A set of overlapping DNA fragments that are obtained from a
single genetic source. These contigs are used to deduce the original DNA
sequence.
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4.Sequencing: DNA fragments that have been amplified using the BAC
are sequenced to obtain the base pairs of each fragment. These are then
used to deduce the original sequence of the intact DNA by aligning the
fragments having overlapping end sequences.
1 Part 3, step 1:
2
Genomic DNA
Restriction
endonuclease
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Fragmentation
& insertion into
BAC
Contigs aligned
Sequence
alignment
Final DNA sequence
Action
The pieshaped
object must
break the
blue line
into several
small
pieces.
Description of the action
(Please redraw all figures.)
First show the blue line on the left top. The pieshaped object must then move along this blue line
and cut it up into smaller pieces. These must then
be straightened out and aligned next to each
other. The sequence must then be shown to
appear with the pink regions highlighted followed
by the final sequence shown above.
Source: Biochemistry by A.L.Lehninger, 4th edition (ebook)
Audio Narration
The genomic DNA is cleaved using a suitable
restriction endonuclease and inserted into the
bacterial artificial chromosome. The amplified
sequences are sequenced using an automated
sequencer and then mapped by aligning the
overlapping fragments to obtain the original DNA
sequence.
1 Part 3, step 2:
GCCTTTAAGGATCCGGA
TTGCAAATCCCGATTCG
GGCGTTATGGCTTGGAA
2
CCCGGATGCCTGGTCCA
Available genome
database
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MS sequencing
Ala-Leu-Val-Cys-Trp-Tyr-Ala-Gly-Gly-TyrHis-Pro-Met-Arg-Ile-Lys-Lys-Glu-Ser-ProThr-Thr-Val-Val-Gln
Protein sequence
Protein
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DATABASE
SEARCH –
Corresponding
protein sequence
obtained from
genomic sequences
Action
Description of the action
As shown in First show the computer screen with all the
sequences. Next show the green protein below
animation.
followed by the ‘MS’ apparatus. The protein must
be shown to move through the tube and from the
other end, the sequence shown must appear. This
sequence must then be shown to enter the
computer screen and the green star with text must
appear out of it.
Audio Narration
Genome sequences of several organisms, including
humans, have been successfully completed and these
genome databases are extremely useful in correlating
gene and protein sequences. Several databases are
now readily available which can easily help in identifying
gene sequence of a protein that has been sequenced by
mass spectrometry.
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Interactivity option 1:Step No:1
A genomic DNA sequence was cut into 5 fragments using a suitable restriction endonuclease. After
amplification in the BAC, sequencing of these fragments gave the following sequences:
1. ATTTGCAAATCCCGGAAT 2. GCCTTTAAGGATTTG 3. GGAATCCCGGATGCCTATAT
4. CGGGCGTTATGGCTTAC 5. TATATGGCCCAATACGCGGGC
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What is the sequence of the intact original DNA?
A) GCCTTTAAGGATTTGCAAATCCCGGAATCCCGGATGCCTATATGGCCCAATACGCGGG
CGTTATGGCTTAC
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B) ATTTGCAAATCCCGGAATGCCTTTAAGGATTTGGGAATCCCGGATGCCTATATCGGGCGTTAT
GGCTTACTATATGGCCCAATACGCGGGC
C) GCCTTTAAGGATTTGCAAATCCCGGAATTATATGGCCCAATACGCGGGCGTTATGGCTTACGG
AATCCCGGATGCCTATAT
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D) GCCTTTAAGGATTTGCAAATCCCGGAATGGCCCAATACGCGGGCGTTATGGCTTACCCC
GGATGCCTATAT
Interacativity Type
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Choose the correct
option.
Options
User should be
allowed to choose
one of the four
options but can
continue to choose
until he arrives at the
right answer.
Boundary/limits
Results
The correct answer (A)
must turn green if chosen
while remaining must turn
red if selected. User should
be directed to next slide,
step 2, upon choosing
correct answer.
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Interactivity option 1:Step No:2
By analyzing the end sequence overlaps and aligning the fragments, the entire original DNA
sequence can be deduced.
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GCCTTTAAGGATTTG
ATTTGCAAATCCCGGAAT
GGAATCCCGGATGCCTATAT
TATATGGCCCAATACGCGGGC
CGGGCGTTATGGCTTAC
3
GCCTTTAAGGATTTGCAAATCCCGGAATCCCGGATGCCTATATGGCCCAATACGCGGG
CGTTATGGCTTAC
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5
1
Questionnaire
1. Which of the following reagents is used for Edman degradation process?
Answers: a) Dansyl chloride b) Urea c) Phenylisothiocyanate d) Methyisothiocyanate
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2. Which of the following is not a mass analyzer in MS?
Answers: a) TOF b) MALDI
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c) Quadrupole d) Ion trap
3.What pH range can be used to separate a crude protein extract containing 100 proteins of
varying isoelectric points?
Answers: a) pH 2-3 b) pH 4-5 c) pH 8-9 d) pH 3-11
4.There are three fragments A, B and C of lengths 30, 40 and 50 nucleotides respectively. The
region of overlap between fragment A and B is of 15 nucleotides while that between A and
C is of 10 nucleotides. What is the total length of the intact DNA segment?
Answers: a) 100 b) 95 c) 120 d) 85
Links for further reading
Books:
Discovering Genomics, Proteomics & Bioinformatics, 2nd edition, A.Malcolm Campbell & Laurie
J.Heyer
Research papers:
Gorg, A. et al. The current state of two-dimensional electrophoresis with immobilized pH
gradients. Electrophoresis 2000, 21, 1037-1053.
Gorg, A. et al. Two-dimensional polyacrylamide gel electrophoresis with immobilized pH
gradients in the first dimension (IPG-Dalt): the current state of the art and the controversy of
vertical versus horizontal systems. Electrophoresis 1995, 16, 1079-1086.
Gorg, A et al. 2-DE with IPGs. Electrophoresis 2009, 30, S122-S132.