Protein chemistry to proteomics

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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

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Master Layout (Part 1)
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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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Mass analyzer
Intact protein to be analyzed
Mass spectrometry
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Detector
Ionization source
Edman degradation
Labeling
Labeling
Release
Polypeptide chain
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Labelled amino acid
Release
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Definitions of the componentsPart 2 Mass
spectrometric analysis Vs Edman degradation
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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. 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 componentsPart 2 Mass
spectrometric analysis Vs Edman degradation
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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 (FT-ICR), 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. 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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Part 1, step 1
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The development of these soft ionization
techniques which could be used for protein
samples was a major turning point for proteomic
studies.
Koichi Tanaka and John Bennett Fenn were awarded
the Nobel Prize in Chemistry in 2002 for
development of these techniques!
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Sample ionization
Laser beam
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Electron beam
ESI
MALDI
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Action
Audio Narration
Description of the action
Small circles must be show to enter tube and then
the coil must be zoomed into to show the figures
below.
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.
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.
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Part 1, step 2
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Edman degradation
Release
Release
...
Polypeptide chain
Labeling
Labelled amino acid
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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
Rapid sequencing
Intact protein to be analyzed
MS data analysis
Mass analyzer
Detector
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Ionization source
Action
Audio Narration
Description of the action
(Please redraw all figures.) As shown in
animation.
First show the chain with circles on top. The 1st
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.
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.
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Master Layout (Part 2)
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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
Separation based on pI
2-D Gel electrophoresis
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IPG strip
Tube gel
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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 componentsPart 2 IPG
strips Vs tube gels
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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.
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Part 2, step 1
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2-DE of same protein sample carried out using
tube gels
Tube gel
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Decreasing molecular weight
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SDS-PAGE
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Action
Audio Narration
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.
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
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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
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IPG strip
Decreasing molecular weight
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SDS-PAGE
Action
Audio Narration
Description of the action
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.
As shown in animation.
(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.
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Master Layout (Part 3)
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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
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Contigs identified mapped
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Sequencing
Sequence overlaps reveal final sequence
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Source Biochemistry by A.L.Lehninger, 4th
edition (ebook)
12
Definitions of the componentsPart 3
Completion of several genome sequence projects
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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. 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.
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Part 3, step 1
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Fragmentation insertion into BAC
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Genomic DNA
Restriction endonuclease
Contigs aligned
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Sequence alignment
Final DNA sequence
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Action
Audio Narration
Description of the action
The pie-shaped object must break the blue line
into several small pieces.
(Please redraw all figures.) First show the blue
line on the left top. The pie-shaped 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.
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.
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Source Biochemistry by A.L.Lehninger, 4th
edition (ebook)
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Part 3, step 2
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DATABASE SEARCH Corresponding protein sequence
obtained from genomic sequences
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Available genome database
MS sequencing
Ala-Leu-Val-Cys-Trp-Tyr-Ala-Gly-Gly-Tyr-His-Pro-Me
t-Arg-Ile-Lys-Lys-Glu-Ser-Pro-Thr-Thr-Val-Val-Gln
Protein
Protein sequence
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Action
Audio Narration
Description of the action
First show the computer screen with all the
sequences. Next show the green protein below
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.
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.
As shown in animation.
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Interactivity option 1Step No1
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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 What is the sequence of
the intact original DNA?
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A) GCCTTTAAGGATTTGCAAATCCCGGAATCCCGGATGCCTATATGGCC
CAATACGCGGG CGTTATGGCTTAC
B) ATTTGCAAATCCCGGAATGCCTTTAAGGATTTGGGAATCCCGGATGC
CTATATCGGGCGTTAT GGCTTACTATATGGCCCAATACGCGGGC
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C) GCCTTTAAGGATTTGCAAATCCCGGAATTATATGGCCCAATACGCGG
GCGTTATGGCTTACGG AATCCCGGATGCCTATAT
D) GCCTTTAAGGATTTGCAAATCCCGGAATGGCCCAATACGCGGGCGTT
ATGGCTTACCCC GGATGCCTATAT
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Boundary/limits
Interacativity Type Options
Results
User should be allowed to choose one of the four
options but can continue to choose until he
arrives at the right answer.
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.
Choose the correct option.
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Interactivity option 1Step No2
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By analyzing the end sequence overlaps and
aligning the fragments, the entire original DNA
sequence can be deduced.
GCCTTTAAGGATTTG
ATTTGCAAATCCCGGAAT
2
GGAATCCCGGATGCCTATAT
TATATGGCCCAATACGCGGGC
CGGGCGTTA-TGGCTTAC
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GCCTTTAAGGATTTGCAAATCCCGGAATCCCGGATGCCTATATGGCCCAA
TACGCGGG CGTTATGGCTTAC
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Questionnaire
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• 1. Which of the following reagents is used for
  Edman degradation process?
• Answers a) Dansyl chloride b) Urea c)
  Phenylisothiocyanate d)? Methyisothiocyanate
• 2. Which of the following is not a mass analyzer
  in MS?
• Answers a) TOF b) MALDI 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

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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.