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Proteomics Tutorial


Title Page Proteins make up the bodies of organisms What is a proteome? What is Protein? How to Separate Proteins How about protein structure? How to Identify Proteins – PowerPoint PPT presentation

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Title: Proteomics Tutorial

Title Page
Proteins make up the bodies of organisms

What is a proteome?
What is Protein?
How to Separate Proteins
How about protein structure?
How to Identify Proteins
Protein Synthesis
Proteins build up information networks in
What is protein?
Proteins are fundamental components of all
living cells. They exhibit an enormous amount of
chemical and structural diversity, enabling them
to carry out an extraordinarily diverse range of
biological functions. Proteins help us
digest our food, fight infections, control body
chemistry, and in general, keep our bodies
functioning smoothly. Proteins make up the
skin, muscle, hair, bones and other organs in
your body. They are primarily composed of a set
of 20 building blocks, called amino acids.
Proteins contain from ten to several thousand
amino acids linked by peptide bonds in long
Proteins perform various functions in our bodies!
Scientists know that the critical feature
of a protein is its ability change shape. If it
has a missing part, it may be prevented from
doing its job.
Amino Acids make up Proteins!
The monomeric building blocks of proteins
are 20 amino acids, all of which have a
characteristic structure consisting of a central
a carbon atom (C) bonded to four
different chemical groups an amino (NH2) group,
a carboxyl (COOH) group, a hydrogen (H) atom, and
one variable group, called a side chain, or R
group.  Amino acids are the alphabet in the
protein language when combined in a specific
order, they make up meaningful structures
(proteins) with varied and specific functions.
Amino acids have distinct shapes, sizes, charges
and other characteristics. Many amino
acids are synthesized in your body from breakdown
products of sugars and fats, or are converted
from other amino acids by the action of specific
enzymes. However, a few of them, called essential
amino acids, cannot be synthesized or converted
in your body and have to be obtained from the
food you eat. Phenylalanine is one such
essential amino acid. It is closely related to
another amino acid, tyrosine, which just has an
additional hydroxyl (OH) group. Liver cells
contain an enzyme called phenylalanine
hydroxylase, which can add this group and convert
phenylalanine to tyrosine. Thus as long as this
enzyme is functional and there is a reasonable
supply of phenylalanine, tyrosine can be
synthesized in your body and does not have to be
included in the food that you eat.
Essential amino acids for humans
Humans can produce 10 of the 20 amino acids. The
others must be supplied by food. Failure to
obtain enough of even 1 of the 10 essential amino
acids of those that we cannot make, results in
degradation of the body's proteinsmuscle and so
forthto obtain the one amino acid that is
needed. Unlike fat and starch, the human body
does not store excess amino acids for later
usethe amino acids must be in the food every
day. The 10 amino acids that we can
produce are alanine, asparagine, aspartic acid,
cysteine, glutamic acid, glutamine, glycine,
proline, serine and tyrosine. Tyrosine is
produced from phenylalanine, so if the diet is
deficient in phenylalanine, tyrosine will be
required as well. The essential amino acids are
arginine (required for the young, but not for
adults), histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, threonine, tryptophan,
and valine. These amino acids are required in the
diet. Plants, of course, must be able to make all
the amino acids.
Damaged Protein
  • Sometimes a protein twists into the
    wrong shape or has a missing part, preventing it
    from doing its job. Many diseases, such as
    Alzheimers and Mad Cow, result from proteins
    that have adopted an incorrect structure.

What is a Proteome? 
The term proteome refers to the entire protein
complement of an organism. For example, the
proteome of yeast consists of about 6000
different proteins the human proteome is only
about five times as large, comprising about
32,000 different proteins.  
By comparing protein sequences and structures,
scientists can classify many proteins in an
organisms proteome and deduce their functions by
homology with proteins of known
function. Although the three-dimensional
structures of relatively few proteins are known,
the function of a protein whose structure has not
been determined can often be inferred from its
interactions with other proteins, from the
effects resulting from genetically mutating it,
from the biochemistry of the complex to which it
belongs, or from all three.   
Dr. Marc Wilkins University of New South Wales,
Sydney, Australia Defined the Concept of the
Proteome and Coined the Term
From Genomics to Proteomics (1)
Genomics has provided a vast amount of
information forming a basis to link genetic
variations with diseases. It is now recognized,
however, that there are a number of reasons why
gene sequence information and the pattern of gene
activity in a cell do not provide a complete and
accurate profile of a protein's abundance or its
final structure and state of activity.
From Genomics to Proteomics (2)
After transcription from DNA to RNA, the gene
transcript can be spliced in different ways prior
to translation into protein. Following
translation, most proteins are chemically changed
through post-translational modification, mainly
through the addition of carbohydrate and
phosphate groups. Such modification plays a vital
role in modulating the function of many proteins
but is not directly coded by genes.
As a consequence, the information from a single
gene may encode many different proteins, and that
is before they undergo post translational
modifications. It is clear from a growing number
of data that genomic information very often does
not provide an accurate profile of protein
abundance, structure and activity. Since it is
proteins and, to a much lesser extent, other
types of biological molecules that are directly
involved in both normal and disease-associated
biochemical processes, a more complete
understanding of disease may be gained by looking
directly at the proteins present within a
diseased cell or tissue, and this is achieved
through the proteome and proteomics.
What is Proteomics? 
  • Proteomics is the scientific
    discipline which studies proteins and searches
    for proteins that are associated with a disease
    by means of their altered levels of expression
    and/or post-translational modification between
    control and disease states. It enables
    correlations to be drawn between the range of
    proteins produced by a cell or tissue and the
    initiation or progression of a disease state and
    the effect of therapy.
  • Proteome research permits the
    discovery of new protein markers for diagnostic
    purposes and of novel molecular targets for drug
  • The abundance of information
    provided by proteome research is entirely
    complementary, with the genetic information being
    generated from genomics. Proteomics will make a
    key contribution to the development of functional
    genomics. The combination of proteomics and
    genomics will play a major role in biomedical
    research and will have a significant impact on
    the development of future generations of
    diagnostic and therapeutic products.

How the Proteome of an Organism is Found
Protein Gel Electrophoresis
SDS - Polyacrylamide Gel Electrophoresis
Treatment with SDS, a negatively charged
detergent, dissociates multimeric proteins and
denatures all the polypeptide chains (Step1).
During electrophoresis, the SDS-protein complexes
migrate through the polyacrylamide gel (Step 2).
Small proteins are able to move through the pores
more easily, and faster, than larger proteins.
Thus the proteins separate into bands according
to their sizes as they migrate through the gel.
The separated protein bands are visualized
by staining aining with a dye (Step 3). 
Two-dimensional Gel Electrophoresis
In this technique, proteins are first separated
on the basis of their charges by isoelectric
focusing (step1). The resulting gel strip
is applied to an SDS-polyacrylamide gel and the
proteins are separated into bands by mass (step
3). In this two- dimensional gel of a protein
extract from cultured cells, each spot represents
a single polypeptide. Polypeptides can
be detected by dyes, as here, or by other
techniques such as autoradiography. Each
polypeptide is characterized by its isoelectric
point (pI) and molecular weight.
Gel Image Analysis Software
The SDS-PAGE or 2DE resulted Gel images can be
analyzed by specific software. The software can
automatically detected the protein spots,matched
them between gels, determine the MW and pI of
proteins on gel, and batch process multiple
analyses for high-throughput, quantitation and
statistical analysis differential expression
analysis of sets of gels.
Chromatography (1)
Liquid chromatographic techniques separate
proteins on the basis of mass, charge, or
affinity for a specific ligand.
(a) Gel filtration chromatography separates
proteins that differ in size. A mixture of
proteins is carefully layered on the top of a
glass cylinder packed with porous beads. Smaller
proteins travel through the column more slowly
than larger proteins. Thus different proteins
have different elution volumes and can
be collected in separate liquid fractions from
the bottom.
Mikhail Semenovich Tswett (1872 - 1919) Father
of Chromatography
Chromatography (2)
(b) One-exchange  chromatography separates
proteins that differ in net charge in columns
packed with special beads that carry either
a positive charge (shown here) or a negative
charge. Proteins having the same net charge as
the beads are repelled and flow through the
column, whereas proteins having the
opposite charge bind to the beads. Bound
proteinsin this case, negatively chargedare
eluted by passing a salt gradient (usually of
NaCl or KCl) through the column. As the ions bind
to the beads, they desorbe the protein.
(c) In antibody-affinity chromatography, a
specific antibody is covalently attached to beads
packed in a column. Only protein with high
affinity for the antibody is retained by the
column all the nonbinding proteins flow through.
The bound protein is eluted with an acidic
solution, which disrupts the antigenantibody
Mass Spectrometry
Mass Spectrometry measures molecular or atomic
Time-of-Flight MS (1)
The molecular weight of proteins and peptides can
be determined by Time-Of-Flight Mass
In a laser-desorption mass spectrometer, pulses
of light from a laser ionize a protein or peptide
mixture that is absorbed on a metal target (1).
An electric field accelerates the molecules in
the sample toward the detector (2 and 3). The
time to the detector is inversely proportional to
the mass of a molecule. For molecules having the
same charge, the time to the detector is
inversely proportional to the mass. The molecular
weight is calculated using the time of flight of
a standard.  
Time-Of-Flight MS (2)
Peptide Mass Fingerprinting (PMF) using MALDI-TOF
In 2002, American Society for Mass Spectrometry
awarded Distinguished Contribution in Mass
Spectrometry Award to Henzel, Stults and
Watanabe for their proposal of PMF technology in
Protein Identification by Database Searching
Database Searching Result