Title Page
Proteins make up the bodies of organisms
What is Protein?
What is a proteome?
How about protein
structure?
How to Separate
Proteins
Protein Synthesis
How to Identify
Proteins
Proteins build up information networks in organisms
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 chains.
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 proteins—muscle and so
forth—to obtain the one amino acid that is needed. Unlike fat
and starch, the human body does not store excess amino
acids for later use—the 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 Alzheimer’s 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 organism’s
proteome and deduce their functions by homology
with proteins of known function. Although the threedimensional 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
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 (1)
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 posttranslational 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 discovery.
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
Arne Tiselius (1902-1971, Swedish)
Father of Electrophoresis
1948 Nobel Prize for Protein
Electrophoresis
SDS - Polyacrylamide Gel Electrophoresis (SDS-PAGE)
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 SDSpolyacrylamide 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 proteins—
in this case, negatively
charged—are 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 antigen–antibody complexes.
Mass Spectrometry
Mass Spectrometry measures molecular or atomic weight
Time-of-Flight MS (1)
The molecular weight
of proteins and peptides can
be determined by Time-OfFlight Mass Spectrometry.
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)
MALDI-TOF-MS
Laser Source
Sample target
TOF
Peptide Mass Fingerprinting (PMF) using MALDI-TOF MS
William J Henzel, Colin Watanable and John T Stults
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 1989.
Protein Identification by Database Searching
http://www.matrixscience.com/search_form_select.html
Database Searching Result