Protein, Proteomics and Proteomic
Techniques
(II)
Dwi Candra Pratiwi, MSc
Program Studi Ilmu Kelautan
Fakultas perikanan dan Ilmu kelautan
Universitas Brawijaya
2014
Protein
Proteins are polymers of amino
acids.
Each type of protein molecule has a
unique 3-dimensional structure
determined principally by the amino
acids sequence.
This results in the enormous
diversity of protein structure that
catalyze the thousands of different
processes required by a cell.
A typical protein molecule consist of
one or more polypeptide chains.
Most of amino acids have the basic
structure :
a carbon atom (the carbon) to which
is attached an amino group, a carboxyl
group and a side chain or R-group.
The building blocks of a polypeptide
are 20 different amino acids
1. Alanin
2. Arginine
3. Aspartic acid
4. Asparagine
5. Cystein
6. Glutamine
7. Glutamic acid
8. Glycine
9. Histidine
10.Isoleucin
11.Leucine
12.Lysine
13.Methionine
14.Phenylalanine
15.Proline
16.Serine
17.Tryptophan
18.Tyrosine
19.Thereonin
20.Valine
Amino acids inorangehave hydrophobic side chain R
groups.
Amino acids ingreenare considered to be hydrophilic
because they have electronegative groups on the side chain
except tyrosine which because of the phenyl ring side chain is
also hydrophobic in character
Two amino acids inpink, Glu and Asp, have two carboxylic
acids in the side chain, are hydrophilic and contribute
one negative charge to a polypeptide chain at
neutral pH
The basic amino acids inlight blue are also very
hydrophilic and are positively charged at neutral
pH.
It should be clear from this that amino acid side chains
which contribute to overall charge on a protein are either
acidic or basic at neutral pH
Proteomics
Proteomics definition
Proteomics is a science that
focuses on the study of proteins :
their roles, their structures, their
localization, their interactions,
and other factors.
www.lexicon-biology.com
The omics nomenclature
Genomics
DNA (Gene)
Transcription
Transcriptomics
RNA
Translation
Functional
Genomics
Proteomics
PROTEIN
Enzymatic
reaction
Metabolomics
METABOLITE
Unlike the genome, which is relatively
static, the proteome changes
constantly in response to tens of
thousands of intra- and
extracellular environmental signals.
The proteome varies with health or
disease, the nature of each tissue,
the stage of cell development, and
effects of drug treatments.
3 Kinds of Proteomics
Functional Proteomics
The identification of protein functions, activities or
interactions at a global or organism wide scale
Expressional Proteomics
The analysis of global or organism wide
changes in protein expression
Structural Proteomics
The high throughput, or high volume expression
and structure determination of proteins by Xray,
NMR or computer based methods
Components of
Expressional Proteomics
Protein Separation
Mass Spectroscopy
Bioinformatics
Pathway
Step 1: Sample preparation
Step 2: Separation
Step 3: Mass spectrometry
Sample preparation
Sample preparation
Sample preparation involves
everything that lies between the
sample and 1st dimension of the
2D SDS gel
Cells and cell cultures multiply
Homogenation and protein isolation
Contaminant removal/ cleanup
Fractionation
Cleanup and
fractionation
General Purpose
Cleanup
Improve Resolution
Improve Reproducibility
Fractionation
Reduce Complexity
Improve Range of
Detection
Enrich low-abundance
proteins
www.expressionproteomics.com
Separation
SDS PAGE
(Sodium dodecyl Sulfate Polyacrylamide Gel
Electrophoresis)
Sampel preparation
Preparing acrylamid gel
(Stacking)
Preparing acrylamid gels
(Resolving)
Running SDS page
2D-SDS PAGE gel
The first dimension
(separation by isoelectric focusing)
- gel with an immobilised pH gradient
- electric current causes charged
proteins to move until it reaches the
isoelectric point
The second dimension
(separation by mass)
-pH gel strip is loaded onto a SDS gel
-SDS denatures the protein (to make
movement solely dependent on mass,
not shape) and eliminates charge.
Can Resolve: ~1500-2500 proteins
Staining Technology
Staining
Silver
Coomassie blue
Fluorescent dyes
Sypro Ruby
Radioisotopic labeling
Mass Spectrometry
What is a Mass
Spectrometer?
A Mass Spectrometer is a machine that
weitghs molecules !
0 units
What is a Mass
Spectrometer?
A Mass Spectrometer is a machine that
weighs molecules !
12 units
What is a Mass
Spectrometer?
A Mass Spectrometer is a machine that
weighs molecules !
12 units
8
10 11 12 13 14 15 16
What is a Mass
Spectrometer?
A Mass Spectrometer is a machine that
weighs molecules !
14 units
8
10 11 12 13 14 15 16
What is a Mass
Spectrometer?
12 units
Number of counts
A Mass Spectrometer is a machine that
weighs molecules !
10 11 12 13 14 15 16
mass
How does a mass spectrometer
work?
Create ions
Ionization
method
MALDI
Electrospr
ay
(Proteins must be
charged and dry)
Separate
ions
Detect ions
Mass
Mass analyzer
spectrum
MALDI-TOF
Database
Quadrapole
analysis
MALDI-QqTOF
AA seq and MW
QqTOF
AA seq and protein
modif.
Proteomics
Applications
Why Proteomics?
Proteins are the active biological
agents in cells
DNA sequences dont show how
proteins function or how biological
processes occur
Proteins undergo post transcriptional
modifications
3D structures affect protein function
Alternative splicing
The Human Proteome Initiative. (2007) http://ca.expasy.org/sprot/hpi/hpi_desc.html Retrieved
March 24, 2009.
Challenges
Analyses of complex mixtures are not
comprehensive
Difficult to prepare a pure sample
Protein expression is very sensitive
to environmental conditions
Difficult to use ion currents to
determine peptide abundance
Protein Profiling
Generate large scale proteome
maps
Annotate and correct genomic
sequences
Analyze protein expression as a
function of cellular state
Conclusion
Proteomics is extremely valuable for
understanding biological processes
and advancing the field of systems
biology.
The ultimate goal of systems biology
is the integration of data from these
observations into models that might,
eventually, represent and simulate
the physiology of the cell.
GOOD LUCK
