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Outline
• What is proteomics?
• Why study proteins?
• Discuss proteomic tools and methods
What is proteomics?
Proteomics is the analysis of the protein complement to the
genome
Genomics
Proteomics
Gene
Transcript Protein
Wikipedia, http://en.wikipedia.org
“..the large-scale study of proteins…while it is often viewed as the “next step”, proteomics is much more complicated than genomics.
…while the genome is a rather constant entity, the proteome differs from cell to cell and is constantly changing through its biochemical interactions with the genome and the environment.
One organism will have radically different protein expression in different parts of its body, in different stages of its life cycle and in different environmental conditions.”
Proteomics is multidisciplinary
Proteomics
Molecular Biology
Biology Analytical Chemistry
Protein Biochemistry
Bioinformatics
Proteomics Research
•Basic research:To understand the molecular mechanisms underlying life.
•Applied research: Clinical testing for proteins associated with pathological states (e.g. cancer).
Applications of Proteomics
Proteomics
Structural Proteomics
Proteome Mining
Post-translational Modifications
Protein Expression
Profiling
Functional Proteomics
Protein-protein
Interactions
Glycoyslation
Phosphorylation
Proteolysis
Yeast two-hybrid
Co-precipitation
Phage Display
Drug Discovery
Target ID
Differential Display
Yeast Genomics
Affinity Purified Protein
Complexes
Mouse Knockouts
Medical Microbiology
Signal Transduction
Disease Mechanisms
Organelle Composition
Subproteome Isolation
Protein Complexes
For example: Hemoglobin
Picks up oxygen in the lungs, travels through the blood, and delivers it to the
cells.
O2
hemoglobin
Hbβ
Hbα
HbβHbα
ATG GTG CAC CTG ACT CCT GAG GAG …
ATG GTG CAC CTG ACT CCT GTG GAG …
EEM V H L T P … EVM V H L T P …
Normal Hbβ Mutated Hbβ
Sickle cell disease is caused by a single amino
acid change.
Summary – what is proteomics?
•Involves the study of proteins
•Proteomics is multidisciplinary
•Proteomics is being applied to both basic and clinical research
Why study proteins?
What are PROTEINS?
Proteins are large, complex molecules that serve diverse
functional and structural roles within cells.
TransportHemoglobi
nCarries O2
DefenseAntibodyFights Viruses
EnzymeProteaseDegrades Protein
SupportKeratin
Forms Hair and Nails
MotionActinContracts Muscles Regulatio
nInsulinControls Blood Glucose
Proteins do most of the work in the cell
Proteins are comprised of amino acid building blocks
R
O
OH
C
N H
H
Acid
Base
VariableCH
+
H2O
Dipeptide
Peptide Bond
Amino acid 1 Amino acid 2R1
OCC
N O
H
H2 H
R2
H
CC
N O
OH
H H
R1
O
CC
R2O
C C
H
H
NH2NH OH
Asparagine
Glutamate
LeucinePhenylalanine
Cysteine
Histidine
Methionine
Threonine
Arginine
Glutamine
IsoleucineTryptophan
Alanine
Glycine
Proline
Tyrosine
Aspartate
Lysine
Serine
Valine
Each amino acid has unique chemical properties.
non-polar hydrophobic
acidicbasic
polar hydrophilic
Proteins are chains of amino acids.
C
O
OH
N
H
H
NH
H
Short chains of amino acids are called peptides.
Proteins are polypeptide molecules that contain many
peptide subunits.
GAUA U G G C C U G G
5’
3’
Gene
Messenger Ribonucleic Acid (mRNA)
Amino Acid-
transfer RNA
Ribosome
tRNA
Ala
tRNA
Trp
Met
tRNA
Empty tRNA
MetEmpty tRNA
MetAla
Nucleus
Cytoplasm
Large Subunit
Small Subunit
MetAla
Trp
Ribonucleotides
A UG C
Codon 1 A U G =Methionine
CG CCodon 2 = Alanine
U GGCodon 3 Tryptophan=
U GCodon 4 Stop=A
Translation is the synthesis of proteins in the cell.
http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html
Proteins have specific architecture
Proteins arrive at their final structure in an
ordered fashion
J. E. Wampler, 1996, http://bmbiris.bmb.uga.edu/wampler/tutorial/prot0.html
Summary – why study proteins?
•Biological workhorses that carry out most of the functions within the cell
•Serve diverse functional and structural roles
•Composed of amino acids that are covalently linked by peptide bonds
•Synthesized during the translation process
•Must fold correctly to perform their functions
Proteomic tools and methods
Proteomic tools to study proteins
• Protein isolation
• Protein separation
• Protein identification
Protein Isolation
How are proteins isolated?
• Mechanical Methods– grinding – break open cell– centrifugation – remove insoluble debris
• Chemical Methods– detergent – breaks open cell compartments– reducing agent – breaks specific protein
bonds– heat – break peptide bonds to “linearize”
protein
Protein isolation procedure
Find a samplePick it
Grind sample in buffer
Transfer to tube
Heat the sampleCentrifuge to remove insoluble material
“pure” protein solution
Recover supernatant Keep solution for gel analysis
Protein X
“pure” protein solution
Isolated Protein X
Summary – protein isolation
•Proteins can be isolated from a variety of samples
•Proteomics includes the use of both mechanical and chemical methods to isolate proteins
•Opening cell or cellular compartments•Breaking bonds and “linearizing” proteins•Removal cell debris
Protein Separation
SDS-PAGE
Why separate proteins?
“PURE” Protein Solution
Tube 1
Decreased Protein IDIncreased Complexity
Tube 2
Increased Protein IDDecreased Complexity
How to separate proteins?
Separating intact proteins is to take advantage of their diversity in physical properties, especially isoelectric point and molecular
weight
Methods of Protein Separation
• Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis (SDS-PAGE)
• Isoelectric Focusing (IEF)
SDS-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) is a
widely used technique to separate proteins in solution
SDS-PAGE separates only by molecular weight
• Molecular weight is mass one molecule
• Dalton (Da) is a small unit of mass used to express atomic and molecular masses.
PAGE is widely used in
• Proteomics• Biochemistry• Forensics• Genetics• Molecular biology
Polyacrylamide gels separate proteins and small
pieces of DNA
• Major components of polyacrylamide gels
• Acrylamide – matrix material/ NEUROTOXIN
• Bis-acrylamide - cross-linking agent/ NEUROTOXINS
• TEMED - catalyst
• Ammonium persulfate - free radical initiator
HN
HN
O O
Bisacrylamide(cross-linking agent)
NH 2
O
Acrylamide(matrix material)
SO4
TEMED(catalyst)
Ammonium persulfate(free radical initiator)
Polyacrylamide (non-
toxic)
PolymerizationN N
Polyacrylamide (non-
toxic)
Polyacrylamide
O
NH
CH2
NH
O
O
NH
CH2
NH
O
CONH2 CON H2
CONH2
CONH
Bis-acrylamidecross links
Sodium dodecyl sulfate - SDS
The anionic detergent SDS unfolds or denatures proteins
• Uniform linear shape
• Uniform charge/mass ratio
Cathode (-)
Anode (+)Standard Sample1 Sample2
One-dimensional polyacrylamide gel
electrophoresis (SDS-PAGE)
During SDS-PAGE proteins separate according to their
molecular weight
BromophenolBlue dye front
Cathode (-)
Anode (+)Standard Sample1 Sample2
20 kDa
100 kDa75 kDa
50 kDa
37 kDa
25 kDa
150 kDa
Image of Real SDS-PAG
20 kDa
250 kiloDaltons
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
Cathode
Anode
Separation of Protein X
BromophenolBlue dye front
Cathode (-)
Anode (+)Standard Sample1 Sample2
20 kDa
100 kDa75 kDa
50 kDa
37 kDa
25 kDa
150 kDa
Protein X
11 kDa
25 kDa
Two-dimensional gel electrophoresis (2-DGE)
Most widely used protein separation technique in proteomics
Capable of resolving thousands of proteins from a complex sample (i.e. blood, organs, tissue…)
1st dimension - isoelectric focusing2nd dimension - SDS-PAGE
Isoelectric focusing (IEF) is separation of proteins according to native charge.
isoelectric point -pH at which net charge is zero
1st Dimension-Isoelectric Focusing
2-DGEprotein samples
IEF1st dimension
SDS-PAGE
2nd
dimension
Neutral at pH 3
20 kDa
100 kDa75 kDa
50 kDa
37 kDa
25 kDa
150 kDa
11 kDa
pH gradient 103
pI
mass
100
75
50
25
3 10
Arabidopsis developing leaf
kDa
2-DG
4 5 6 7 8 9
2-DGE
SDS-PAGE
2nd
dimension
20 kDa
100 kDa75 kDa
50 kDa
37 kDa
25 kDa
150 kDa
11 kDa
103 4 5 6 7 8 9
Protein X25 kDapI 5
1-DGE vs. 2-DGE
1-DGE (SDS-PAGE)• High reproduciblity• Quick/Easy• Separates solely based
on size• Modest resolution,
dependent on complexity of sample
2-DGE• Modest reproducibility • Slow/Demanding• Separates based on pI
and size• High resolution, not
dependent on complexity of sample
Summary – protein separation
•Protein separation takes advantage physical properties such as isoelectric point and molecular weight
•SDS-PAGE is a widely used technique to separate proteins
•1-DGE is a quick and easy method to separate protein by size only
•2-DGE combines isoeletric focusing (IEF) and SDS-PAGE to separate proteins by pI and size
Protein identification
mass spectrometry
Peptide mass fingerprintin
g
intact protein x
protein digestion
mass spectrometry
m/z
inte
nsi
ty 952.09841895.90571345.6342 899.87432794.9761
mass
Protein ID
Make proteolytic peptide fragments - Digest the protein into peptides (using trypsin)
Measure peptide masses - “Weigh” the peptides in a mass spectrometer
Match peptide masses to protein or nucleotide sequence database - Compare the data to known proteins and look for a match
Protein digestion
We use the enzyme TRYPSIN to digest (cut) proteins into peptides – trypsin cuts after Lysine (K) and Arginine (R)
????????K?????R????????????????K?????R????????????????K?????R????????Protein X
????????K?????R????????????????K?????R????????????????K?????R????????
How does mass spectrometry identify unknown proteins?
Basics of mass spectrometry
• determination of mass to charge ratio (m/z)
• Mass spectrometer = very accurate weighing scales– third or fourth decimal place
????????K
?????R
????????
We then “weigh” these peptides with a Mass
Spectrometer
Mass Spectrometer
????????K
?????R
????????
We then “weigh” these peptides with a Mass
Spectrometer
692.31 Da
1106.55 Da
1002.37Da
Mass of peptides should be compared to theoretical masses of
known peptides
?????R = 692.31 Da
????????K = 1106.55 Da
???????? = 1002.37Da
Computation of theoretical masses of known peptides
knownComputer
Peptides• WEGETMILK 1106.55• ADEMTYEK 1105.23• PLMEHGAK 1089.50• LMEHHH 782.25• ASTEER 692.31• DMGEYIILES 1056.92• EGEDMPAFY 1002.35• CYHGMEI 984.36• EFPKLYSEK 900.56• YSEPYSSIIR 1102.34• IESPLMIA 864.35• AEFLYSR 600.21• DLMILIYR 864.97• METHIPEEK 795.36• KISSMER 513.21• PEPTIDEK 456.23• MANYCQWS 792.15• TYSMEDGHK 678.46• YMEPSATFGHR 995.46• GHLMEDFSAC 896.35• HHFAASTR 564.88• ALPMESS 469.12
Proteome = all protein sequences
Digest Proteome with simulated
Trypsin
Mass of peptides compared to theoretical masses of all peptides
known, using a computer program.
?????R = 692.31 Da
????????K = 1106.55 Da
???????? = 1002.37Da
Computer Peptides
• WEGETMILK 1106.55• ADEMTYEK 1105.23• PLMEHGAK 1089.50• LMEHHH 782.25• ASTEER 692.31• DMGEYIILES 1056.92• EGEDMPAFY 1002.35• CYHGMEI 984.36• EFPKLYSEK 900.56• YSEPYSSIIR 1102.34• IESPLMIA 864.35• AEFLYSR 600.21• DLMILIYR 864.97• METHIPEEK 795.36• KISSMER 513.21• PEPTIDEK 456.23• MANYCQWS 792.15• TYSMEDGHK 678.46• YMEPSATFGHR 995.46• GHLMEDFSAC 896.35• HHFAASTR 564.88• ALPMESS 469.12
Mass of peptides matched to theoretical masses known
peptides, using a computer program.
?????R = 692.31 Da
????????K = 1106.55 Da
???????? = 1002.37Da
Computer Peptides
• WEGETMILK 1106.55• ADEMTYEK 1105.23• PLMEHGAK 1089.50• LMEHHH 782.25• ASTEER 692.31• DMGEYIILES 1056.92• EGEDMPAFY 1002.35• CYHGMEI 984.36• EFPKLYSEK 900.56• YSEPYSSIIR 1102.34• IESPLMIA 864.35• AEFLYSR 600.21• DLMILIYR 864.97• METHIPEEK 795.36• KISSMER 513.21• PEPTIDEK 456.23• MANYCQWS 1002.37• TYSMEDGHK 678.46• YMEPSATFGHR 995.46• GHLMEDFSAC 896.35• HHFAASTR 564.88• ALPMESS 469.12
The unknown peptides have been identified
?????R = 692.31 Da
????????K = 1106.55 Da
???????? = 1002.37Da
WEGETMILK
ASTEER
MANYCQWS
Protein X has been identified
????????K?????R????????????????K?????R????????????????K?????R????????WEGETMILK AFTEER MANYCQWS
Summary – tools to study proteins?
•Proteins are digested into peptides
•Peptides are analyzed with a mass spectrometer
•Match observed peptide masses to theoretical masses of all peptides in database
•Assemble those peptide matches into a protein identification
Concluding points about Proteomics
-Proteomics is the analysis of all proteins
-Interdisciplinary research
-Essential to both basic and clinical research
-Protein are the workhorses of the cell
- Discovery research – drugs and diseases
-Proteomics tools allow identification of proteins
Questions