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
Gene
Genomics
Transcript
Protein
Proteomics
One organism
will have
radically
different
protein
…while
the genome
is a rather
constant
entity,
the
“..the
large-scale
study
proteins…while
it
often
expression
in different
parts
body,
in is
different
proteome
differs
from of
cell
to of
cellitsand
is constantly
viewed
the
“next
is environmental
much with
morethe
stages as
ofthrough
its life
cycle
andproteomics
in different
changing
itsstep”,
biochemical
interactions
complicated
thanenvironment.
genomics.
conditions.”
genome
and the
Wikipedia, http://en.wikipedia.org
Proteomics is multidisciplinary
Protein
Biochemistry
Biology
Analytical
Chemistry
Proteomics
Bioinformatics
Molecular
Biology
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
Medical
Microbiology
Drug Discovery
Target ID
Proteome
Mining
Differential Display
Signal
Transduction
Protein
Expression
Profiling
Disease
Mechanisms
Posttranslational
Modifications
Phosphorylation
Proteolysis
Proteomics
Yeast Genomics
Affinity Purified
Protein Complexes
Glycoyslation
Yeast two-hybrid
Functional
Proteomics
Structural
Proteomics
Mouse Knockouts
Organelle
Composition
Subproteome
Isolation
Proteinprotein
Interactions
Co-precipitation
Phage Display
Protein
Complexes
For example: Hemoglobin
Picks up oxygen in the lungs, travels through
the blood, and delivers it to the cells.
Hbβ
Hbα
O2
Hbα
Hbβ
hemoglobin
Sickle cell disease is caused
by a single amino acid change.
Mutated Hbβ
Normal Hbβ
ATG GTG CAC CTG ACT CCT GAG GAG …
M V H L
T P
E
E…
ATG GTG CAC CTG ACT CCT GTG GAG …
M V H L
T P
V
E…
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.
Proteins do most of the work
in the cell
Enzyme
Protease
Degrades Protein
Motion
Actin
Contracts Muscles
Transport
Hemoglobin
Carries O2
Regulation
Insulin
Controls Blood Glucose
Defense
Antibody
Fights Viruses
Support
Keratin
Forms Hair and
Nails
Proteins are comprised of amino
acid building blocks
O
Acid
Amino acid 1
R1
C OH
R
Amino acid 2
R2
H C C
CH
H2N
O
H C C O
+
H
OH
Variable
N OH
H
N H
H Base
R1
H
O
R2
O
C
C H C
C
H
OH
H2N
N
Peptide Bond
H2O
Dipeptide
Each amino acid has unique
chemical properties.
basic
acidic
Histidine
Aspartate
Lysine
Glutamate
Arginine
non-polar hydrophobic
Valine
Alanine
Isoleucine
Leucine
Proline
Methionine
Phenylalanine
polar hydrophilic
Serine
Glycine
Tryptophan
Cysteine
Glutamine
Tyrosine
Asparagine
Threonine
Proteins are chains of amino acids.
O
C OH
N H
H
Short chains of amino acids are
called peptides.
Proteins are polypeptide molecules
that contain many peptide subunits.
N H
H
Gene
3’
Nucleus
Messenger
Ribonucleic Acid
(mRNA)
Trp
tRNA
Ala
tRNA
Met
Met
Ribosome
Large
Subunit
Met
5’
Amino Acidtransfer
RNA
tRNA
Ala
Empty tRNA
Trp
Empty tRNA
A
U
G
G
C
C
U
G
G
U
A
G
Small Subunit
Cytoplasm
Ribonucleotides
Codon 1
A
U
G = Methionine
Codon 3
U
G
G = Tryptophan
Codon 2
G
C
C
Codon 4
U
A
G = Stop
= Alanine
A
G
C
Translation is the synthesis of proteins in the cell.
U
Proteins have specific architecture
http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html
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 sample
Pick it
Grind sample in buffer
Transfer to tube
Centrifuge to remove Heat the sample
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
Increased Complexity
Decreased Protein ID
Tube 2
Decreased Complexity
Increased Protein ID
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
Acrylamide
(matrix material)
NH2
O
Bisacrylam ide
(cross-linking agent)
H
N
O
H
N
O
TEMED
Polymerization
N
N
(catalyst)
Am monium persulfate
(free radical initiator) SO4
Polyacrylamide
(non-toxic)
Polyacrylamide
C ON2H
Polyacrylamide
(non-toxic)
O
C ON2H
O
NH
C H2
Bis-acrylamide
cross links
NH
O
NH
C H2
NH
C ON2H
O
C ONH
Sodium dodecyl sulfate - SDS
The anionic detergent SDS unfolds or
denatures proteins
• Uniform linear shape
• Uniform charge/mass
ratio
One-dimensional polyacrylamide
gel electrophoresis (SDS-PAGE)
Cathode (-)
Anode (+)
Standard
Sample1
Sample2
During SDS-PAGE proteins separate
according to their molecular weight
Cathode (-)
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
20 kDa
Anode (+)
Standard
Sample1
Sample2
Bromophenol
Blue dye front
Image of Real SDS-PAG
Cathode
250 kiloDaltons
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
20 kDa
Anode
Separation of Protein X
Cathode (-)
150 kDa
100 kDa
75 kDa
50 kDa
37 kDa
Protein X 25 kDa
25 kDa
20 kDa
11 kDa
Anode (+)
Standard
Sample1
Sample2
Bromophenol
Blue dye front
Two-dimensional gel
electrophoresis (2-DGE)
1st dimension - isoelectric focusing
2nd dimension - SDS-PAGE
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
Focusing
Isoelectric focusing (IEF) is separation of
proteins according to native charge.
isoelectric point -pH at which net charge is zero
2-DGE
pH gradient
3
1st dimension
IEF
10
protein
samples
Neutral at pH 3
150 kDa
100 kDa
75 kDa
2nd
dimension
SDS-PAGE
50 kDa
37 kDa
25 kDa
20 kDa
11 kDa
2-DG
kDa 3
100
4
5
6
7
mass
75
50
25
Arabidopsis developing leaf
8
9
10
pI
2-DGE
3
4
5
6
7
8
9
10
150 kDa
100 kDa
75 kDa
50 kDa
2nd dimension
37 kDa
SDS-PAGE
25 kDa
20 kDa
11 kDa
Protein X
25 kDa
pI 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 SDSPAGE to separate proteins by pI and size
Protein identification
mass spectrometry
Peptide mass
fingerprinting
Measure peptide masses “Weigh” the peptides in a
mass spectrometer
protein digestion
mass spectrometry
intensity
Make proteolytic peptide
fragments - Digest the
protein into peptides (using
trypsin)
intact protein x
m/z
mass
Match peptide masses to
protein or nucleotide
sequence database - Compare
the data to known proteins
and look for a match
952.0984
1895.9057
1345.6342
899.8743
2794.9761
Protein ID
Protein digestion
We use the enzyme TRYPSIN to digest (cut) proteins
into peptides – trypsin cuts after Lysine (K) and
Arginine (R)
Protein X
????????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
We then “weigh” these peptides
with a Mass Spectrometer
????????K
?????R
????????
Mass Spectrometer
We then “weigh” these peptides
with a Mass Spectrometer
????????K
1106.55 Da
?????R
692.31 Da
????????
1002.37Da
Mass of peptides should be compared to
theoretical masses of known peptides
????????K = 1106.55 Da
?????R = 692.31 Da
???????? = 1002.37Da
Computation of theoretical masses of
known peptides known
Proteome = all protein sequences
Digest Proteome with
simulated Trypsin
Computer Peptides
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WEGETMILK
ADEMTYEK
PLMEHGAK
LMEHHH
ASTEER
DMGEYIILES
EGEDMPAFY
CYHGMEI
EFPKLYSEK
YSEPYSSIIR
IESPLMIA
AEFLYSR
DLMILIYR
METHIPEEK
KISSMER
PEPTIDEK
MANYCQWS
TYSMEDGHK
YMEPSATFGHR
GHLMEDFSAC
HHFAASTR
ALPMESS
1106.55
1105.23
1089.50
782.25
692.31
1056.92
1002.35
984.36
900.56
1102.34
864.35
600.21
864.97
795.36
513.21
456.23
792.15
678.46
995.46
896.35
564.88
469.12
Mass of peptides compared to
theoretical masses of all peptides
known, using a computer program.
Computer Peptides
????????K = 1106.55 Da
?????R = 692.31 Da
???????? = 1002.37Da
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WEGETMILK
ADEMTYEK
PLMEHGAK
LMEHHH
ASTEER
DMGEYIILES
EGEDMPAFY
CYHGMEI
EFPKLYSEK
YSEPYSSIIR
IESPLMIA
AEFLYSR
DLMILIYR
METHIPEEK
KISSMER
PEPTIDEK
MANYCQWS
TYSMEDGHK
YMEPSATFGHR
GHLMEDFSAC
HHFAASTR
ALPMESS
1106.55
1105.23
1089.50
782.25
692.31
1056.92
1002.35
984.36
900.56
1102.34
864.35
600.21
864.97
795.36
513.21
456.23
792.15
678.46
995.46
896.35
564.88
469.12
Mass of peptides matched to
theoretical masses known peptides,
using a computer program.
Computer Peptides
????????K = 1106.55 Da
?????R = 692.31 Da
???????? = 1002.37Da
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WEGETMILK
ADEMTYEK
PLMEHGAK
LMEHHH
ASTEER
DMGEYIILES
EGEDMPAFY
CYHGMEI
EFPKLYSEK
YSEPYSSIIR
IESPLMIA
AEFLYSR
DLMILIYR
METHIPEEK
KISSMER
PEPTIDEK
MANYCQWS
TYSMEDGHK
YMEPSATFGHR
GHLMEDFSAC
HHFAASTR
ALPMESS
1106.55
1105.23
1089.50
782.25
692.31
1056.92
1002.35
984.36
900.56
1102.34
864.35
600.21
864.97
795.36
513.21
456.23
1002.37
678.46
995.46
896.35
564.88
469.12
The unknown peptides have been
identified
????????K = 1106.55 Da
?????R = 692.31 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