What is proteomics?

Proteomics is the analysis of the

protein complement to the genome

Gene Transcript Protein

Genomics Proteomics
Proteomics is multidisciplinary

Protein
Biochemistry
Analytical
Biology
Chemistry

Proteomics

Molecular
Bioinformatics
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 Signal Disease
Microbiology Transduction Mechanisms

Protein
Drug Discovery Glycoyslation
Expression
Profiling Post-
Proteome
Target ID translational Phosphorylation
Mining
Modifications
Differential Display Proteolysis

Proteomics
Yeast Genomics Yeast two-hybrid
Protein-
Affinity Purified Functional protein Co-precipitation
Protein Complexes Proteomics Interactions
Mouse Knockouts Structural Phage Display
Proteomics

Organelle Subproteome Protein

Composition Isolation 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.
Normal Hbβ Mutated Hbβ

ATG GTG CAC CTG ACT CCT GAG GAG … ATG GTG CAC CTG ACT CCT GTG GAG …

M V H L T P E E… 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
Transport
Degrades Protein
Hemoglobin
Motion Carries O2
Actin
Contracts Muscles
Regulation
Insulin
Controls Blood Glucose
Support
Keratin
Defense Forms Hair and
Antibody Nails
Fights Viruses
Proteins are comprised of amino
acid building blocks
O Amino acid 1 Amino acid 2
Acid
R1 R2
C OH
H C C O + H C C O

R
Variable
CH H2N OH H N OH
H
H2O
N H
H O R2 O

H Base R1 C C H C C
Dipeptide
H2N H N OH
Peptide Bond
 Each amino acid has unique
chemical properties.
basic
acidic

Histidine Aspartate
Glutamate
Lysine
Arginine

non-polar hydrophobic

Valine
Proline
Alanine Isoleucine Phenylalanine
Leucine Methionine Tryptophan

polar hydrophilic

Serine Cysteine

Glutamine
Glycine Tyrosine Asparagine Threonine
Proteins are chains of amino acids.
O
C OH

N H
H
Short chains of amino acids are
called peptides.
N H
Proteins are polypeptide molecules H
that contain many peptide subunits.
 Gene

Nucleus 3’

Messenger
Trp Ribonucleic Acid
tRNA (mRNA)
Ala
tRNA
Met
Amino Acid-
tRNA
Met transfer
5’ Ribosome
Large Subunit Ala RNA
Met Trp
Empty tRNA
Empty tRNA

A U G G C C U G G U A G
Small Subunit
Cytoplasm Ribonucleotides A G C U

Codon 1 A U G = Methionine Codon 3 U G G = Tryptophan

Codon 2 G C C = Alanine Codon 4 U A G = Stop

Translation is the synthesis of proteins in the cell.

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 Grind sample in buffer

Pick it

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 Tube 2
Increased Complexity Decreased Complexity
Decreased Protein ID 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 H
N N
Polymerization
N N
O O
TEMED
(catalyst)

Am monium persulfate
(free radical initiator) SO4
Polyacrylamide
(non-toxic)
Polyacrylamide
C ON2H C ON2H

Polyacrylamide
(non-toxic) O O

NH NH
Bis-acrylamide
C H2 cross links C H2

NH NH

O 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

Bromophenol
Anode (+) Blue dye front
Standard Sample1 Sample2
 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

25 kDa Protein X 25 kDa

20 kDa
11 kDa
Bromophenol
Anode (+) Blue dye front
Standard Sample1 Sample2
 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
protein
3 pH gradient 10
samples
1st dimension
IEF Neutral at pH 3

150 kDa
100 kDa
75 kDa
50 kDa
2nd dimension
37 kDa
SDS-PAGE
25 kDa

20 kDa
11 kDa
 2-DG
kDa 3 4 5 6 7 8 9 10 pI
100

75
mass

50

25

Arabidopsis developing leaf

 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) 2-DGE
• High reproduciblity • Modest reproducibility
• Quick/Easy • Slow/Demanding
• Separates solely based • Separates based on pI and
on size size
• Modest resolution, • High resolution, not
dependent on complexity dependent on complexity
of sample 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 intact protein x
fingerprinting protein digestion

Make proteolytic peptide

fragments - Digest the mass spectrometry
protein into peptides (using
trypsin)

intensity
Measure peptide masses -
“Weigh” the peptides in a m/z

mass spectrometer
mass
952.0984
1895.9057
Match peptide masses to 1345.6342
899.8743
protein or nucleotide 2794.9761

sequence database - Compare

the data to known proteins
and look for a match 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
Computer Peptides
Proteome = all protein sequences •
•
WEGETMILK
ADEMTYEK
1106.55
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

Digest Proteome with •

•
MANYCQWS
TYSMEDGHK
792.15
678.46

simulated Trypsin •
•
YMEPSATFGHR
GHLMEDFSAC
995.46
896.35
• HHFAASTR 564.88
• ALPMESS 469.12
 Mass of peptides compared to
theoretical masses of all peptides
known, using a computer program.
Computer Peptides
• WEGETMILK 1106.55
• ADEMTYEK 1105.23
• PLMEHGAK 1089.50

????????K = 1106.55 Da •
•
LMEHHH
ASTEER
782.25
692.31
• DMGEYIILES 1056.92
• EGEDMPAFY 1002.35
• CYHGMEI 984.36

?????R = 692.31 Da •
•
EFPKLYSEK
YSEPYSSIIR
900.56
1102.34
• IESPLMIA 864.35
• AEFLYSR 600.21
• DLMILIYR 864.97
• METHIPEEK 795.36

???????? = 1002.37Da
• 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.
Computer Peptides
• WEGETMILK 1106.55
• ADEMTYEK 1105.23
• PLMEHGAK 1089.50

????????K = 1106.55 Da •
•
LMEHHH
ASTEER
782.25
692.31
• DMGEYIILES 1056.92
• EGEDMPAFY 1002.35
• CYHGMEI 984.36

?????R = 692.31 Da •
•
EFPKLYSEK
YSEPYSSIIR
900.56
1102.34
• IESPLMIA 864.35
• AEFLYSR 600.21
• DLMILIYR 864.97
• METHIPEEK 795.36
• KISSMER 513.21
???????? = 1002.37Da • 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

????????K = 1106.55 Da WEGETMILK

?????R = 692.31 Da ASTEER

???????? = 1002.37Da 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