Outline What is proteomics? Why study proteins? Discuss proteomic tools and methods

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