Proteomics

&

Neurodegenerative Disorders

Simonetta Sipione9-21 Medical Sciences Building

Email: ssipione@ualberta.ca

Genome Transcriptome Proteome Metabolome

DNA mRNA proteins metabolites

The full complement of

proteins produced by a

particular genome

The full complement

of expressed mRNA

The complete complement of

all small molecule (<1500 Da)

metabolites found in a specific

cell, organ or organism.

Genome Transcriptome Proteome Metabolome

DNA mRNA proteins metabolites

Functional Genomics

Databases

Mathematical modeling

Analysis

Transcriptomics

Proteomics

Metabolomics

Geno

mics

Systems

Biology

Classical protein biochemistry

versus proteomics…

Proteomics Applications

Protein expression

profiling

Clinical proteomics Disease proteomics

Protein-protein interactions

Biomarkers

discovery

Drug

development

Disease

mechanisms

Proteomics Flow Chart

Biological Sample

Protein Separation

Preparation for

quantitation

Mass spectrometry

analysis

Data analysis

Protein identification

& quantification

2D-electrophoresis

Excision &

trypsin digestion

Liquid chromatography

Protein separation methods

Two-dimensional gel electrophoresis (2-DE)

One- and two-dimensional liquid chromatography

(1D-LC and 2D-LC)

Biological Sample

Protein Separation

Preparation for

quantitation

Mass spectrometry

analysis

Data analysis

Separation by 2D-electrophoresis

Maldi-TOF

Mass spectrometry

&

Trypsin digestion

Separation by 2D-electrophoresis

Isoelectric focusing(pH)

SD

S-P

AG

E

(siz

e)

Sample A

Sample B

Labeling with

fluorescent dyes

(Cy3, Cy5 etc.)

Sample A

Sample B

Differential in-gel electrophoresis

(2D-DIGE)

2D-electrophoresis

Gel imaging & data analysis

Excision and trypsin digestion

Mass spectrometry

Separation by 1D and 2D liquid chromatography

Mass spectrometry

1D-LC: peptide mixture are separated based

on hydrophobicity.

2D-LC: isoelectric chromatography followed by

separation by hydrophobicity (MudPIT -

“multidimensional protein identification technology”)

Mass Spectrometry Techniques

Matrix-assisted laser desorption ionization

(MALDI) time-of-flight (TOF) mass

spectrometry (MS) (MALDI-TOF MS)

Electrospray ionization (ESI) MS (1D-LC and 2D-LC)

Biological Sample

Protein Separation

Preparation for

quantitation

Mass spectrometry

analysis

Data analysis

Mass Spectrometry Techniques

www.udel.edu/.../ websites/Lloyd/MALDI

MALDI-TOF MS

www.chm.bris.ac.uk/ ms/theory/esi-ionisation.html

Mass Spectrometry Techniques

ESI MS

Micro-Sequencing by Tandem Mass Spectrometry

(MS/MS)

• Ions of interest are selected in the first mass analyzer

• Collision Induced Dissociation (CID) is used to fragment the selected ions by colliding the ionswith gas (typically Argon for low energy CID)

• The second mass analyzer measures the fragment ions

• Fragmentation of peptides (amino acid chains) typically occurs along the peptide backbone. Eachresidue of the peptide chain successively fragments off, both in the N->C and C->N direction.

MALDI-MS

ESI-MSMS

Argon

Collision Cell

Sequence Nomenclature for Mass Ladder

H2NHC C

O

R1

HN

HC

R2

C

O

HN

HC

R3

C

O

HN CH

R4

C

O

OH

a1 a3a2 b3b2b1 c2c2c1

x1 x3x2 y3y2y1 z2z2z1

1598

14241166 965

723529401

TGPNLHGFGRR

GR FGRGFGR etc

Second Stage (fragmentation) Mass Spectrum

m/z75 2000

GDVEKGKKIFVQKC

AQCHTVEKGGKHKT

GPNLHGLFGRKTGQ

APGFTYTDANKNKGI

TWKEETLMEYLENP

KKYIPGTKMIFAGIKK

KTEREDLIAYLKKAT

NE

TGPNLHGLFGR

Protein Sequence

Data analysis and

database search

(MASCOT Engine)

AAT = !1-anti-trypsin ABC = !B-crystallin

Quantitative proteomics

Biological Sample

Protein Separation

Preparation for

quantitation

Mass spectrometry

analysis

Data analysis

ICAT

Sample A

Labeling of Cys with

ICAT reagent

Sample A“heavy”

Quantitative Proteomics:

Isotope-coded affinity tags (ICAT)

Digestion & LC/MS/MS separation

Sample B

2H2H2H

1H1H1H

+ +

Sample B“light”

Quantitative Proteomics:

Isotope-coded affinity tags (ICAT)

Analysis of protein interactions

In situ proteomics

MALDI-TOF MS

Matrix application

Frozen section MS images

Laser

Nature Medicine 7, 493 - 496 (2001)

In situ proteomics

Nature Medicine 7, 493 - 496 (2001)

Further reading

David DC, Hoerndli F, Gotz J. Functional Genomics meets neurodegenerative disorders.

Part I: transcriptomic and proteomic technology.

Prog Neurobiol. 2005 Jun;76(3):153-68.

Hoerndli F, David DC, Gotz J. Functional Genomics meets neurodegenerative disorders.

Part II: application and data integration.

Prog Neurobiol. 2005 Jun;76(3):169-88.