PROTEOMICS LECTURE. Genomics DNA (Gene) Functional Genomics TranscriptomicsRNA Proteomics PROTEIN Metabolomics METABOLITE Transcription Translation Enzymatic.

PROTEOMICSLECTURE

Genomics DNA (Gene)

FunctionalGenomics

Transcriptomics RNA

Proteomics PROTEIN

Metabolomics METABOLITE

Transcription

Translation

Enzymatic reaction

The “omics” nomenclature…

GenTranscriptProteMetabol

~ome Sequence of a complete set of

GenesTranscriptsProteinsMetabolites

=

GenProte

~omics = Analysis of the GenomeProteome

A few definitions…

Why study protein expression?(The steps of gene expression control)

Nucleus Cytosol

DNA

PrimaryRNA

transcriptmRNA mRNA

protein Modified protein

Transcriptional control

RNA Processing

control

RNATransport

control

Inactive mRNARNA

Degradationcontrol

Translation control

Post-translationalcontrol

(Gygi et al., Mol. Celll. Biol., 1990, p.1720-1730)

Applications of Proteomics• Mining: identification of proteins

(catalog the proteins)• Protein-expression profile:

identification of proteins in a particular state of the organism

• Protein-network mapping: protein interactions in living systems

• Mapping of protein modifications: how and where proteins are modified.

Proteins classes for Analysis

• Membrane• Soluble proteins• Nuclear• Chromosome-

associated• Phosphorylated• Glycosylated• Complexes

General flow for

proteomics analysis

SEPA

RA

TIO

N

IDE

NTIF

ICA

TIO

N

Current Proteomics Technologies• Proteome profiling/separation

– 2D SDS PAGE (two-dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis)

– 2-D LC/LC (LC = Liquid Chromatography)– 2-D LC/MS (MS= Mass spectrometry)

• Protein identification– Peptide mass fingerprint– Tandem Mass Spectrometry (MS/MS)

• Quantative proteomics- ICAT (isotope-coded affinity tag)

1) Sample loading

2) Remove the cover sheet from the IEF gel

3)Place the strip gel in the focusing tray

4) Place the strip on the top of the SDS-

PAGE gel

2D-SDS PAGE gel

The first dimension (separation by isoelectric focusing)- gel with an immobilised pH gradient- electric current causes charged proteins to move until it reaches the isoelectric point (pH gradient makes the net charge 0)

2D-SDS PAGE gel

Isoelectric point (pI)

• Separation by charge:

4

5

6

7

8

9

10

Sta

ble

pH

g

rad

ien

t

High pH: protein is negatively charged

Low pH:Protein is positively charged

At the isolectric point the protein has no net charge and therefore no longer migrates in the electric field.

The first dimension (separation by isoelectric focusing)- gel with an immobilised pH gradient- electric current causes charged proteins to move until it reaches the isoelectric point (pH gradient makes the net charge 0)

The second dimension (separation by mass)-pH gel strip is loaded onto a SDS gel-SDS denatures and linearises the protein (to make movement solely dependent on mass, not shape)

2D-SDS PAGE gel

2D-SDS PAGE gel

2D-gel technique example

Advantages vs. Disadvantages

• Good resolution of proteins

• Detection of posttranslational modifications

• Not for hydrophobic proteins

• Limited by pH range

• Not easy for low abundant proteins

• Analysis and quantification are difficult

2D - LC/LC

Study protein complexes without gel electrophoresis

Peptides all bind to cation exchange column

Peptides are separated by hydrophobicity on reverse phase column

Successive elution with increasing salt gradients separates peptides by charge

Complex mixture is simplified prior to MS/MS by 2D LC

(trypsin)

Polypeptides enter the column in the mobile phase…

…the hydrophobic “foot” of the polypeptides adsorb to the hydrophobic (non polar) surface of

the reverse-phase material (stationary phase) where they remain until…

…the organic modifier concentration rises to critical concentration and desorbs the polypeptides

Reverse Phase column

2D - LC/MS

Methods for protein

identification

Mass Spectrometry (MS) Stages

• Introduce sample to the instrument• Generate ions in the gas phase• Separate ions on the basis of differences

in m/z with a mass analyzer • Detect ions

How the protein sequencing works?

• Use Tandem MS: two mass analyzer in series with a collision cell in between

• Collision cell: a region where the ions collide with a gas (He, Ne, Ar) resulting in fragmentation of the ion

• Fragmentation of the peptides in the collision cell occur in a predictable fashion, mainly at the peptide bonds (also phosphoester bonds)

• The resulting daughter ions have masses that are consistent with known molecular weights of dipeptides, tripeptides, tetrapeptides…

Ser-Glu-Leu-Ile-Arg-Trp

Collision Cell

Ser-Glu-Leu-Ile-Arg

Ser-Glu-Leu

Ser-Glu-Leu-Ile

Etc…

Isolates individual peptide fragments for 2nd mass spec – can obtain peptide sequence

Compare peptide sequence with protein

databases

(trypsin)

Tandem Mass Spectrometry

Advantages vs. Disadvantages

• Determination of MW and aa. Sequence

• Detection of posttranslational modifications

• High-throughput capability

• High capital costs

• Requires sequence databases for analysis

Protein identification by Peptide Mass fingerprint

• Use MS to measure the masses of proteolytic peptide fragments.

• Identification is done by matching the measured peptide masses to corresponding peptide masses from protein or nucleotide sequence databases.

Mass spectrometry – method of separating molecules based on mass/charge ratio

Compare peptide m/z with protein

databases

eg. MALDI-TOF

(trypsin)

Mass spectometry (MS)

Protein Identification by MS

Artificial spectra built

Artificially trypsinated

Database of sequences

(i.e. SwissProt)

Spot removed from gel

Fragmented using trypsin

Spectrum of fragments generated

MATCHLi

bra

ry

ISOTOPE-CODED AFFINITY TAG (ICAT): a quantitative method

• Label protein samples with heavy and light reagent

• Reagent contains affinity tag and heavy or light isotopes

Chemically reactive group: forms a covalent bond to the protein or peptideIsotope-labeled linker: heavy or light, depending on which isotope is usedAffinity tag: enables the protein or peptide bearing an ICAT to be isolated by affinity chromatography in a single step

Example of an ICAT Reagent

S OI

NH

**

* *

O

OON

H

O

O

NH

NH

Biotin Affinity tag: Binds tightly to streptavidin-agarose resin

Linker: Heavy version will have deuteriums at *Light version will have hydrogens at *

Reactive group: Thiol-reactive group will bind to Cys

How ICAT works?

Proteolysis (eg trypsin)

Lyse & Label

MIX

Affinity isolation on streptavidin

beads

QuantificationMS

IdentificationMS/MS

100

m/z200 400 600

0

100

550 570 5900

m/z

Light

Heavy

NH2-EACDPLR-COOH

Advantages vs. Disadvantages

• Estimates relative protein levels between samples with a reasonable level of accuracy (within 10%)

• Can be used on complex mixtures of proteins

• Cys-specific label reduces sample complexity

• Peptides can be sequenced directly if tandem MS-MS is used

• Yield and non specificity• Slight chromatography

differences• Expensive• Tag fragmentation• Meaning of relative

quantification information

• No presence of cysteine residues or not accessible by ICAT reagent