PROTEOMICS LECTURE

The omics nomenclature

Genomics DNA (Gene) Transcription

Transcriptomics
Functional Genomics Proteomics

RNA
Translation PROTEIN Enzymatic reaction

Metabolomics

METABOLITE

A few definitions
Gen Transcript Prote Metabol ~ome = Sequence of a complete set of Genes Transcripts Proteins Metabolites

Gen Prote

~omics =

Analysis of the

Genome Proteome

Why study protein expression?

(The steps of gene expression control)
(Gygi et al., Mol. Celll. Biol., 1990, p.1720-1730)

Nucleus

Cytosol
RNA Degradation control Inactive mRNA

DNA

Primary RNA transcript

mRNA

mRNA Translation control

Transcriptional control

RNA Processing control

RNA Transport control

protein

Modified protein

Post-translational control

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

IDENTIFICATION

General flow for proteomics analysis

SEPARATION

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)

2D-SDS PAGE gel

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)

Isoelectric point (pI)

Separation by charge:
4

5 6
7 8 9 10

Low pH: Protein is positively charged

High pH: protein is negatively charged

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

Stable pH gradient

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)

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-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
(trypsin)

Peptides all bind to cation exchange column Successive elution with increasing salt gradients separates peptides by charge Peptides are separated by hydrophobicity on reverse phase column

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

Reverse Phase column

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

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-Ile Ser-Glu-Leu

Etc

Tandem Mass Spectrometry

(trypsin)

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

Compare peptide sequence with protein databases

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 spectometry (MS)

(trypsin)

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

eg. MALDI-TOF

Compare peptide m/z with protein databases

Protein Identification by MS
Spot removed from gel Fragmented using trypsin Spectrum of fragments generated

Library

MATCH

Artificial spectra built

Artificially trypsinated

Database of sequences (i.e. SwissProt)

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 peptide
Isotope-labeled linker: heavy or light, depending on which isotope is used Affinity tag: enables the protein or peptide bearing an ICAT to be isolated by affinity chromatography in a single step

Example of an ICAT Reagent

Biotin Affinity tag: Binds tightly to streptavidin-agarose resin Reactive group: Thiolreactive group will bind to Cys

O
NH NH

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

H N S O

* *

O O

O
*

H N I O

How ICAT works?

Affinity isolation on streptavidin beads Lyse & Label

Quantification MS
Light
Heavy

Identification MS/MS
NH2-EACDPLR-COOH 100

100

MIX

Proteolysis (eg trypsin)

0
550

m/z

570

590

200

m/z

400

600

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