Lecture 10

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

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

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

Lecture 10

Uploaded by

Copyright:

Available Formats

Lecture 10

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Lecture 10

Uploaded by

Copyright:

Available Formats

PROTEOMICS LECTURE

The omics nomenclature

Why study protein expression?

Primary RNA transcript

mRNA Translation control

RNA Processing control

RNA Transport control

Proteins classes for Analysis

General flow for proteomics analysis

Current Proteomics Technologies

2D-SDS PAGE gel

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

Isoelectric point (pI)

Low pH: Protein is positively charged

High pH: protein is negatively charged

2D-SDS PAGE gel

The second dimension

2D-SDS PAGE gel

2D-gel technique example

Advantages vs. Disadvantages

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

Reverse Phase column

Methods for protein identification

Mass Spectrometry (MS) Stages

How the protein sequencing works?

Tandem Mass Spectrometry

Compare peptide sequence with protein databases

Advantages vs. Disadvantages

Protein identification by Peptide Mass fingerprint

Mass spectometry (MS)

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

Compare peptide m/z with protein databases

Artificial spectra built

Database of sequences (i.e. SwissProt)

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

Example of an ICAT Reagent

How ICAT works?

Proteolysis (eg trypsin)

Advantages vs. Disadvantages

You might also like