Genome of the week - Enterococcus faecalis

• E. faecalis - urinary tract infections, bacteremia, endocarditis.

• Organism sequenced is vancomycin resistant.– Vancomycin is often last available antibiotic -

resistance to this drug often means no other antibiotics will work.

• Major cause of nosocomial infections.• Possible transfer of vanomycin resistance genes

to more serious pathogens is a major concern.

Genome of the week - Enterococcus faecalis

• Over 25% of the E. faecalis genome consists of foreign DNA.– Phages, insertions sequences, transposons.– Likely contributed to the acquisition of resistance to

multiple antibiotics.

• Over 35 PTS systems– Responsible for transporting sugars into the cell.– Most found in any sequenced genome, likely utilize

undigested sugars in the intestine.

Genomics DNA (Gene)

FunctionalGenomics

Transcriptomics RNA

Proteomics PROTEIN

Metabolomics METABOLITE

Transcription

Translation

Enzymatic reaction

The “omics” nomenclature…

Why study protein expression?

DNA

PrimaryRNA

transcriptmRNA mRNA

protein Modified protein

Transcriptional control

RNA Processing

control

RNATransport

control

Inactive mRNARNA

Degradationcontrol

Translation control

Post-translationalcontrol

• 2D gel electrophoresis– Method for separating and visualizing proteins– Separation by charge and mass

• Mass spectrometry– High throughput analysis and identification of

proteins.– Fragmentation of proteins– Analysis of peptides

• Book - pages 273-300.

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)

What determines the charge of a protein?

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 the protein (to make movement solely dependent on mass, not shape) and eliminates charge.

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

Current Mass Spec Technologies• Proteome profiling/separation

– 2D SDS PAGE - identify proteins– 2-D LC/LC - high throughput analysis of lysates(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)– ITRAQ

Mass Spectrometry (MS)• 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

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)

2D - LC/MS

Methods for protein

identification

Identifying proteins

• Trypsin - digest your protein – Digests after R and K amino acids.

• Run peptide fragments on mass spec

• Digest the protein database “in silico”

• Compare mass spec data with theoretical data.

• What must be true to identify your protein?

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

How 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

• 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…

Advantages vs. Disadvantages

• Determination of MW and aa. Sequence

• Detection of posttranslational modifications

• High-throughput capability

• High capital costs

• Requires sequence databases for analysis

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

**

* *

OO

ONH

O

O

NHNH

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