Implementation of radiotracers use in methods for differential analysis of protein

expression

Mauro FasanoCentre of NeuroScience

and DBSFUniversity of Insubria

at Busto Arsiziomauro.fasano@uninsubria.it

Outline

• Background, state of the art(adapted from my presentation at the Varese meeting)

• The toy-project

• Update in protein arrays technology

Proteome

• The set of proteins encoded by the genome• The set of all p. isoforms, their

modifications, their interactions• The set of p. as above in relationship to a

given state (disease, treatment, time, etc.)

40000 Genes 40000 Genes 1 Million Proteins 1 Million Proteins

40000 Genes 40000 Genes 1 Million 1 Million ProteinsProteins

• Post-translational modifications

• Splice variants

• Proteolytical processing

Proteome is dynamic…

The classical approach

Sample solubilization

IEF

SDS - PAGE

Display of results

Gel analysis

Mass spectrometry (MALDI)

The classical approach

Sample solubilization

IEF

SDS - PAGE

Display of results

Gel analysis

Mass spectrometry (MALDI)

UREA 7M, THIOUREA 2M, CHAPS 4%

The classical approach

Sample solubilization

IEF

SDS - PAGE

Display of results

Gel analysis

Mass spectrometry (MALDI)

The classical approach

Sample solubilization

IEF

SDS - PAGE

Display of results

Gel analysis

Mass spectrometry (MALDI)

The classical approach

Sample solubilization

IEF

SDS - PAGE

Display of results

Gel analysis

Mass spectrometry (MALDI)

•Immunoblotting

•Coomassie Blue or silver staining

The classical approach

Sample solubilization

IEF

SDS - PAGE

Display of results

Gel analysis

Mass spectrometry (MALDI)

The classical approach

Sample solubilization

IEF

SDS - PAGE

Display of results

Gel analysis

Mass spectrometry (MALDI)

Protein identification

• Immunoblot (against selected proteins)

• Digest, MS & database query

• Digest & MS/MS

Immunoblot(Western blot)

EE

Reagent

Excited product

LIGHTLIGHT

Database query with peptide masses

MS/MS

Limits of the classical approach

• Too many proteins in the sample• Only the most abundant proteins are displayed and

identified• Small & hydrophobic proteins are not adequately

separated• Low-abundance proteins are hindered by more

abundant ones• Gels are not always reproducible

Making it simpler…

• Subcellular fractionation

• Affinity tags

• Enrich for specific post-translational modifications

• Protein-protein interaction (Interactomics)

Quantitative proteomics

1. Differential Gel Electrophoresis

2. Gel-free MS with ICAT

DIGE ®

DIGE ®

Isotope-coded affinity tagging

Phosphoproteomics

• Enrichment of phosphopeptides• Immunoblot (anti-pSer, etc.)

or

• in vitro labeling with 32P

The toy project

• Task 1:Task 1: real-time acquisition of western blots with radiolabelled probes

• Task 2:Task 2: development of an antibody array to perform simultaneous multiple Western blots

• Task 3:Task 3: differential in-gel electrophoresis by using tracers at different energy (32P and 33P)

Real-time western blots

B

S*S*

Real-time western blots

Mic

rostrip

sen

sor

Miniaturized multi-western array

B

S*S*

Spotting antibodies on a membrane

120000 spots on a

25 x 75 mm slide

(64 spots per mm2)

RadioDIGE

+

+

P P 32P

P P 33P

32P

32P

32P

32P

33P

33P

Healthy

Diseased

Mix and separate by 2D-PAGE

32P

33P

Other Matters

• Multiplexing protein-protein interactions

– Assays in solution– Zeptosens CeLyA– Biacore affinity chip

– Peptide chips

Cell Lysate Assay

Witterswil, Switzerland-based Zeptosens has developed a method based on planar waveguides (PWGs), modifying the standard glass-slide substrate with a thin film of tantalum pentoxide (Ta2O5). This high-refractive-index material guides laser light on the surface of the chip only, permitting selective detection of captured labels.

The Biacore Approach

Biacore, Uppsala, Sweden

The Jerini approach

• Take the enzyme (e.g., kinase)• Scan databases for potential targets• Synthesize and array 20000 peptides on

a chip• Incubate with the kinase and 32P-ATP

Jerini peptide technol., Berlin

Further readings

• Nature Insight in the 13 March 2003 issue (Vol. 422, p. 191 and ff.)

• Proteomics in multiplex. Nature 429, 101-107 (2004)

• Protein Microarrays Mature. The Scientist 18, 42 (August 2004 issue)