Chemical proteomics in plantthe methodical preview

René Lenobel

Group of proteomics, Laboratory of Growth Regulators

8. 9. 2011, Olomouc

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Chemical proteomics – basic background

o Background• Incubation of a sorbent modified with specific ligand with a protein lysate. After washing step, retained proteins are released by elution buffer or by denaturation. Collected proteins are analyzed using proteomic methods.

o Application• Study of proteins with similar specificity• Analysis and identification of putative cell targets of immobilized ligands

WASH

SDS-PAGE

„Shotgun“

approach

LC-MS/MS

Bioinformatic analysis

Lysis

Incubation

Aff. sorbent

Ligand Spacer Matrix

Elution

Chemical proteomics:Unbiased but not so straightforward approach

Ligand design, its synthesis and immobilization on a solid matrix.

Preventing of protein degradation, omitting strong detergents.

Resolving between specific and nonspecific interactors• Strong washing buffers (high salt conc, weak detergents)• Using chemical competitors or unspecific control matrices (similar compounds without specific binding to desired proteins)• Quantitative proteomic methods (stable isotope labeling, label-free quantitation)

Chemical proteomics with cytokininsExperimental design

Wash

Elution

SDS-PAGE

„Shotgun“

approach

LC-MS/MS

Lysis

Incubation

Affinity matrix

Ligand Spacer Matrix

Bioinformatic

analysis

N

NN

NH

NH

NH

NH2

ON O

O OH

N

O

O

N

NN

NH

NH

NH

NH

N O

O OH

N

N N

NH

NH

6-benzylaminopurine (BAP)

Cytokinin-binding protein 1 (CBP-1)

Wheat seeds (T. Aestivum)

BAP affinity sorbent synthesis

N

NN

NH

NH

NH

NH2

ON O

O OH

N

O

O

N

NN

NH

NH

NH

NH

N O

O OH

N

N N

NH

NH

2(6-aminobuthylimino)-BAP (C2-BAP) NHS-Fast Flow Sepharose

19 atoms, 30 Å

BAP

Small preview of our results

Competitor NO 5mM ATP 5mM ATP, 0,1mM BAPDatabase   CBP-custom    

T. Aestivum UniGene EST  

Competitor NO 5 mM ATP5 mM ATP

0,1 mM BAP NO 5 mM ATP5 mM ATP

0,1 mM BAP

Peptide number 9 10 5 8 8 4

Sequence coverage 19,8% 15,4% 9,4% 40% 26% 17%

Mascot score 376,6 445,2 251,4 281 332 202

Spectral count 40 37 19 35 34 17

emPAI - - - 1,58 1,03 0,68

Database   T. Aestivum UniGene EST    

NCBInr Green plants  

Competitor NO 5 mM ATP5 mM ATP

0,1 mM BAP NO 5 mM ATP5 mM ATP

0,1 mM BAP

Number of ID proteins 308 203 192 197 116 122

200 kDa

150 kDa

100 kDa

75 kDa

50 kDa

37 kDa

25 kDa

20 kDa

200 kDa

150 kDa

100 kDa

75 kDa

50 kDa

37 kDa

25 kDa

20 kDa

200 kDa

150 kDa

100 kDa

75 kDa

50 kDa

37 kDa

25 kDa

20 kDa

catalytic activity27%

nucleotide binding

23%

transferase activity

5%

unknown36%

hydrolase activity

9%

Putative targets of BAP

N N

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NH2

O

NH3+

SCH3

O

O

OH OH

N N

NN

NH2

O

NH3+

S

O

O

OH OH

N N

NN

NH2

O

OH OH

OHNH3+

SH

O

ONH3+

S

O

O

CH3

R-H R-CH3 + H+

OH2

-CH3

ATP

+

S-adenosyl-L-homocystein hydrolase

Methionin synthetase

S-adenosyl-L-methionine synthase

Acknowledgement

LRR – proteomic group

Ivo Chamrád

Radim Simerský

Lucie Švehlová

prof. ing. Miroslav Strnad, CSc.

LRR – organic synthesis group

Lucie Szüčová

Václav Mik

Jindřich Kania

Marek Zatloukal

And many other people for fruitfull discussions and advices

Thank you for your attention