L’applicazione della spettrometria di massa allo studio della chemorecezione degli insetti: dai feromoni alle proteine chemosensoriali

Francesca R. Dani C.I.S.M.

C.I.S.M.

PRIN 2002

PRIN 2006

European Network ENAROMATIC

PRIN 2008

C.I.S.M.

Collaborazione con Prof. P. Pelosi e Dr. A. Felicioli, Univ. Pisa

Dr. A. della Torre, Univ La Sapienza

sensillarpores

sensillar lymph

Dendrites of the olfactory receptorneuron

1 µm1 µm

longitudinal section of a chemosensillum

OLFACTORY SENSILLAC.I.S.M.

C.I.S.M.

Proteins involved in the Perireceptor Events

C.I.S.M.OBPs Odorant Binding Protein and CSPs Chemosensory Proteins

Mapping the expression of soluble olfactory proteins in the honeybee.

Traditional proteomic approach: digestion of 2D gel spots and micro LC-ESI LTQ ORBITRAP analyses

Dani et al. 2010. J. Proteomic Research

WORKER ANTENNAE

12 OBPs and

2 CSPs

LARVAE

3 OBPs and

1 CSP

C.I.S.M.

21 OBP and 6 CSP genes identified in the genome

Mapping the expression of soluble olfactory proteins in the honeybee: MALDI profiling

Dani et al. 2010. J. Proteomic Research

C.I.S.M.

Mapping the expression of soluble olfactory proteins in the mandibular gland of honeybee

WORKER MANDIBULAR GLANDS QUEENS MANDIBULAR GLANDS

Is the queen specific OBP a carrier for the queen pheromone?

C.I.S.M.

Mapping the expression of soluble olfactory proteins in Anopheles gambiae: MALDI profiling

C.I.S.M.

OBP-1

OBP-9 *

Dani et al. 2008. Plos one

PRIN 2006European Network ENAROMATIC

*OBP-9 was identified in a topdown experiment

Mapping the expression of soluble olfactory proteins in Anopheles gambiae

C.I.S.M.

OBP9

CSP SAP-3

CSP

57 OBP and 7 CSP genes identified in the genome

ANTENNAE FROM 1100 MALES

1 5 7 6 . 5 1 5 7 7 . 0 1 5 7 7 . 5 1 5 7 8 . 0 1 5 7 8 . 5 1 5 7 9 . 0m / z

0

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

1 0 0

Rel

ativ

e Ab

unda

nce

1 5 7 7 . 4 2 0 6 11 5 7 7 . 3 3 7 1 1

1 5 7 7 . 5 0 4 1 0

1 5 7 7 . 2 5 3 6 11 5 7 7 . 5 8 7 5 9

1 5 7 7 . 6 7 1 0 81 5 7 7 . 1 7 0 1 0

1 5 7 7 . 7 5 4 5 6

1 5 7 7 . 0 8 6 5 9

1 5 7 7 . 8 3 8 0 4

1 5 7 7 . 0 0 3 0 7

1 5 7 7 . 9 2 1 5 1

1 5 7 6 . 9 1 9 5 51 5 7 8 . 0 0 4 9 8

1 5 7 8 . 0 8 8 4 51 5 7 6 . 8 3 6 0 2

1 5 7 8 . 1 7 1 9 2

1 5 7 6 . 7 5 2 4 8 1 5 7 8 . 2 5 5 3 8

1 5 7 8 . 3 3 8 8 41 5 7 6 . 6 6 8 9 4 1 5 7 8 . 4 2 2 3 0

1 5 7 6 . 5 8 5 4 0 1 5 7 8 . 5 8 9 2 1 1 5 7 8 . 9 2 3 0 2

1 5 7 7 . 5 0 4 8 1

1 5 7 7 . 6 7 1 7 41 5 7 7 . 3 3 7 8 6 1 5 7 7 . 7 5 5 2 0

1 5 7 7 . 2 5 4 3 8 1 5 7 7 . 8 3 8 6 6

1 5 7 7 . 9 2 2 1 11 5 7 7 . 1 7 0 9 0

1 5 7 7 . 0 8 7 4 0

1 5 7 7 . 0 0 3 9 0

1 5 7 7 . 3 3 6 8 4

1 5 7 7 . 2 5 3 3 71 5 7 7 . 5 0 3 7 7

1 5 7 7 . 5 8 7 2 3

1 5 7 7 . 0 8 6 4 11 5 7 7 . 6 7 0 6 9

1 5 7 7 . 7 5 4 1 41 5 7 7 . 0 0 2 9 2

1 5 7 6 . 9 1 9 4 3

N L :2 . 2 9 E 4

C 8 2 5 H 1 3 1 1 N 2 1 5 O 2 4 9 S 2 2 * 3 . 0 0 + C 8 2 5 H 1 3 0 9 N 2 1 5 O 2 4 9 S 2 2 * 7 . 0 0 : p ( g s s , s / p : 4 0 ) C h r g 1 2R : 1 0 0 0 0 0 R e s . P w r . @ F W H MN L :2 . 2 9 E 4

C 8 2 5 H 1 3 1 1 N 2 1 5 O 2 4 9 S 2 2 : C 8 2 5 H 1 3 1 1 N 2 1 5 O 2 4 9 S 2 2p ( g s s , s / p : 4 0 ) C h r g 1 2R : 1 0 0 0 0 0 R e s . P w r . @ F W H MN L :2 . 2 9 E 4

C 8 2 5 H 1 3 0 9 N 2 1 5 O 2 4 9 S 2 2 : C 8 2 5 H 1 3 0 9 N 2 1 5 O 2 4 9 S 2 2p ( g s s , s / p : 4 0 ) C h r g 1 2R : 1 0 0 0 0 0 R e s . P w r . @ F W H M

Analysis of OBP47, a 13 Cys OBPC.I.S.M.

Native

MW: 18922.71

Alkylated

MW: 18981.18

//

//

CH2 CO

NH2MW: 58

O B P 4 7 B a tte r ic a H R # 1 -1 7 R T : 0 .0 0 -0 .9 4 AV: 1 7 N L : 1 .2 7 E 6T : F T M S + p E S I F u l l m s [1 2 0 0 .0 0 -2 0 0 0 .0 0 ]

1 5 7 6 .6 1 5 7 6 .8 1 5 7 7 .0 1 5 7 7 .2 1 5 7 7 .4 1 5 7 7 .6 1 5 7 7 .8 1 5 7 8 .0 1 5 7 8 .2 1 5 7 8 .4m /z

0

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

1 0 0

1 0 5

1 1 0

Rel

ativ

e Ab

unda

nce

1 5 7 7 .4 2 6 1 3z= 1 2

1 5 7 7 .3 4 2 9 8z= 1 2

1 5 7 7 .5 0 9 8 7z= 1 2

1 5 7 7 .5 9 2 9 0z= 1 2

1 5 7 7 .1 7 6 3 3z= 1 2

1 5 7 7 .6 7 6 2 4z= 1 2

1 5 7 7 .0 9 2 8 3z= 1 2

1 5 7 7 .7 5 9 0 3z= 1 2

1 5 7 7 .0 0 9 6 6z= 1 2 1 5 7 7 .8 4 3 7 7

z= 1 2

1 5 7 6 .9 2 5 7 5z= 1 2 1 5 7 7 .9 2 6 8 1

z= 1 2

1 5 7 8 .0 0 9 7 0z= 1 2

1 5 7 6 .8 4 2 2 6z= 1 2

1 5 7 8 .0 9 4 1 0z= 1 21 5 7 6 .7 5 7 8 2

z= 1 2 1 5 7 8 .1 7 6 3 0z= 1 2

1 5 7 8 .4 2 5 2 7z= 1 2

1 5 7 6 .5 0 7 5 0z= ?

High resolution mass spectrum

Simulation of OBP47 spectrum with 6or 5 disulfidebridges (7:3)

Z=12

Identification of compounds evoking antennogramresponses to plant extracts in A. gambiaeC.I.S.M.

GC-FID

EAGAbundance

6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00

carv

acro

l

para

-cym

ene

gam

ma-

terp

inen

e

GC-MS

Proteins involved in the Perireceptor Events

PBPs and OBPs Pheromone and Odorant Binding Protein

CSPs Chemosensory Proteins

C.I.S.M.