Mass Analysis + Database search

Mass fingerprint analysisMass fingerprint analysis

Post-source decay of metastable ionsPost-source decay of metastable ions

linear ion trap3x10-5 Torr

Q0

N2 CAD Gas Exit lens

Aux AC

Q1 Q2 Q3

Precursor ion selection

Ion accumulation

-Palmitoiloma de Saccaromyces cerevisae

Dolores Bernal (Dept. Bioquímica y Biología Molecular, UV)

-Palmitoiloma de Saccaromyces cerevisae

Dolores Bernal (Dept. Bioquímica y Biología Molecular, UV)

Protein profile of yeasts under cold stressSonia Rodríguez-Vargas, Francisca Randez-GilDepartamento de Biotecnología. Instituto de Agroquímica y Tecnología de los Alimentos (CSIC) Valencia.

Protein profile of yeasts under cold stressSonia Rodríguez-Vargas, Francisca Randez-GilDepartamento de Biotecnología. Instituto de Agroquímica y Tecnología de los Alimentos (CSIC) Valencia.

50

60

70

kDa

45

40

30

25

15

4.5 6.05.0 7.0pI 9.5

1242.64

Peptide fragmented by

MS/MS

MAEGVFQGAIGIDLGTTYSCVATYESSVEIIANEQGNRVTPSFVAFTPEERLIGDAAKNQAALNPRNTVFDAKRLIGRRFDDESVQKDMKTWPFKVIDVDGNPVIEVQYLEETKTFSPQEISAMVLTKMKEIAEAKIGKKVEKAVITVPAYFNDAQRQATKDAGAISGLNVLRIINEPTAAAIAYGLGAGKSEKERHVLIFDLGGGTFDVSLLHIAGGVYTVKSTSGNTHLGGQDFDTNLLEHFKAEFKKKTGLDISDDARALRRLRTAAERAKRTLSSVTQTTVEVDSLFDGEDFESSLTRARFEDLNAALFKSTLEPVEQVLKDAKISKSQIDEVVLVGGSTRIPKVQKLLSDFFDGKQLEKSINPDEAVAYGAAVQGAILTGQSTSDETKDLLLLDVAPLSLGVGMQGDMFGIVVPRNTTVPTIKRRTFTTCADNQTTVQFPVYQGERVNCKENTLLGEFDLKNIPMMPAGEPVLEAIFEVDANGILKVTAVEKSTGKSSNITISNAVGRLSSEEIEKMVNQAEEFKAADEAFAKKHEARQRLESYVASIEQTVTDPVLSSKLKRGSKSKIEAALSDALAALQIEDPSADELRKAEVGLKRVVTKAMSSR

Ssb1p

GS

I

A

G

L

LEUCEMIA LINFOIDE CRÓNICA

Miguel Marín (Javier García-Conde)

Servicio de Hematología y Oncología, HospitalClínico Universitario de Valencia

LEUCEMIA LINFOIDE CRÓNICA

Miguel Marín (Javier García-Conde)

Servicio de Hematología y Oncología, HospitalClínico Universitario de Valencia

LEUCEMIA MIELOIDE CRÓNICA

Quique Andreu, Felipe Prósper (Clínica Universitaria de Pamplona) y Nacho Pérez-Roger (Universidad Cardenal Herrera, CEU, Valencia)

LEUCEMIA MIELOIDE CRÓNICA

Quique Andreu, Felipe Prósper (Clínica Universitaria de Pamplona) y Nacho Pérez-Roger (Universidad Cardenal Herrera, CEU, Valencia)

C03701: FACTORES DE RIESGO, EVOLUCIÓN Y TRATAMIENTO DE LAS ENFERMEDADES CARDIOVASCULARES Y SUS MECANISMOS MOLECULARES Y CELULARES

Red temática "Cardiovascular”, Instituto de Salud Carlos III (Nodo IBV: V. Andrés, J.J. Calvete, M. Casado)

C03701: FACTORES DE RIESGO, EVOLUCIÓN Y TRATAMIENTO DE LAS ENFERMEDADES CARDIOVASCULARES Y SUS MECANISMOS MOLECULARES Y CELULARES

Red temática "Cardiovascular”, Instituto de Salud Carlos III (Nodo IBV: V. Andrés, J.J. Calvete, M. Casado)

Proteínas de pared celular deCandida albicans

(Rafael Sentrandreu, Dept.Microbiología y Ecología,Facultad de Farmacia, UV)

Proteínas de pared celular deCandida albicans

(Rafael Sentrandreu, Dept.Microbiología y Ecología,Facultad de Farmacia, UV)

SESQXASEXAQXSGFDVXR

X= Ile/Leu

E

Proteínas vegetales(alfalfa)

Jesús Jorrín

Universidad de Córdoba

Proteínas vegetales(alfalfa)

Jesús Jorrín

Universidad de Córdoba

Mass (m/z)

(1859.8)

Proteína de paciente

(Vicente Felipo, FVIB)

Proteína de paciente

(Vicente Felipo, FVIB)

Tubulin α2

845.0

743.8

288.1175.1 FV

D

XE

PT

V

XDXR

y1

(171)FVDXEPTVXDXRProteína de cerebelo de rata/NMDA (anti-Pser/Thr)

(Vicente Felipo, FVIB)

Proteína de cerebelo de rata/NMDA (anti-Pser/Thr)

(Vicente Felipo, FVIB)

mass (m/z)

Ratón transgénico α-antitripsina humana

(Salvador AliñoFacultad de Farmacia)

Ratón transgénico α-antitripsina humana

(Salvador AliñoFacultad de Farmacia)

Lectina del alga roja Hypnea musciformis

Lectina del alga roja Hypnea musciformis

A, DTT refolded

B, A + VP

C, GSH/GSSG refolded + VP

D, C35S (or C61S) + VP

A, DTT refolded

B, A + VP

C, GSH/GSSG refolded + VP

D, C35S (or C61S) + VP(44549-43518)/105.3= 9.8 Cys

(44331-43518)/105.3=

7.8 Cys

(44455-43518)/105.3=

8.7 Cys

α3 Colágeno IV y enfermedad autoinmune de Goodpasture

Juan Saus, Quique Pérez-Payá(FVIB)

α3 Colágeno IV y enfermedad autoinmune de Goodpasture

Juan Saus, Quique Pérez-Payá(FVIB)

RGDKGDWGD

RGD, WGD

MLD

RGDVGDMGD

MLDECD

ECD RGD

5 µg

5 µg

IC50 = 0.8 nM

60 ± 9 79 ± 11

179 ± 23

178 ± 5

84% Inhibition

Effect on the adhesion of α1-K562 tocoll IV ( ) and α2-K562 to coll I (o).

Effect on the adhesion of α1-K562 tocoll IV ( ) and α2-K562 to coll I (o).

AI, Angiogenesis index = nº vessel branch points

AI, Angiogenesis index = nº vessel branch points

Effect of obtustatin (6) and PBS ( ) on Lewis lung carcinoma growth in C57BL/ 6 mice

Effect of obtustatin (6) and PBS ( ) on Lewis lung carcinoma growth in C57BL/ 6 mice

n = 4

4395.3

TK

S

Monleón,D., Moreno-Murciano,M.P., Kovacs,H.,Marcinkiewicz,C., Calvete,J.J. & Celda,B.

Concerted motions of the integrin-binding loop and the C-terminal tail of the non-RGD disintegrin obtustatin

J. Biol. Chem. (2003) in press

Monleón,D., Moreno-Murciano,M.P., Kovacs,H.,Marcinkiewicz,C., Calvete,J.J. & Celda,B.

Concerted motions of the integrin-binding loop and the C-terminal tail of the non-RGD disintegrin obtustatin

J. Biol. Chem. (2003) in press

5Å35º

100-300 picosec100-300 picosec

Venómica

Paula Juárez, Libia SanzIBV

Venómica

Paula Juárez, Libia SanzIBV

Characterization of the protein components of the venom of Sistrurus barbouriCharacterization of the protein components of the venom of Sistrurus barbouri

The protein composition of the venom of Sistrurus barbouri was analyzed by 2D-SDS-PAGE electrophoresis.

Std 8 10 12 14 1566 45 36 29 24 20 14

In-gel digestionIn-gel digestion

MALDI-TOF mass fingerprinting

MALDI-TOF mass fingerprinting

In solutionIn solution

15

11/12

13/14

9/10

4/5

6/7

QUANTITATION OF FREE CYSTEINE RESIDUES AND DISULPHIDE BONDS

Mass of the native protein

NSH = (MVP-MNAT) /105.3Number of free cysteines

Mass of nonreduced protein incubatedwith 4-vinylpyridine

Mass of alkylating group

NCYS = [ (MPE - MVP ) /106.3] + NSH

Total number of cysteines

Mass of reduced and pyridylethylated protein

NS-S = ( NCYS - NSH ) / 2

Number of disulphide bonds

MNAT and MVPMNAT and MVP

MPEMPE

No free cysteine residueNo free cysteine residue

(15483-13980)/106= 14.1 Cys

14/2 = 7 Disulphide bonds

(15483-13980)/106= 14.1 Cys

14/2 = 7 Disulphide bonds

This suggested that protein 15 might be ametalloproteinase of the ADAM family

This suggested that protein 15 might be ametalloproteinase of the ADAM family

Protein 15 contained a blocked N-terminus.Protein 15 contained a blocked N-terminus.

The protein contained 1 free cysteine and17 disulphide bonds

The protein contained 1 free cysteine and17 disulphide bonds

The proteins in the major peaks were identified by combination of N-terminal sequencing and mass spectrometric determination of molecular masses and cysteine content

The proteins in the major peaks were identified by combination of N-terminal sequencing and mass spectrometric determination of molecular masses and cysteine content

A The doubly-charged ion of1742.9 was analyzed by MS/MS

The doubly-charged ion of1742.9 was analyzed by MS/MS

MALDI-TOF tryptic mass fingerprint of thecarboxyamidomethylated-protein 15 in solution. MALDI-TOF tryptic mass fingerprint of thecarboxyamidomethylated-protein 15 in solution.

(b1) b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14

y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1

K K H D N A Q I/L I/L T A I/L D F KB

b14b13y111333.1

b12

1220.1

b11

1149.0

b10

b9b6/y6

623.0

b5

b4

394.0

b3

257.1

b2

b1

y2 y3

522.0

y4y7

MS/MS spectra of the doubly-chargedmonoisotopic ion at m/z 871.0 showing major b and y ions from which the corresponding amino acid sequence (B) was deduced.

MS/MS spectra of the doubly-chargedmonoisotopic ion at m/z 871.0 showing major b and y ions from which the corresponding amino acid sequence (B) was deduced.

The sequence of peptide at m/z 871.0:The sequence of peptide at m/z 871.0:

K K H D N A Q I/L I/L T A I/L D F K

shows strong homology to peptide 43-58 of jararhagin:shows strong homology to peptide 43-58 of jararhagin:

K K H D N A Q L L T A I D F N

Further indicating that protein 15 of Sistrurus barbourivenom is a jarrarhagin-like metalloproteinase

Further indicating that protein 15 of Sistrurus barbourivenom is a jarrarhagin-like metalloproteinase

CONCLUSIONS

Although the lack of genome sequences of snake species is a handicap for the identificacion of venom proteins by MALDI-TOF mass fingerprinting, MS/MS fragmentation of selected ions yielded sufficient sequence information to identify an homologue protein from a related snake venom metalloprotease.

Although the lack of genome sequences of snake species is a handicap for the identificacion of venom proteins by MALDI-TOF mass fingerprinting, MS/MS fragmentation of selected ions yielded sufficient sequence information to identify an homologue protein from a related snake venom metalloprotease.

The venom proteome of the pigmy rattlesnake Sistrurus barbouriis composed of proteins belonging to only a few known proteins families, including:

- Phospholipases A2- PI Zn2+ metalloproteinases- Disintegrins- CRISP- Serine proteinases- ADAM

The venom proteome of the pigmy rattlesnake Sistrurus barbouriis composed of proteins belonging to only a few known proteins families, including:

- Phospholipases A2- PI Zn2+ metalloproteinases- Disintegrins- CRISP- Serine proteinases- ADAM