Mass Analysis + Database search

Transcripción

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2 Mass Analysis + Database search

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4 Mass fingerprint analysis Post-source decay of ofmetastable ions ions

5 Ion accumulation N 2 CAD Gas Aux AC Exit lens Q0 Q1 Q2 Q3 Precursor ion selection linear ion trap 3x10-5 Torr

6 Palmitoiloma -Palmitoilomade desaccaromyces cerevisae cerevisae Dolores Dolores Bernal Bernal (Dept. (Dept. Bioquímica Bioquímicay Biología BiologíaMolecular, UV) UV) kda Peptide Protein profile profile of of yeasts yeasts under under cold cold stress stress fragmented by Sonia SoniaRodríguez-Vargas,, MS/MS Francisca Randez-Gil Departamento de de Biotecnología. Instituto de de Agroquímica y Tecnología de de los los Alimentos (CSIC) (CSIC) Valencia. Ssb1p MAEGVFQGAIGIDLGTTYSCVATYESSVEIIANEQGNR VTPSFVAFTPEERLIGDAAKNQAALNPRNTVFDAKRLI GRRFDDESVQKDMKTWPFKVIDVDGNPVIEVQYLEETK TFSPQEISAMVLTKMKEIAEAKIGKKVEKAVITVPAYF NDAQRQATKDAGAISGLNVLRIINEPTAAAIAYGLGAG KSEKERHVLIFDLGGGTFDVSLLHIAGGVYTVKSTSGN THLGGQDFDTNLLEHFKAEFKKKTGLDISDDARALRRL RTAAERAKRTLSSVTQTTVEVDSLFDGEDFESSLTRAR FEDLNAALFKSTLEPVEQVLKDAKISKSQIDEVVLVGG STRIPKVQKLLSDFFDGKQLEKSINPDEAVAYGAAVQG AILTGQSTSDETKDLLLLDVAPLSLGVGMQGDMFGIVV PRNTTVPTIKRRTFTTCADNQTTVQFPVYQGERVNCKE NTLLGEFDLKNIPMMPAGEPVLEAIFEVDANGILKVTA VEKSTGKSSNITISNAVGRLSSEEIEKMVNQAEEFKAA DEAFAKKHEARQRLESYVASIEQTVTDPVLSSKLKRGS KSKIEAALSDALAALQIEDPSADELRKAEVGLKRVVTK AMSSR A 15 G I L S G pi

7 LEUCEMIA LEUCEMIA MIELOIDE MIELOIDE CRÓNICA CRÓNICA Quique Quique Andreu, Andreu, Felipe FelipePrósper (Clínica (Clínica Universitaria Universitariade de Pamplona) Pamplona) y Nacho NachoPérez- Roger Roger (Universidad (Universidad Cardenal CardenalHerrera, CEU, CEU, Valencia) Valencia) LEUCEMIA LEUCEMIA LINFOIDE LINFOIDE CRÓNICA CRÓNICA Miguel MiguelMarín Marín (Javier (JavierGarcía-Conde) Servicio Serviciode dehematología Hematologíay Oncología, Oncología, Hospital Hospital Clínico Clínico Universitario Universitariode de Valencia Valencia C03701: C03701: FACTORES FACTORES DE DE RIESGO, RIESGO, EVOLUCIÓN EVOLUCIÓN Y TRATAMIENTO TRATAMIENTO DE DE LAS LAS ENFERMEDADES ENFERMEDADES CARDIOVASCULARES Y SUS SUS MECANISMOS MECANISMOS MOLECULARES MOLECULARES Y CELULARES CELULARES Red Red temática temática "Cardiovascular, "Cardiovascular, Instituto Instituto de de Salud Salud Carlos Carlos III III (Nodo (Nodo IBV: IBV: V. V. Andrés, Andrés, J.J. J.J. Calvete, Calvete, M. M. Casado) Casado)

8 Proteínas Proteínasde de pared paredcelular celularde de Candida Candida albicans albicans (Rafael (RafaelSentrandreu, Dept. Dept. Microbiología Microbiologíay Ecología, Ecología, Facultad Facultadde defarmacia, UV) UV)

9 SESQXASEXAQXSGFDVXRE X= Ile/Leu

10 Proteínas Proteínas vegetales vegetales (alfalfa) (alfalfa) Jesús Jesús Jorrín Jorrín Universidad Universidadde decórdoba

11 Proteína Proteínade depaciente (Vicente (VicenteFelipo, FVIB) FVIB) (1859.8) Mass (m/z)

12 Proteína Proteínade decerebelo cerebelode de rata/nmda rata/nmda (anti- (anti- Pser/Thr) Pser/Thr) (Vicente (VicenteFelipo, FVIB) FVIB) R y1 X D X V P T (171)FVDXEPTVXDXR X E F V D Tubulin α2

13 Ratón Ratón transgénico transgénicoα- α- antitripsina antitripsina humana humana (Salvador (SalvadorAliño Facultad Facultadde defarmacia) mass (m/z)

14 Lectina Lectinadel del alga algaroja Hypnea Hypnea musciformis musciformis

15 ( )/105.3= 9.8 Cys A, A, DTT DTT refolded refolded B, B, A + VP VP C, C, GSH/GSSG GSH/GSSG refolded refolded + VP VP D, D, C35S C35S (or (or C61S) C61S) + VP VP ( )/105.3= 7.8 Cys ( )/105.3= 8.7 Cys

16 α3 α3colágeno ColágenoIV IV y enfermedad enfermedad autoinmune autoinmunede degoodpasture Juan JuanSaus, Quique Quique Pérez-Payá Pérez-Payá (FVIB) (FVIB)

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18 ECD MLD RGD RGD, WGD MLD RGD VGD MGD ECD RGD KGD WGD

19 5 µg 84% Inhibition Effect Effect on on the the adhesion adhesion of of α1-k562 α1-k562 to to coll coll IV IV ( ) ( ) and and α2-k562 α2-k562 to to coll coll I I (o). (o). 60 ± 9 79 ± 11 IC50 = 0.8 nm 179 ± 23 5 µg 178 ± 5 AI, AI, Angiogenesis Angiogenesis index index = = nº nº vessel vessel branch branch points points

20 Effect Effect of of obtustatin obtustatin (6) (6) and and PBS PBS ( ) ( ) on on Lewis Lewis lung lung carcinoma carcinoma growth growth in in C57BL/ C57BL/ 6 6 mice mice n = 4

21 4395.3

22 T K S

23 Monleón,D., Monleón,D., Moreno-Murciano,M.P., Kovacs,H., Kovacs,H., Marcinkiewicz,C., Marcinkiewicz,C., Calvete,J.J. Calvete,J.J. & Celda,B. Celda,B. Concerted Concerted motions motions of of the theintegrin-binding loop loop and and the the C-terminal C-terminal tail tail of of the the non-rgd non-rgddisintegrin obtustatin obtustatin J. J. Biol. Biol. Chem. Chem. (2003) (2003) in in press press 5Å 35º picosec

24 Venómica Paula Juárez, Libia Sanz IBV

25 Characterization of of the the protein components of of the thevenom of of Sistrurus barbouri Std The protein composition of the venom of Sistrurus barbouri was analyzed by 2D-SDS-PAGE electrophoresis.

26 In-gel In-gel digestion digestion MALDI-TOF MALDI-TOFmass fingerprinting fingerprinting In In solution solution

27 15 11/12 13/14 6/7 9/10 4/5

28 QUANTITATION OF FREE CYSTEINE RESIDUES AND DISULPHIDE BONDS Number of free cysteines N SH = (M VP -M NAT ) /105.3 No No free freecysteine residue residue Mass of the native protein Mass of alkylating group M NAT and NAT andm VP VP Mass of nonreduced protein incubated with 4-vinylpyridine ( )/106= ( )/106= Cys 14/2 14/2 = 7 Disulphide Disulphide bonds bonds Total number of cysteines N CYS = [ (M PE -M VP ) /106.3] + N SH Mass of reduced and pyridylethylated protein M PE PE Number of disulphide bonds N S-S = ( N CYS -N SH ) / 2

29 Protein Protein15 15contained containeda blocked blockedn-terminus. The The protein protein contained contained1 free freecysteine and and 17 17disulphide bonds bonds This This suggested suggested that that protein protein15 15might mightbe be a metalloproteinase metalloproteinase of of the theadam ADAMfamily The proteins in inthe major peaks were identified by bycombination of ofn-terminal sequencing and mass spectrometric determination of ofmolecular masses and cysteine content

30 A The The doublychargecharged ion ion of of doubly was was analyzed analyzedby by MS/MS MS/MS MALDI-TOF tryptic mass fingerprint of of the carboxyamidomethylated-protein in in solution.

31 y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 B K K H D N A Q I/L I/L T A I/L D F K (b1) b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14 y2 b2 b b y3 y4 b4 b6/y6 b MS/MS MS/MS spectra spectra of of the the doubly-charged doubly-charged monoisotopic monoisotopic ion ion at at m/z m/z showing showing major major b b and and y y ions ions from from which which the the corresponding corresponding amino amino acid acid sequence sequence (B) (B) was was deduced. deduced. y7 b10 b12 b9 b11 b13 b14 y

32 The The sequence sequence of of peptide peptide at atm/z 871.0: 871.0: K K H D N A Q I/L I/L T A I/L D F K shows shows strong strong homology homology to to peptide peptide of of jararhagin: jararhagin: K K H D N A Q L L T A I D F N Further Further indicating indicating that that protein protein15 15of of Sistrurus Sistrurus barbouri barbouri venom venom is isa jarrarhagin-like jarrarhagin-like metalloproteinase metalloproteinase

33 CONCLUSIONS Although the the lack lack of of genome sequences of of snake species is is a handicap for for the the identificacion of of venom proteins by by MALDI-TOF mass fingerprinting, MS/MS fragmentation of of selected ions ions yielded sufficient sequence information to to identify an an homologue protein from a related snake venom metalloprotease. The venom proteome of of the pigmy rattlesnake Sistrurus barbouri is is composed of of proteins belonging to to only a few known proteins families, including: --Phospholipases A2 A2 -PI Zn Zn metalloproteinases --Disintegrins -CRISP --Serine proteinases --ADAM