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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)
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)
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)
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
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
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
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