Curso Espectrometría de masas, Electrospray
Trampa Iónica y Maldi-Tof
UNIVERSIDAD FRANCISCO DE VITORIA
MALDI-TOF MS
y
Análisis de Proteínas
Alberto Jorge García
Laboratorio de Proteómica
Centro de Biología Molecular Severo Ochoa
Universidad Autónoma de Madrid- CSIC
Identificación
Proteína
Proteómica:
Proteoma
Péptidos
BASE DE
DATOS
Fragmentos
BASE DE
DATOS
Proteína
Identificación
Péptido
ESTRATEGIA INTEGRADORA PARA
EL ANÁLISIS DEL PROTEOMA
Digestión “in situ”
en paralelo
SDS-PAGE
Bandas de proteínas
2D-IEF-SDS-PAGE
Extracto de péptidos
MALDI-TOF
mapa de péptidos
Identificación
de proteína
m-desalado automático
aislamiento y
fragmentación
Electrospray-trampa iónica
Secuenciación
“de novo”
Mapa de péptidos (MALDI-TOF)
Extracto crudo digestión “en gel”
14000
Intensidad
12000
10000
8000
6000
4000
2000
0
1000
1500
2000
m/z
2500
3000
Identificación de la proteína a partir del mapa de péptidos
(“peptide-mass fingerprinting”)
Búsqueda en MASCOT
(D.Pappin, ICRF, Londres)
1. gi|191618 Mass: 223549 Score: 87
(M76598) alpha cardiac myosin heavy chain [Mus musculus]
Observed
Mr(expt) Mr(calc)
Delta
Start
End Miss
1061.02
1060.01
1060.13
-0.12
1366 - 1374
0
1085.02
1084.01
1084.32
-0.31
436 443
0
1239.27
1238.26
1238.28
-0.01
1253 - 1262
0
1346.49
1345.48
1345.52
-0.04
24 34
0
1442.63
1441.62
1441.61
0.02
1680 - 1691
0
1489.17
1488.16
1488.57
-0.41
1117 - 1128
0
1602.80
1601.79
1601.82
-0.02
955 968
1
1703.76
1702.75
1702.90
-0.14
1423 - 1436
0
1741.93
1740.92
1740.98
-0.06
726 741
0
1840.03
1839.02
1838.99
0.03
1179 - 1195
0
1896.08
1895.07
1895.02
0.06
1198 - 1214
0
1979.38
1978.37
1978.18
0.19
1620 - 1636
0
2107.39
2106.38
2106.35
0.03
1619 - 1636
1
2277.72
2276.71
2276.54
0.17
1063 - 1081
1
2343.63
2342.62
2342.42
0.20
887 906
0
2809.57
2808.56
2809.04
-0.48
1000 - 1024
1
2837.05
2836.04
2836.07
-0.02
1654 - 1678
1
No match to: 1771.94
Peptide
ANSEVAQWR
MFNWMVTR
TLEDQANEYR
LEAQTRPFDIR
NNLLQAELEELR
IEELEEELEAER
DIDDLELTLAKVEK
LQNEIEDLMVDVER
ILNPAAIPEGQFIDSR
DLEEATLQHEATAAALR
HADSVAELGEQIDNLQR
MEGDLNEMEIQLSQANR
KMEGDLNEMEIQLSQANR
LTQESIMDLENDKLQLEEK
NDLQLQVQAEQDNLNDAEER
ALQEAHQQALDDLQAEEDKVNTLTK
DTQLQLDDAVHANDDLKENIAIVER
MALDI-TOF/MS
 Transfer into the gas phase
(desorb)
 Ionize
 Apply electromagnetic
fields
Ion Source
Analyse ion movements
Determine m/z
Detector
MALDI-TOF/MS
Laser
Sample Target
Time-of-Flight
Clock
Detector
Molecular Mass
MALDI-TOF/MS
matrix
analyte
sample solution
dry samples
insert target and
perform analysis
sample
deposition
Ionización/Desorción
Sample embedded in
light-absorbing matrix
LASER-excitation of
matrix molecules
Sample desorption and
protonation
LASER
H+
Matrix for Proteins: sinapinic acid, dihydroxybenzoic acid etc.
Matrix for Peptides: 4-hydroxy-a-cyanocinnamic acid, DHB.
it co-crystallizes, absorbs laser energy, evaporates and acts as acid
Matrix Assisted Laser Desorption / Ionization (MALDI)
Laser
beam
Matrix
molecules
Matrix ions
+
Matrix molecules
+
+
+
Cationized analyte
Analyte molecules
Cortesía de Bruker Daltonics
Tres niveles de preparación de
muestras:
– Sensibilidad normal: método estándar.
– Alta sensibilidad: método “anchor-chip” con
matriz HCCA y cristalización homogénea de
Bruker.
– Muy alta sensibilidad: método “anchor-chip”
con matriz DHB y cristalización heterogénea.
Preparación de la muestra: anchor-DHB
Fundamentos del TOF
MALDI-TOF
Laser
Source
Reflector
detector
Reflector
Linear
detector
Resolución isotópica
a. i .
6000
5000
4000
3000
2000
1000
0
1106
1111
1116
1121
1126
m/ z
Voltaje de extracción
Extracción retardada
Extracción retardada
Decomposition occurs in the flight tube
Laser
Source
Decay can
occur at any
point along here
Reflector
detector
Reflector
Linear
detector
Fundamentals of PSD
1. PSD refers to a method of detecting and measuring
the masses of fragment ions formed from a selected
precursor ion during the flight time.
2. Fragment ions are mainly formed by unimolecular
decomposition after the precursor ions are fully
accelerated (after they exit the source—hence postsource decay)
3. Fragment ions are separated in the reflector.
PSD fragment ion velocities are
the same as their precursors
+
+
All three of these species
travel at the same velocity in
the flight tube until they reach
the mirror.
Why? Velocity is determined by initial acceleration. Initial
energy = 20 keV. Bond energies = ~ 10 eV, so breaking a
bond has a very minor effect on velocities.
Neutral fragments are not
detected
Reflector
detector
Fragmented
precursor ion
+
+
0 V.
Reflector
+20 kV
Fragment ions take different
paths
in
the
reflector
Reflector
detector
Intact
precursor ion
+
+
Fragment ion
formed by PSD
0 V.
Reflector
+20 kV
PSD
ISD