MALDI-TOF MSy

Análisis de Proteínas

Alberto Jorge GarcíaLaboratorio de Proteómica

Centro de Biología Molecular Severo OchoaUniversidad Autónoma de Madrid- CSIC

Curso Espectrometría de masas, Electrospray Trampa Iónica y Maldi-Tof

UNIVERSIDAD FRANCISCO DE VITORIA

Proteoma

Proteína

Péptidos

Fragmentos

BASE DEDATOS

BASE DEDATOS

Identificación Proteína

Identificación Péptido

Proteómica:

ESTRATEGIA INTEGRADORA PARA EL ANÁLISIS DEL PROTEOMA

SDS-PAGE

2D-IEF-SDS-PAGEBandas de proteínasDigestión “in situ”

en paralelo

MALDI-TOF mapa de péptidos

m-desalado automático

Electrospray-trampa iónica

aislamiento y fragmentación

Identificación de proteína

Secuenciación “de novo”

Extracto de péptidos

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nsid

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m/z

Mapa de péptidos (MALDI-TOF)Extracto crudo digestión “en gel”

1. gi|191618 Mass: 223549 Score: 87 (M76598) alpha cardiac myosin heavy chain [Mus musculus]Observed Mr(expt) Mr(calc) Delta Start End Miss Peptide 1061.02 1060.01 1060.13 -0.12 1366 - 1374 0 ANSEVAQWR 1085.02 1084.01 1084.32 -0.31 436 - 443 0 MFNWMVTR 1239.27 1238.26 1238.28 -0.01 1253 - 1262 0 TLEDQANEYR 1346.49 1345.48 1345.52 -0.04 24 - 34 0 LEAQTRPFDIR 1442.63 1441.62 1441.61 0.02 1680 - 1691 0 NNLLQAELEELR 1489.17 1488.16 1488.57 -0.41 1117 - 1128 0 IEELEEELEAER 1602.80 1601.79 1601.82 -0.02 955 - 968 1 DIDDLELTLAKVEK 1703.76 1702.75 1702.90 -0.14 1423 - 1436 0 LQNEIEDLMVDVER 1741.93 1740.92 1740.98 -0.06 726 - 741 0 ILNPAAIPEGQFIDSR 1840.03 1839.02 1838.99 0.03 1179 - 1195 0 DLEEATLQHEATAAALR 1896.08 1895.07 1895.02 0.06 1198 - 1214 0 HADSVAELGEQIDNLQR 1979.38 1978.37 1978.18 0.19 1620 - 1636 0 MEGDLNEMEIQLSQANR 2107.39 2106.38 2106.35 0.03 1619 - 1636 1 KMEGDLNEMEIQLSQANR 2277.72 2276.71 2276.54 0.17 1063 - 1081 1 LTQESIMDLENDKLQLEEK 2343.63 2342.62 2342.42 0.20 887 - 906 0 NDLQLQVQAEQDNLNDAEER 2809.57 2808.56 2809.04 -0.48 1000 - 1024 1 ALQEAHQQALDDLQAEEDKVNTLTK 2837.05 2836.04 2836.07 -0.02 1654 - 1678 1 DTQLQLDDAVHANDDLKENIAIVERNo match to: 1771.94

Identificación de la proteína a partir del mapa de péptidos(“peptide-mass fingerprinting”)

Búsqueda en MASCOT(D.Pappin, ICRF, Londres)

MALDI-TOF/MSTransfer into the gas phase

(desorb) IonizeApply electromagnetic

fields

Analyse ion movementsDetermine m/z

DetectorIon Source

MALDI-TOF/MS

Laser

Sample Target DetectorClock

Time-of-Flight Molecular Mass

matrix analyte sample solution

sample deposition

dry samples

insert target andperform analysis

MALDI-TOF/MS

Sample embedded inlight-absorbing matrix

LASER

LASER-excitation ofmatrix molecules

H+

Sample desorption andprotonation

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

Ionización/Desorción

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+

Cationized analyte

Analyte moleculesMatrix

molecules

Laser beam

Matrix molecules

Matrix ions

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Matrix Assisted Laser Desorption / Ionization (MALDI)

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

Laser

ReflectorSource

Lineardetector

Reflector detector

MALDI-TOF

Resolución isotópica

1106 1111 1116 1121 1126 m/z

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Voltaje de extracción

Extracción retardada

Extracción retardada

Laser

ReflectorSource

Lineardetector

Reflector detector

Decay can occur at any point along here

Decomposition occurs in the flight tube

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 post-source decay)

3. Fragment ions are separated in the reflector.

Fundamentals of PSD

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 detectedReflector

detector

Reflector +20 kV0 V.

Fragmentedprecursor ion

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Fragment ions take different paths in the reflector

Reflectordetector

Reflector +20 kV0 V.

Fragment ion formed by PSD

Intact precursor ion

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PSDISD