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MALDI-TOF MS y Análisis de Proteínas

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.

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MALDI-TOF MS y Análisis de Proteínas

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  1. Curso Espectrometría de masas, Electrospray Trampa Iónica y Maldi-Tof UNIVERSIDAD FRANCISCO DE VITORIA MALDI-TOF MSyAnálisis de Proteínas Alberto Jorge García Laboratorio de Proteómica Centro de Biología Molecular Severo Ochoa Universidad Autónoma de Madrid- CSIC

  2. Identificación Proteína BASE DE DATOS BASE DE DATOS Proteómica: Péptidos Proteoma Fragmentos Proteína Identificación Péptido

  3. ESTRATEGIA INTEGRADORA PARA EL ANÁLISIS DEL PROTEOMA SDS-PAGE Digestión “in situ” en paralelo Bandas de proteínas 2D-IEF-SDS-PAGE Extracto de péptidos mapa de péptidos MALDI-TOF Identificación de proteína m-desalado automático aislamiento y fragmentación Secuenciación “de novo” Electrospray-trampa iónica

  4. Mapa de péptidos (MALDI-TOF) Extracto crudo digestión “en gel” Intensidad m/z

  5. 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 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 DTQLQLDDAVHANDDLKENIAIVER No match to: 1771.94

  6. Ion Source Detector MALDI-TOF/MS • Analyse ion movements • Determine m/z Transfer into the gas phase (desorb) Ionize Apply electromagnetic fields

  7. MALDI-TOF/MS Laser Clock Detector Sample Target MolecularMass Time-of-Flight

  8. MALDI-TOF/MS sample solution matrix analyte dry samples sample deposition insert target and perform analysis

  9. Ionización/Desorción 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

  10. Matrix ions + Laser beam Matrix molecules + + Cationized analyte Matrix molecules + Analyte molecules Matrix Assisted Laser Desorption / Ionization (MALDI) Cortesía de Bruker Daltonics

  11. 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.

  12. Preparación de la muestra: anchor-DHB

  13. Fundamentos del TOF

  14. MALDI-TOF Lineardetector Reflector detector Laser Reflector Source

  15. Resolución isotópica

  16. Voltaje de extracción

  17. Extracción retardada

  18. Extracción retardada

  19. Decomposition occurs in the flight tube Lineardetector Reflector detector Laser Decay can occur at any point along here Reflector Source

  20. Fundamentals of PSD • PSD refers to a method of detecting and measuring the masses of fragment ions formed from a selected precursor ion during the flight time. • Fragment ions are mainly formed by unimolecular decomposition after the precursor ions are fully accelerated (after they exit the source—hence post-source decay) • Fragment ions are separated in the reflector.

  21. + + 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.

  22. + + Neutral fragments are not detected Reflectordetector Fragmentedprecursor ion 0 V. +20 kV Reflector

  23. + + Fragment ions take different paths in the reflector Reflectordetector Intact precursor ion Fragment ion formed by PSD 0 V. +20 kV Reflector

  24. PSD ISD

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