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Spettrometria di massa

Spettrometria di massa. Applicazioni in biologia. Spettrometria di massa. Tecnica analitica separativa Ioni in fase gassosa. Spettrometro di massa. Rivelatore. Sorgente. Analizzatore. m/z. Sorgenti. Ionizzazione diretta EI, CI Desorbimento FAB MALDI Nebulizzazione ESI.

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Spettrometria di massa

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  1. Spettrometria di massa Applicazioni in biologia

  2. Spettrometria di massa • Tecnica analitica separativa • Ioni in fase gassosa

  3. Spettrometro di massa Rivelatore Sorgente Analizzatore m/z

  4. Sorgenti • Ionizzazione diretta • EI, CI • Desorbimento • FAB • MALDI • Nebulizzazione • ESI

  5. Ionizzazione diretta

  6. Matrix Assisted Laser Desorption Ionization (MALDI) Laser Piastra (target) hn • 1. Campione (A) miscelato con matrice (M) e asciugato sulla piastra. • 2. Il Laser eccita la matrice. • 3. Il campione è protonato e ionizzato per trasferimento energetico dalla matrice: • MH+ + A  M + AH+. AH+ Variable Ground Grid Grid +20 kV

  7. 40000 30000 20000 10000 0 Spetto MALDI/TOF di IgG MH+ Relative Abundance (M+2H)2+ (M+3H)3+ 50000 100000 150000 200000 m/z

  8. + + + + + + + + To MS + – + + + + + + – + + + Electrospray Ionization (ESI)* Vacuum Interface charged droplets form High Voltage – + + Sample Flow + + Nebulizer Gas Ions released Spray * Broad range of implementations based on flow rate and polarity of compound class

  9. Spettro ESI del Tripsinogeno (MW 23983) M + 15 H+ 1599.8 M + 16 H+ M + 14 H+ 1499.9 1714.1 Relative Abundance M + 13 H+ 1845.9 1411.9 1999.6 2181.6 m/z Mass-to-charge ratio

  10. 1599.8 1499.9 1714.1 1845.9 1411.9 1999.6 2181.6 Calcolo accurato della massa (MR = 23983 Da) • (M+n+3)/(n+3) = 1499.9 • (M+n+2)/(n+2) = 1599.8 • (M+n+1)/(n+1) = 1714.1 • (M+n)/n = 1845.9 n = 13 • (M+16)/16 = 1499.9  23982.4 • (M+15)/15 = 1599.8  23982.0 • (M+14)/14 = 1714.1  23983.4 • (M+13)/13 = 1845.9  23983.7

  11. Mass spectrometer LC Fraction collector 0.18mm X 15cms C18 column 7:1 flow split 4µl/min La sorgente ESI può essere collegata all’uscita di un sistema di separazione 400nl/min Further MS analysis

  12. Analizzatori di massa • Time of Flight (TOF) • Quadrupolo • Trappola ionica • Fourier Transform (FT)

  13. time-of-flight (TOF) Ion Source Flight Tube 20-25 kV Detector Principle: If ions are accelerated with the same potential at a fixed point and a fixed initial time and are allowed to drift, the ions will separate according to their mass to charge ratios.

  14. Appendix 5: time-of-flight mass analyzer Ion Source Flight Tube Detector The ions enter the flight tube with the lighter ions travelling faster than the heavier ions to the detector

  15. Appendix 5: time-of-flight mass analyzer Ion Source Flight Tube Detector The lighter ions strike the detector before the heavier ions. This “time of flight” (TOF) can be converted to mass

  16. Reflector detector Lineardetector Laser +20 kV 0 V. +20 kV Ions follow this path Source Reflector TOF reflector

  17. Quadrupolo Uses a combination of Radio Frequency(RF) and Direct Current(DC) voltages to operate as a mass filter. • Has four parallel metal rods. • Lets one mass pass through at a time. • Can scan through all masses or sit at one fixed mass.

  18. m1 m2 m4 m3 m2 m1 m4 m3 mass scanning mode m1 m2 m2 m2 m2 m2 m4 m3 single mass transmission mode Quadrupolo

  19. Tandem MS

  20. MS/MS

  21. Sample ionized Mass selected Analyze fragments Fragmentation MS/MS -Tandem MS Informazioni strutturali (es. Sequenza) Applying two or more steps of mass analysis separated in space or time (in the same instrument) to select an analyte (ion) of interest from a mixture and generate fragments from it to give structural information.

  22. Frammentazione di peptidi xn-i = Most common yn-i yn-i-1 vn-i wn-i zn-i -HN-CH-CO-NH-CH-CO-NH- CH-R’ Ri i+1 ai R” i+1 bi bi+1 ci di+1 low energy fragments high energy fragments

  23. ApplicazionePeptide Mass Fingerprinting (PMF) 1D or 2D gel silver-stained MALDI-TOF Protein ID Excise spot, destain, wash, digest, extract peptides Search all spectra against protein databases Run gel, stain Spot onto plate and mass analyze

  24. Peptide Mass Fingerprinting (MS) peptide fragments intact protein enzyme MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT

  25. Peptide Mass Fingerprinting intact protein peptide fragments enzyme 1265.65 15000 1394.77 1083.54 842.512 10000 Counts 1742.96 900.392 1100.6 1042.53 5000 1457.75 823.501 962.494 1507.75 1777.15 2396.28 1624.04 1542.01 1248.39 1357.91 1940.58 2495.04 2010.53 0 1000 1200 1400 1600 1800 2000 2200 2400 Mass (m/z) Process data and peak detect spectrum … 900.3921 1083.5423 1265.6489 1394.7688 1507.7522 1542.0116 1777.1544 … Create mass list from spectrum

  26. Identificazione della proteina

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