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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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Spettrometria di massa l.jpg

Spettrometria di massa

Applicazioni in biologia


Spettrometria di massa2 l.jpg

Spettrometria di massa

  • Tecnica analitica separativa

  • Ioni in fase gassosa


Spettrometro di massa l.jpg

Spettrometro di massa

Rivelatore

Sorgente

Analizzatore

m/z


Sorgenti l.jpg

Sorgenti

  • Ionizzazione diretta

    • EI, CI

  • Desorbimento

    • FAB

    • MALDI

  • Nebulizzazione

    • ESI


Ionizzazione diretta l.jpg

Ionizzazione diretta


Slide6 l.jpg

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


Slide7 l.jpg

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


Electrospray ionization esi l.jpg

+

+

+

+

+

+

+

+

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


Slide10 l.jpg

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


Calcolo accurato della massa m r 23983 da l.jpg

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 = 1845.9n = 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


La sorgente esi pu essere collegata all uscita di un sistema di separazione l.jpg

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


Analizzatori di massa l.jpg

Analizzatori di massa

  • Time of Flight (TOF)

  • Quadrupolo

  • Trappola ionica

  • Fourier Transform (FT)


Slide15 l.jpg

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.


Slide16 l.jpg

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


Slide17 l.jpg

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


Slide18 l.jpg

Reflector detector

Lineardetector

Laser

+20 kV

0 V.

+20 kV

Ions follow this path

Source

Reflector

TOF reflector


Quadrupolo l.jpg

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.


Quadrupolo20 l.jpg

m1

m2

m4

m3

m2

m1

m4

m3

mass scanning mode

m1

m2

m2

m2

m2

m2

m4

m3

single mass transmission mode

Quadrupolo


Tandem ms l.jpg

Tandem MS


Ms ms l.jpg

MS/MS


Ms ms tandem ms l.jpg

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.


Frammentazione di peptidi l.jpg

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


Applicazione peptide mass fingerprinting pmf l.jpg

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


Peptide mass fingerprinting ms l.jpg

Peptide Mass Fingerprinting (MS)

peptide fragments

intact protein

enzyme

MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS

PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW

EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK

SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE

SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL

LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE

PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK

GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED

HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT


Peptide mass fingerprinting l.jpg

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


Identificazione della proteina l.jpg

Identificazione della proteina


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