Spettrometria di massa
Applicazioni in biologia
Spettrometria di massa
• Tecnica analitica separativa
• Ioni in fase gassosa
Spettrometro di massa
Sorgente
Analizzatore
m/z
Rivelatore
Sorgenti
• Ionizzazione diretta
– EI, CI
• Desorbimento
– FAB
– MALDI
• Nebulizzazione
– ESI
Ionizzazione diretta
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.
AH+
3. Il campione è protonato
e ionizzato per
trasferimento energetico
dalla matrice:
Variable Ground
+20 kV
Grid
Grid
MH+ + A  M + AH+.
Spetto MALDI/TOF di IgG
MH+
Relative Abundance
40000
30000
(M+2H)2+
20000
10000
(M+3H)3+
0
50000
100000
m/z
150000
200000
Electrospray Ionization (ESI)*
Vacuum Interface
charged
droplets form
High Voltage
–
+
+
Sampl
e Flow
+
+
++++
– +
++–
+
+
+
+
+
+
+
+
+
+
+
Nebulizer Gas
Spray
* Broad range of implementations based on
flow rate and polarity of compound class
Ions
released
To MS
Spettro ESI del Tripsinogeno (MW 23983)
M + 15 H+
Relative Abundance
1599.8
M + 16 H+
M + 14 H+
1499.9
1714.1
M + 13 H+
1845.9
1411.9
1999.6
2181.6
m/z
Mass-to-charge ratio
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
1599.8
1499.9
1714.1
1411.9
1845.9
1999.6
2181.6
•
•
•
•
(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 può essere collegata
all’uscita di un sistema di separazione
0.18mm X 15cms C18 column
400nl/min
7:1 flow split
Mass spectrometer
4µl/min
LC
Fraction collector
Further MS analysis
Analizzatori di massa
• Time of Flight (TOF)
• Quadrupolo
• Trappola ionica
• Fourier Transform (FT)
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.
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
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
TOF reflector
Laser
Reflector
detector
Linear
detector
0 V.
+20 kV
+20 kV
Source
Ions follow this
path
Reflector
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.
Quadrupolo
m2
m4
m1
m4
m3
m2
m2
m2
m1
m3
mass scanning mode
m2
m4
m1
m3
m2
single mass transmission mode
m2
Tandem MS
MS/MS
MS/MS -Tandem MS
Informazioni strutturali (es. Sequenza)
Sample
ionized
Mass
selected
Fragmentation
Analyze
fragments
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
xn-i
= Most common
yn-i
vn-i
yn-i-1
wn-i
zn-i
-HN-CH-CO-NH-CH-CO-NHRi
CH-R’
i+1
ai
R”
i+1
bi
low energy fragments
ci
di+1
bi+1
high energy fragments
Applicazione
Peptide Mass Fingerprinting (PMF)
1D or 2D gel
silver-stained
Run gel,
stain
MALDI-TOF
Excise spot,
destain, wash,
digest, extract
peptides
Spot onto plate and
mass analyze
Protein ID
Search all spectra
against protein
databases
Peptide Mass Fingerprinting (MS)
peptide
fragments
intact
protein
enzyme
MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS
PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW
EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK
SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE
SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL
LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE
PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK
GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED
HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT
2495.04
2396.28
2010.53
1940.58
1624.04
1457.75
1507.75
1542.01
1357.91
1248.39
1100.6
1042.53
962.494
900.392
5000
1742.96
1777.15
1394.77
1265.65
842.512
intact
peptide
protein enzyme fragments
10000
823.501
Counts
15000
1083.54
Peptide Mass Fingerprinting
0
1000
1200
1400
1600
1800
2000
2200
2400
Mass (m/z)
Process data and peak detect spectrum
Create mass list from spectrum
…
900.3921
1083.5423
1265.6489
1394.7688
1507.7522
1542.0116
1777.1544
…
Identificazione della proteina