1. Tandem MS

2. Linked System

3. Mass Spectrometry Linked systems
The determination of a mixture of compounds requires the separation of the components prior to detailed analysis.
This is typically achieved by two different approaches:

4. LC-MS system

5. CROMATOGRAMMA TIC (TOTAL ION COURRENT)
600
1000
Intensità
230
Intensità
m/z
Somma di tutte le intensità degli ioni presenti nello spettro di massa selezionato per generare il particolare cromatogramma TIC
123000
Tempo

6. LC-MS analysis of complex mixtures
B: 898.5 E: A B E

7. What is MS/MS? Fragmentation
MS/MS means using two mass analyzers to select an ion from a mixture, then fragment it to give structural information
Tandem mass spectrometry selects one of the intense peaks observed in the single stage mass spectrum and further fragments all peptides with the selected mass to charge ratio. The tandem mass spectrum typically contains mass to charge ratio information about fragments of a a single peptide.
Fragmentation
Selected ion

8. The masses of all the pieces give an MS/MS spectrum
What is MS/MS?
Peptide
mixture
1 peptide
selected for MS/MS +
MS/MS + + + +
The masses of all the pieces give an MS/MS spectrum
Have only masses to start

9. Interpretation of an MSMS spectrum to derive structural information is analogous to solving a puzzle+ + + + +
Use the fragment ion masses as specific pieces of the puzzle to help piece the intact molecule back together

10. NanoLC MS/MS MS MS/MS MS trace MS/MS trace
m/z
600
200
1000 y2 b3 y3 y4 y5 b7 y7 b8 y6 b9 y9
y10
b11
b12
MS/MS
MS trace
MS/MS trace 30 40 50
Time [min]
100 fmol BSA injected on column. BPC of m/z , and typical MS/MS spectrum (right inset).

11. Tandem in Space
Separation in space is achieved by coupling two mass analyzers
A parent ion is selected by the first analyzer (MS 1) and separated from the other ions in the sample
Each selected ion is activated by a collision process
The resulting set of product ions are analyzed by the second mass analyzer (MS 2)

12. Tandem in Time
Separation in time is achieved by using a single mass analyzer able to perform two steps of mass analysis
The ion of interest is first isolated in the ion trap
The ion is then activated by collisions and its dissociated products are then mass analyzed
Both the ion trap and the ion cyclotron resonance can be used in this way

13. Tandem MS - nomenclatura
Parent ion : (Ione precursore) uno ione che subisce una decomposizione o una variazione di carica.
Daughter Ion: (Ione prodotto) ione formato da una qualsiasi reazione dello ione precursore.
Fragment ion : (Ione Frammentio) ione formato dalla frammentazione dello ione precursore.
Neutral loss: specie neutra formata dalla frammentazione di uno ione precursore.

14. Collisions Induced Fragmentation
CID + + Ar
Collisions Induced Fragmentation +
Accelerated
Precursor Ion +
Product Ions
(And Neutrals)

15. Instruments for MS/MS
Instruments where one or more analyzers are placed in series
Triple Quadrupole
Four Sectors
Q-TOF
TOF-TOF
Trapping Instruments
Quadrupole Ion Trap (LIT)
- Ion Trap
FT-ICR

16. Post Source Decay
Thus, it came rather as a surprise that roughly the same kind of information,as in the ‘protein ladder’ sequencing, can be obtained as a by-product in reflectron TOF spectrometers
Simply by the analysis of the abundant post source decay (PSD) fragments
These fragments are formed in the field-free drift region after MALDI
Need a reflectron TOF spectrometer and a precursor ion selector

19. The ion originates fragment ions
Product Ion Scan
m/z
FIXED
CID
SCAN
ESI
DETECTOR Q1 Q2 Q3
Selective Mass
Filter
Originates
Scan Fragment
Ions
The ion originates fragment ions

20. Triple Stage Quadrupole
DETECTOR Q0 Q1 Q2 Q3
ION SOURCE Ar

21. QSTARTM ESI QQ TOF or MALDI QQ TOF
Sample Q0 Q1 Q2
Effective Flight
Path = 2.5 m
Ion Mirror
(reflector)

28. B q B m =  m So we can calculate the mass of the ion We know the
Once the ion is trapped,
So we can calculate the mass of the ion
the magnet bends it into
We know the
a circular path.
Magnetic Field B
q B m = 
We measure the frequency m
“Light” Ions have a High frequency
“Heavy” Ions have a Low frequency

29. Differential Amplifier
Time (ms)
800
700
600
500
400
300
200
100
0.05
0.04
0.03
0.02
0.01
-0.01
-0.02
-0.03
-0.04
-0.05
Time-Domain Transient
Image Current
Differential Amplifier
As the spiraling ion gets near
The signal is recorded for
a detect plate, it induces a
a period of time and then
current that is detected by
displayed by the software
our instrument.

30. Time-Domain Transient
Time (ms)
800
700
600
500
400
300
200
100
0.05
0.04
0.03
0.02
0.01
-0.01
-0.02
-0.03
-0.04
-0.05
Time-Domain Transient
Image Current
A Fourier Transform then
converts the “time” domain
signal into all the frequencies
that compose the “time” signal
We know how frequency relates
to mass, so we convert to the
“Mass Spectrum”
Frequency (kHz)
300
250
200
150
100 50 FT
Frequency Spectrum m z A  = + B 2
m/z
1400
1300
1200
1100
1000
900
800
700
600
500
Mass Spectrum