1. What is MSMS?
MS/MS means using two mass analyzers (combined in one instrument) to select an analyte (ion) from a mixture, then generate fragments from it to give structural information.
Mixture of ions
Single ion
Fragments
Ion source
MS-1
MS-2

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

3. 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

4. Applicazioni della MS/MS
Maggior contenuto di informazioni
Studio della struttura
Informazioni addizionali per determinare la struttura di uno ione
Rivelazione selettiva di uno ione
Drastica riduzione delle interferenze
Studio delle reazioni ione-molecola

5. Mass Spectrometry +COH +CH3 CH3COCH3 CH3+COCH3 CH3C+OCH3 +COCH3 Sample
Tandem MS
9/6/2018
Mass Spectrometry
CH3COCH3
Sample
Inlet
CH3+COCH3
Ionization
& Adsorption
of Excess Energy
Mass Analysis
CH3C+OCH3
+COCH3
+CH3
+COH
Fragmentation
(Dissociation)
Detection
A mass spectrometer is comprised of a number of discrete components.
Firstly, the sample needs to be introduced into the mass spectrometer. In all the work in this presentation, a solution of the sample to be analysed is infused or injected into the mass spectrometer.
In order to then analyse molecules by mass spectrometry, it is necessary to generate gas-phase ions to ionise the sample. There are a variety of methods of ionisation that can be used to generate the species of interest, but the one we will be focusing on today is electrospray ionisation.... but we'll cover that in more detail in a couple of slides, or so. Predominantly, though, the mechanism of ionisation is molecular protonation, or the addition of another cationic (e.g. sodium or ammonium ion) species to the molecule of interest.
Depending on how much energy is applied during the ionisation process, we may or may not induce fragmentation in the molecule. Typically, electrospray is a soft ionisation technique which means that we see predominantly molecular related species in the mass spectrum.
Once we have generated the ions, we separate them out according to, strictly, their mass to charge ratios. As most of the ions we will be considering today will appear as the singly charged species, what we are in effect measuring is the mass of the molecules and fragments generated in the ionisation process.
The ions separated out are then detected at the end of the instrument, the information is passed to a computer, and we see the mass spectrum. The mass spectrum is typically represented with the mass (or m/z) of the ions on the abscissa and the relative signal strengths on the ordinate.
Now, how does tandem mass spectrometry differ from single stage mass spectrometry that we have seen in this slide.
Dr. Edward Randell

6. Multidimensional Analyses
Tandem MS
9/6/2018
Multidimensional Analyses
m/z
m/z
response
m/z
chromatogram
time
Dr. Edward Randell

7. Common Methods of Fragmentation
Threshold Dissociation (Weakest bonds are broken)
Collision Induced Dissociation (CID)
Apply an electric field to accelerate precursor ions and induce bond cleavage via collision with neutral gas molecules (Ar, N2)
Infrared Multiphoton Dissociation (IRMPD)
Use infrared laser to break bonds
Electron Capture Dissociation (ECD) (Random)
Multiply-charged cations capture electrons, inducing odd-electron fragmentation

8. CID + + Ar + + Accelerated Precursor Ion Fragmentation through one or
more collisions
Product Ions
(And Neutrals)

9. Instruments for MS/MS
Instruments where one or more analyzers are placed in series
Triple Quadrupole (CID at eV levels)
Four Sector (CID at keV levels)
Quadrupole-Time-of-Flight (Q-TOF) (CID eV)
TOF-TOF
Trapping Instruments
Quadrupole Ion Trap (CID eV, IRMPD, ECD)
FT-ICR (CID, SORI-CID, IRMPD, ECD)

10. 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

13. Ion Trap Mass Analyzer
Top View
Cut away side view

14. Tandem Quadrupole Collision cell MS2 MS1 Tandem MS 9/6/2018
This is a picture taken of the insides of a Quattro micro where we can see the components of MS1 (left), the collision cell (centre) and MS2 (right). This is an area of the instrument that the user would never be expected to access, but it is worth seeing nonetheless.
Dr. Edward Randell

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

16. Full Scan Acquisition Mode
Tandem MS
Collision cell
MS1
MS2
9/6/2018
MS1
MS2
Collision
Cell
Scanning
Rf only, pass all masses
Full Scan Acquisition Mode
So, what kind of experiments can we conduct, and importantly, what type of data can we generate from a tandem quadrupole mass spectrometer?
First, in a full scan acquisition the quadrupole mass filter is scanned over a user-defined mass range. Thus, all ions generated in the source region of the instrument will be sorted, or filtered, by the first quadrupole mass spectrometer. Only those compounds having masses within the user-defined range will be allowed to pass to the detector. These are the only compounds we will monitor in this experiment.
SCANNING MODE: The first quadrupole mass analyzer is Scanning over a mass range. The collision cell and the second quadrupole mass analyzer allow all ions to pass to the detector.
Dr. Edward Randell

17. Collision induced dissociation
Tandem MS
9/6/2018
Collision induced dissociation
Argon gas
Precursor ion
Product ions
This slide demonstrates the principles of collision-induced dissociation. Ions from MS1 – the precursor ions pass from MS1 into the collision region. Inside the collision cell, argon gas is maintained at a known pressure. The ions from MS1 collide with the gas and some of the translational (kinetic) energy of the ions is converted into internal energy.
Molecules are fragile, and if sufficient energy is put into the molecules, they will break at their weakest points. This fragmentation may not be predictable (it is not quantised as in NMR or infra-red spectrometry/spectroscopy), but it is very reproducible.
The ions pass through the collision cell and out of the other side, into MS2, where the fragments are measured.
The fragmentation of the selected precursor ions can be controlled by varying: (1) the speed of the ions as they enter the collision region (the collision energy) or (2) the number of collisions undertaken (the collision gas pressure)
In the collision cell, the TRANSLATIONAL ENERGY of the ions is converted to INTERNAL ENERGY.
Collision conditions (FRAGMENTATION) is controlled by altering:
The collision energy (speed of the ions as they enter the cell)
Number of collisions undertaken (collision gas pressure)
Dr. Edward Randell

18. Product ion scanning Collision cell MS1 MS2 Argon gas
Tandem MS
Collision cell
MS1
MS2
9/6/2018
Argon gas
Product ion scanning
Products
Precursor
…product ion scans. Product ion scans provide useful structural information about compounds as well as aiding in the selection of product ions for quantitation when using Multiple Reaction Monitoring (MRM).
Static (m/z 315.1)
Scanning
The first quadrupole mass analyzer is fixed at the mass-to-charge ratio (m/z) of the precursor ion to be interrogated while the second quadrupole is Scanning over a user-defined mass range.
Dr. Edward Randell

19. Modalità di scansione MS/MS
Scansione dello ione prodotto
Selezione m/z
Scansione
Scansione dello ione precursore
Simbolismo alternativo
Analizzatore di massa fisso
Spettrometro di massa a scansione
Scansione dello ione prodotto
Scansione dello ione precursore
Scansione di perdita neutra
Monitoraggio di reazione selezionata
Scansione
Selezione m/z
Scansione di perdita neutra
Scansione m/z = x
Scansione m/z = x-a
Monitoraggio di reazione selezionata
Selezione del precursore m/z = a
Selezione del frammento m/z = b

20. Product Ion Scan Mode in a
Triple Quadrupole
Q1 Mass selection
Q2 collision cell
Q3 Full Scan A C B
Ion C
C+Ar Products
Products D
Ions in
Source
Q1 only
transmits
Ion C
Fragment Ion C
Q3 Scans for
products

21. 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).

22. Multiple Reaction Monitoring (MRM)
in a Triple Quadrupole
Q1 Mass selection
Q2 collision cell
Q3 Mass Selection A C B
Ion C
C+Ar C1+C2+C3
Ion C2 D
Ions in
Source
Q1 only
transmits
Ion C
Fragment Ion C
Q3 only transmits
Ion C2

23. Neutral Loss Scan Mode in a Triple Quadrupole
Q1 Pass A-H2O
Q2 collision cell
Q3 Pass A
Ion A-H2O
Ion B
Ion C
Ion D
Ion E
Ion F
Ions
A-F A
A-H2O A+ +H2O
Scans across
mass range
Fragment ions one at a time
Scans across
mass range at
18 amu lower
than Q1

24. Neutral loss scanning Collision cell MS1 MS2 Argon gas Scanning (M)
Tandem MS
Collision cell
MS1
MS2
9/6/2018
Argon gas
Neutral loss scanning
Products
Precursors
Scanning (M)
Scanning (M-102)
In a neutral loss scan the two quadrupole mass filters are Scanning synchronously at a user-defined offset. The neutral loss is known to be common to the analytes in a mixture.
Dr. Edward Randell

25. Precursor Ion Scan Mode in a Triple Quadrupole
Q1 Mass selection
Q2 collision cell
Q3 pass only 79
Ion A-PO4
Ion B
Ion C
Ion D
Ion E
Ion F
Ions
A-F
PO3- 79
Scans across
mass range
Fragment ions one at a time
Transmits only 79

26. -Casein Digest Total Ion Chromatogram2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52
Time, min
0.0
5.0e7
1.0e8
1.5e8
2.0e8
2.5e8
3.0e8
3.5e8
4.0e8
4.5e8
5.0e8
5.5e8
6.0e8
6.5e8
7.0e8
7.5e8
Intensity (cps)
Courtesy of the MSCLS at the Univ. of Minn.

27. Negative Precursor Ion Chromatogram
Intensity (cps)
8.00e7
7.00e7
6.00e7
5.00e7
4.00e7
3.00e7
2.00e7
1.00e7
0.00 5 10 15 20 25 30 35 40 45 50 55 60 65
Courtesy of the MSCLS at the Univ. of Minn.
Time, min