Tandem Mass Spectrometry : MS/MS or MSn

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Tandem Mass Spectrometry MS/MS or MSn

• Mass spectrometry is a very powerful method to
  analyse the structure of organic compounds, but
  suffers from 3 major limitations
• Compounds cannot be characterised without clean
  samples
• This technique has not the ability to provide
  sensitive and selective analysis of complex
  mixtures
• For big molecules like peptides spectra are very
  complex and very difficult to interpret

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Tandem MS
M1x, M1y,.
MS
M1x, M1y,.
M1, M2,.
GC-MS
MS-MS
M1, M2
M1x, M1y,.
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GC-MS/MS
M1a, M1b..
M1, M2,.
M1ax, M1ay,.
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Tandem Mass Spectrometry MS/MS or MSn

• The technique has been used for
• Structure elucidation of unknown
• Analysis of complex mixture with minimum sample
  clean up.

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Tandem mass spectrometry (MS/MS)
Tandem Mass Spectrometry MS/MS or MSn

• Enhance selectivity by combining complimentary
  techniques
• Number of Stages
• GC
• GC/MS
• GC/MS/MS
• GC/MS/MS/MS

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Tandem Mass Spectrometry MS/MS or MSn
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Tandem Mass Spectrometry MS/MS or MSn
MS1
MS2
The ion selected by the MS1 is the parent ion and
can be a molecular ion resulting from the primary
fragmentation. DISSOCIATION occurs in the
fragmentation region. The daughter ions are
analysed in the Second Spectrometer (MS2). In
fact, the MS1 can be viewed as an ion source for
MS2.
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Formation of daughter ions
The general equation mp ? md
mn 1 mp is referred to as the parent
ion. Molecular ion is the ion, which has the
mass equivalent to the molecular weight of the
compound, introduced into the ion source. Parent
ion need not to be molecular ions. Both
molecular ions and fragment ions can be selected
as parent ions in MS/MS.
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• MS/MS experiment can be described as
• Mass selection of a parent ion in the first stage
  of analysis within the instrument, and then
• Analysis of the daughter ions often formed in a
  collision- induced dissociation (CID) process, in
  the second stage of analysis.

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Two processes used to provide the energy for
reaction 1. (1) Metastable ions Excess energy
deposited in the parent ion during the ionization
process. Metastable ions An ion with the
appropriate dissociation rate versus internal
energy content may survive long enough to be
extracted from the ion source before it
fragments, but may then fragment prior to
detection. Such ions are called metastable ions.
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• Limitation of using metastable ions
• the percentage of metastable ions relative to all
  ions leaving the ion source is generally very
  small, and the time the ion spend in the reaction
  regions is generally only a small percentage of
  the total transit time through the MS.
• The metastable ions are characterized by a
  relatively narrow range of internal energies,
  therefore there are few fragmentation routes that
  are available for their decomposition.

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(2) Collision-induced dissociation (CID)
Admit a collision gas to a reaction region to
induce dissociations by collision of the parent
ion with the neutral target gas. This process is
called Collision-induced dissociation (CID).
N mp md mn 2 here the
only difference from 1 is the extra energy
imparted to mp , in this case by collision with
neutral target gas N.
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Types of dissociation reactions (1)
N mp ? md mn 2 (2)
Charge striping N mp ? mp2
e- 3 The process involves the loss of an
electron from the parent ion rather than the loss
of neutral fragment. The loss of an electron from
a positive ion usually requires substantially
more energy than does a dissociation process.
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(3) Neutralization-reionization Nx
Ny mp ? mp ? md
mn 4 The parent ion is first neutralized and
subsequently reionized in a collision. Useful
process in detailed ion structural studies. (4)
Charge inversion N mp- ? md
mn 2e- 5 This is actually a combination of
2 and 3.
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Tandem Mass Spectrometry MS/MS or MSn
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Triple Quadrupole Mass Spectrometer
microcapillary
Q1
Q2
Q3
electrospray source
collision cell
detector
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Ion Trap Mass Spectrometer
microcapillary
detector
end cap
end cap
electrospray source
ring electrode
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Instrument design Daughter ion scan The parent
ion mass is fixed and the masses of all daughter
ion formed from the parent ion are measured.
The first stage of analysis selects ions of
particular m/z. This parent ion is passed onto
the reaction region. The daughter ions formed in
the reaction region are then analyzed by mass.
These daughter ions are generally characteristic
of the structure of the parent ions and thus
provide a means of determining that structure
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Parent ion Scan The first spectrometer is
scanned, while the second one is set to the mass
of one of the daughters. Closely related
compounds usually give several of the same
daughters, so that this method of operation
provides a measure of identity and concentration
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Tandem Mass Spectrometry MS/MS or MSn
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MS/MS Product (Daughter) Scan

• Product Ion scans are used to get more
  information unknown fragments.
• Basic question what is?
• First analyzer operates in Quasi SIM at mass X
  to isolate the fragment is question.
• In the collision chamber it is further
  fragmented, which is analyzed in the second
  analyzer which scan from X to 0.

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Protein Identification Strategy
I
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5
4
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Peptides
Time (min)
Separate peptides
Fragment peptide
II
1000
1200
200
400
600
800
m/z
Q2 Collision Cell
Q3
Q1
Tandem mass spectrum
Computer sequence database searching
III
Peptide identification, Protein identification
1200
1200
200
400
600
800
1000
200
400
600
800
1000
m/z
m/z
Theoretical
Acquired
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Tandem MS spectrum
GDIVNLGSIAGR
H2N-NSGDIVNLGSIAGR-COOH
DIVNLGSIAGR
IVNLGSIAGR
VNLGSIAGR
NLGSIAGR
LGSIAGR
GSIAGR
Relative Abundance
SIAGR
IAGR
YMR134W, yeast protein involved in iron metabolism
AGR
GR
R
200
400
600
800
1000
1200
m/z
Q1
Q2
Q3
LC-ESI
685.3 /- 0.3
CID
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MS/MS Product (Daughter) ion Scan
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MS/MS Precursor (Parent) Ion Scan

• Precursor (Parent) Ion scans can be used to find
  the origins of some fragments e.g. the molecular
  ions.
• Basic question What is the origin?
• The second analyser operates in quasi SIM at mass
  x, while the first analyser scans from x1 to
  high masses.
• NOT FOR ION TRAP!!

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MS/MS Precursor (Parent) Ion Scan
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MS/MS Neutral Loss Scan

• Neutral loss scans are used to find compounds
  with special properties in the chromatogram.
• It is essential that the respective compounds do
  a significant cleavage in the collision cell.
• During this experiment both analyzers scan in
  parallel offset with the respective (loss)
  difference.
• The multiplier can only be hit by ions that
  cleave the respective mass in the collision cell.

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MS/MS Neutral Loss Scan
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MS/MS Selected Reaction Monitoring (SRM)

• In SRM specific cleavages are used to quantify
  compounds in complex matrices.
• Basic question were is my quantification signal
  in this jungle of peaks?
• Both analysers work on (co-ordinated) SIM
  programs.

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MS/MS Selected Reaction Monitoring (SRM)
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How many options?