Tandem MS = MS / MS In tandem MS (MSMS) (pseudo-)molecular ions are selected in MS1 ESI-MS give information on the mass of a molecule but none on the structure (pseudo-)molecular ions are selected in MS1 and fragmented by collision with gas. collision induced decay – CID electron transfer decay – ETD (= ECD) The fragment ions are analyzed in a second MS.
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Tandem MS = MS / MS - BOKU · Tandem MS = MS / MS In tandem MS (MSMS ) (pseudo -)molecular ions are selected in MS1 ... Proteomics work with ESI-MS/MS 100 2: TOF MSMS 1087.35ES+ 1553.54
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Tandem MS = MS / MS
In tandem MS (MSMS)
(pseudo-)molecular ions are selected in MS1
ESI-MS give information on the mass of a molecule
but none on the structure
(pseudo-)molecular ions are selected in MS1
and fragmented by collision with gas.collision induced decay – CID
electron transfer decay – ETD (= ECD)
The fragment ions are analyzed in a second MS.
Quadrupole
ESIion
source
Entranceoptics
Massanalyzer
detector
Q0
Q1
Separation of
primary ionsMS
Triple Quadrupole
Collision cell
ESIion
source
Q2
Entranceoptics detector
Q0
Q1 Q3
Separation of
primary ionsMS ion transfer
Separation of
fragment ionsMS / MS
tandem MS
Precursor
ion selection
Difference: Collision energy
just a longer flight path, nothing gained
Target analysis by MS-MS
Collision cell
ESIion
source
Q2
Entranceoptics detector
Q0
Q1 Q3HPLC
Analysis of
fragment ions
MS / MS
Precursor
ion selection
In LC-MS: mass selected
for compounds eluting in a
certain time window
Target analysis by MS-MS
Collision cell
ESIion
source
Q2
Entranceoptics detector
Q0
Q1 Q3HPLC
Detection of
specific
fragment ionsMS / MS
Precursor
ion selection
-
fixed in
time window
Only m/z 147Example: dexamethazon m/z 393
Or: 121,147,237“Transitions“
Reaction monitoring
SRM / MRM
Tandem MS = MS / MS
Target analysis: mass of analyte and mass of fragments are
known beforehand.
MS1 and MS2 are preset on target masses
maximum dwell time, maximum sensitivity
In proteomics applications nothing is known. Precursor mass is
determined by “survey scan“
Precursor mass is selected by operator (off-line) or PC (on-line;
“data-dependent acquisiton“) according to abundance, charge
state and additional information
Tandem MS = MS / MS
Precursor Ion scan:
Neutral loss scan:
Fragment masses indicate structural details
e.g. 365 reveals glycopeptides
Loss of a certain mass by removal of chemical group, e.g. – 18 by H2O
Loss of 98 indicates phosphorylation
Requirement for proteomics applications:
Resolution of multiply charged isotope clusters, high accuracy of MSMS
→ Q-TOF, ion trap, IT-ICR
MS1
quadrupole
MS2
TOFentrancelenses
Collision celloctapole
Hybrid-instruments: Quadrupole-TOF (Q-TOF)
rotary vacuum pumps „rough pumps“
Waters Synapt II:
R in V-mode: 20.000
R in W-mode: 40.000
TOF as MS2 allows
higher resolution, accuracy and
upper mass limit.
rotary vacuum pumps „rough pumps“
turbomolecular pumps for high vacuum inside instrument
PC for control and data aquisition
Server for databank searches
N2-generator (and oil-free compressor)
Argon (collision gas)
Bruker Maxis 4G:
up to 60.000
Hybrid-instruments with Orbitrap analyzers
Combination of ion trap
and Orbitrap analyzer
Newest option:
Combination of quadrupole
with Orbitrap analyzer
Exact mass analysis of unknown compounds
over a wide mass.
Typical application:
Applications of MS-MS
Hybrid instruments or Trap:
Typical application:
peptide identification by MS-MS
structural analysis of biochemicals ....
---> fast "scan" rate of TOF or Trap
MS1 MS2
Q-TOF in MS Mode
entrancelenses
octapole
Primary ions are
collected and sent to
MS1
MS1 MS2
Q-TOF in MS Mode
MS1 does not filter,
all ions pass through
MS1 MS2
Q-TOF in MS Mode
collision cell is inactiv
(ions are slow)
ions pass unaltered
MS1 MS2
Q-TOF in MS Mode
TOF analyses
primary ions
MS1 MS2
Q-TOF in MS/MS Mode
entrancelenses
octapole
Primary ions are
collected and sent to
MS1
MS1 MS2entrancelenses
Collision cell
Q-TOF in MS/MS Mode
MS1 selects parent
ion of a certain mass
(m/z);Others cannot pass
MS1 MS2entrancelenses
Collision cell
Q-TOF in MS/MS Mode
Collision with gas atoms
(e.g. Ar) causes
fragmentation of ions
(collission induced
dissocation = CID)
Collision energy is controlled
by kinetic energy of the
analyte ions.
MS1 MS2entrancelenses
Collision cell
Q-TOF in MS/MS Mode
Daughter ions leave
the collision cell
MS1 MS2entrancelenses
Collision cell
Q-TOF in MS/MS Mode
MS2 (TOF) analyses
fragment ions
De novo sequencing of a peptide of mass 1212.33 from a wasp venom allergen
Proteomics work with ESI-MS/MS
MSMS 607.33 ES+
%
v ulgaris PLA MSMSv ulgaris_PLA _MSMS Max Ent 3 68 [Ev 4631,It50,En1] (0.040,200.00,0.060,14 00.00,2,Cmp)