Tandem MS = MS / MS
ESI-MS give information on the mass of a molecule
but none on the structure
In tandem MS (MSMS)
(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
Entrance
optics
ESI
ion
source
MS
Q0
Mass
analyzer
Q1
Separation of
primary ions
detector
Triple Quadrupole
ESI
ion
source
Entrance
optics
MS
Q0
Q1
Collision
cell
Q2
ion transfer
Q3
detector
Separation of
primary ions
just a longer flight path, nothing gained
MS / MS
tandem MS
Precursor
ion selection
Separation of
fragment ions
Difference: Collision energy
Target analysis by MS-MS
ESI
HPLC
ion
source
Entrance
optics
Q0
Q1
Collision
cell
Q2
Precursor
ion selection
MS / MS
In LC-MS: mass selected
for compounds eluting in a
certain time window
Q3
Analysis of
fragment ions
detector
Target analysis by MS-MS
ESI
HPLC
ion
source
Entrance
optics
MS / MS
Collision
cell
Q2
Q1
Q0
Q3
Precursor
ion selection
fixed in
time window
Detection of
specific
fragment ions
m/z 393
Only m/z 147
Example: dexamethazon
“Transitions“ Or: 121,147,237
Reaction monitoring
SRM / MRM
detector
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:
Fragment masses indicate structural details
e.g. 365 reveals glycopeptides
Neutral loss scan:
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
Hybrid-instruments: Quadrupole-TOF (Q-TOF)
entrance
lenses
MS1
quadrupole
Collision cell
octapole
MS2
TOF
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)
Waters Synapt II:
R in V-mode: 20.000
R in W-mode: 40.000
Bruker Maxis 4G:
up to 60.000
TOF as MS2 allows
higher resolution, accuracy and
upper mass limit.
Hybrid-instruments with Orbitrap analyzers
Combination of ion trap
and Orbitrap analyzer
Newest option:
Combination of quadrupole
with Orbitrap analyzer
Applications of MS-MS
Hybrid instruments or Trap:
Exact mass analysis of unknown compounds
over a wide mass.
Typical application:
peptide identification by MS-MS
structural analysis of biochemicals ....
---> fast "scan" rate of TOF or Trap
Q-TOF in MS Mode
entrance
lenses
MS1
Primary ions are
collected and sent to
MS1
octapole
MS2
Q-TOF in MS Mode
MS1
MS1 does not filter,
all ions pass through
MS2
Q-TOF in MS Mode
MS1
collision cell is inactiv
(ions are slow)
ions pass unaltered
MS2
Q-TOF in MS Mode
MS1
TOF analyses
primary ions
MS2
Q-TOF in MS/MS Mode
entrance
lenses
MS1
Primary ions are
collected and sent to
MS1
octapole
MS2
Q-TOF in MS/MS Mode
entrance
lenses
MS1
MS1 selects parent
ion of a certain mass
(m/z);
Others cannot pass
Collision cell
MS2
Q-TOF in MS/MS Mode
entrance
lenses
MS1
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.
Collision cell
MS2
Q-TOF in MS/MS Mode
entrance
lenses
MS1
Daughter ions leave
the collision cell
Collision cell
MS2
Q-TOF in MS/MS Mode
entrance
lenses
MS1
MS2 (TOF) analyses
fragment ions
Collision cell
MS2
Proteomics work with ESI-MS/MS
De novo sequencing of a peptide of mass 1212.33 from a wasp venom allergen
v ulgaris PLA MSMS
v ulgaris _PLA _MSMS Max Ent 3 68 [Ev 4631,It50,En1] (0.040,200.00,0.060,14 00.00,2,Cmp)
AV I
Y
KI
%
M
C
E
MSMS 607.33 ES+
A
A
E
C
M
bMax
IK
Y
I
V
A
yMax
+AVIYMAECLK+
+
VIYMAECLK
767.36
y6
1043.51
+
866.45
y8
IYMAECLK
620.33
+
y5
YMAECLK
+
MAECLK
theoret.
+
[MH]
979.51
AECLK
930.43
703.37
y7
+
ECLK
420.26 549.29
1044.59
284.21
y4
y3
+
249.17
867.38
2+
b3
[MH 2]
CLK
768.42
1239.77
361.13
704.32
1045.44
601.38
465.67 522.22
1149.67
+
239.18
621.26
311 .11
929.36
1283.761299 .69
LK
M/z
+
120.07 172.12
0
1 00
200
300
400
50 0
600
700
800
900
10 00
Doubly charged precursor → singly charged fragments
1100
1200
1300
Proteomics work with ESI-MS/MS
1553.54
100
MSMS 1087.35 ES+
115
1438.43
1236.32
1753.60
1249.23
77 113
1337.42
1578.42
1476.68
1666.52
1050.31
%
1034.38
890.34
819.24
490.17
762.24
330.15
963.33 1147.51
599.23 721.27
280.10
201.11
508.16
308.08
0
100
1866.71
2067.542179.86
mass
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
FC+H
%
A
Y G
+
308.08
280.10
201.11
C
T T
W
1050.31
1236.32
330.15 490.17 599.23 721.27
508.16
1753.60
1249.23
1337.42
1034.38
890.34
819.24
762.24
963.33
I S
D
1438.43
2000
2100
2200
2: TOF MSMS 1087.35ES+
1553.54
100
0
100
Mexp 2173.70
200
I
1578.42
1666.52
1276.68
1147.51
1866.71
1578.77
1146.40 1235.47 1249.68
2067.542179.86
2185.86
mass
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
FCISIDTTWCAGYCYTR
Doubly charged precursor → singly charged fragments
1700
1800
1900
2000
2100
2200
Peptide fragmentation
Major fragments derived from a peptide (protonated)
y3
+2H
+2H
H2N
y1
y2
+2H
R1
R2
R3
R4
C C N
C C N
C C N
C COOH
H
H
H
H
O
a1 b1
H
O
a2 b2
H
O
H
a3 b3
Doubly charged precursor → singly charged fragments
Fragmentation of a singly charged peptide
y6
R1
H2N
y5
R2
y4
R3
y3
R4
y2
R5
y1
R6
R
C C N
C C N
C C N
C C N
C C N
C
C N
H
H
H
H
H
H
O
O
H
O
H
O
H
O
H
O
H
NH 3
H
C COOH
H
NH 2
H2N
R1
R2
R3
R4
R5
R6
C C N
C C N
C C N
C C N
C C N
C
C N
H
H
H
H
H
H
O
O
H
O
H
O
HH
O
H
O
H
R
H
C COOH
H
NH2
H2N
R1
R2
C C N
C C N
H
H
O
H
H
R4
R5
R6
N
C C N
C C N
C
C N
H
H
H
H
O
R
O
O
neutral b-fragment
H
C
C
R3
H
O
H
O
H
y4-ion
H
C COOH
H
Fragmentation of a doubly charged peptide
NH 3
H3N
R1
R2
R3
R4
R5
R6
C C N
C C N
C C N
C C N
C C N
C
C N
H
H
H
H
H
H
O
O
H
O
H
O
H
H
O
H
O
R
C COOH
H
H
NH 2
H3N
R1
R2
R3
R4
R5
R6
C C N
C C N
C C N
C C N
C C N
C
C N
H
H
H
H
H
H
O
O
H
O
H
O
HH
H
O
H
O
R
C COOH
H
H
Doubly charged precursor → singly charged fragments
H3N
NH2
R1
R2
C C N
C C N
H
H
O
H
O
O
H
C
C
R3
b3-ion
H
H
R4
R5
R6
N
C C N
C C N
C
C N
H
H
H
H
O
O
H
O
H
R
y3-ion
peptide mass + H
H
C COOH
H
Peptide fragmentation
H3N
R1
R2
C C N
C C N
H
H
O
H
O
O
H
C
H2N
C
R3
R1
R2
R3
C C N
C C N
C C O
H
H
H
O
H
O
b3-ions
H
acylium ion
peptide mass - 17
H2N
R1
R2
R3
C C N
C C N
C
H
H
H
O
H
O
H
R3
H2N
C
H
a3-ion
immonium ion
peptide mass - 45
reveal amino acids in peptide
Collision energy
Collision induced or collison activated dissociation of parent ions
(CID or CAD)
Triple quads, ion traps, Q-Tofs and similar mass specs can only provide
low energy (eV – range) fragmentations.
can be modulated within certain range (adjusted to mass of peptide)
Yields relatively simple fragment spectra.
Disadvantage: Leu and Ile cannot be discriminated
High energy CID in BE or TOF-TOF instruments.
Applications of nano-LC / MS-MS
BSA 100fmol, Time=43.0-43.3 min (#339#341), 100%=159692arb
MS
y5
MS/MS
y9
y6
b8
b9
y3
PBC m/z 300 – 2200, all MS
y4
b7
b3
y2
200
600
all MS/MS
30
40
100 fmol BSA injected on column
50
y7
Time [min]
1000
b11
y10
b12
m/z
Protein identification by LC / MS-MS
Archeal histon
Result of the
database search of
silver stained
protein gel spot.
Archeal histon was
unambiguously
found in a
Halobacterium
salinarum (genome)
database using
MASCOTTM.
Important software packages for protein identification:
MASCOT, SEQUEST F. and company derivatives e.g. MassLynx, Proteome Discoverer F..
Data-dependant aquisition
At first, the machine works in the MS mode (survey mode) until
mass is detected which:
- is of sufficient intensity
- is not in exclude list (not background, not trypsin, not keratin)
- is doubly or triply charged
- or is in include list
Then, machine switches into MS/MS mode to acquire CID spectrum of
this compound for e.g. ca. 1 sec
Then, this mass is “locked” for some time to prevent redundancy.
Often, the survey mode detects more than one signal
before switched back to survey.
MSMS 1, MSMS 2, etc.
MS/MS specials
I.) Dependancy of ion type and collision energy
- the larger the more energy required
- charges help fragmentation
- careful choice of collision energy profile
II.) DDA tends to overlook many peptides
Solutions: - increase speed of instrument
- optimize selection criteria
- rerun sample with inclusion list and/or exclusion list
- do no select target ions, fragment them all
MSE
MS/MS specials
Searching for peptide fragment (and mass) in data banks e.g. „Swissprot“
By MASCOT or SEQUEST and related tools yields list of hits with probability
The score depends on:
1.) number of peptides fitting to a particular peptide
“one hit wonders“ usually disregarded
2.) number of fragments fitting to theoretical digest of peptide
(also: those NOT fitting)
3.) size of peptide (the larger, the better)
4.) size of protein (the smaller the better)
5.) allowance of missed cleavage sites
6.) allowance of modifications
the more the worse (search space !)
7.) size of databank (too small is bad !)
Sample inlet systems for ESI
Syringe pump
5 to 50 µL / min
Liquid chromatography
50 to 1000 µL / min
System test
Calibration
“Simple” samples
Most
typical LC-MS
applications
(pharmaceutical,
environmental,
forensic
etc.)
Sample inlet systems for ESI
Nanospray tips
For limited sample
amounts in bioscience
20 nL /min
1-2 µL of sample
give 30 min
of analysis time
40 mm
Nanoflow LC
100 to 1000 nL / min
split
For demanding
life science
applications
Sample inlet systems for ESI
Column i.d.
Flow rate
Technique
4.0 mm
2.0 mm
1.0 mL/min
0.25 mL/min
Conventional HPLC
Small bore LC
1.0 mm
0,0625 mL/min
Micro LC
300 µm
180 µm
5.6 µL/min
2.0 µL/min
Capillary LC
Capillary LC
350 nL/min
Nano LC
75 µm
Sample inlet systems for ESI
S/N= 3800
4.6 mm i.d.
1.0 ml/min
Signal to
Noise ratio
75 µm i.d.
S/N = 1
Sample inlet systems for ESI
4.6 mm i.d
1.0 mm i.d.
UV 206 nm
300 µm i.d.
75 µm i.d.
2 pmol digested myoglobin (each column)
ESI has
a similar, even
stronger
concentration
dependance
Chromatographiesäulen im Vergleich
4 mm
0.18 mm
75 µm
Nano-Elektrospray
Nano-Elektrospray
Diameter 1 mm
Diameter 0.32 mm
Diameter 0.075 mm
Diameter 4 mm, Flow rate 1.6 mL / min
Flow rates ?