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Biochem 523b: Advanced Physical Methods:
Mass Spectrometry, X-ray Crystallography and NMR
A. Mass Spectrometry
Lecture 2
Mass analyzers
Time of flight (MALDI-TOF), TOF-TOF
Quadrupole
Ion trap
Linear ion trap
Q-TOF
FT ICR MS
Orbitrap
Detectors for mass spectrometry
Converts energy of incoming ions into a current signal that will be registered
by the electronic devices and computer of the acquisition system.
When the incoming ions hit the detector, the energy of that impact causes
emission of secondary electrons or photons. The number of secondary
Particles created by an impact depends on the energy and velocity of
the incoming ion.
If all the particles are accelerated to the same kinetic energy as in TOF
Analyzer. The detection sensitivity is lower for high mass (slow) ions
than for low mass (fast) Ions.
To increase sensitivity Ions can be post-accelerated before striking the detector.
Detector should have high efficiency for converting energy of incoming ion to
electrons or photons, a liner response, low noise short recovery time
(to avoid saturation) an minimal variation in transit time (narrow peak width)
Electrons Multiplier: Different Designs
Microchannel plates
Parallel arrays of channel electron multiplier
Ions are counted. The ions counted are often reported in counts per
seconds (Cps).
To minimize statistical errors more ions should be measured by
a) using longer acquisition time
b) adding (averaging) many individual scans
c) have enough ions produced in the source (eg raise source
voltage or T)
d) and efficiently transported through the MS
Detectors can get saturated.
MCPs need periodical replacement
Matrix Assisted Laser Desorption/Ionization
Sample is co-crystallized with matrix (solid)
Formation of singly charged ions
Koichi Tanaka, Nobel Prize 2002
Absorb UV light, crystallize easily, sublimate easily
and transfer proton to analyte
MALDI-TOF…
The energy (E) uptake by an ion of charge q and mass
m is equal to an integer number z of electrons
charges e, and thus q = ez
E = qU = ez U = Ekin = ½ mv2
v=
2 ezU
m
Since we the velocity is related to the time,
we can relate the mass to the time: t = d/v
t =
d
t =
d
m
z
2 ezU
2 eU
m
Time to drift is proportional to square root of m/z
Smaller ions will arrive faster than heavier ions
MALDI Time-of-Flight (TOF)
Heavier ions arrived later
Source
t =
Drift region (d)
d
2 ezU
m
t =
d
2 eU
m
z
MALDI-Time-of-Flight Mass Spectrometer
MALDI TOF (linear)
Mass range = 800-200,000
Sensitivity and accuracy decrease rapidly with size !
MALDI with Reflectron
Laser pulse
Detector
source
Similar Ions may possess slightly different energies and arrive
at different times = broad peaks ie poor resolution
Laser pulse
Ions with more kinetic
energy will penetrate
more deeply
Refocussing
Detector
Ion Mirror
(reflectron)
MALDI –Delayed Extraction
Earlier instruments use continuous extraction of ions,
ie accelerating voltage was applied during and after laser pulse.
Ions are allowed to form in a field free environment. A few hundeds
of nanoseconds later and after the laser pulse has terminated,
a fast pulse extracting voltage is applied. Alternatively
a two stage accelerating voltage can be applied which
diminishes the energy spread.
The resolving power is increased by a factor of 3-4 for linear
MALDI and by a factor of 2-3 for MALDI with reflectron (MALDI-R)
up to 10,000-20,000.
This additional resolution is need for resolving more complex
mixtures and provide sufficient mass accuracy.
Use MCP detectors: velocity (mass) sensitive
Reflectron reduces the ion transmission ie lost in sensitivity.
depending on the reflectron mass above a certain m/z are not observed.
~4,000 on Micromass ~10,000 on Bruker, ABI)
Most MALDI TOF have two detectors one operating in one linear mode
(for higher masses eg proteins) and one in reflectron mode for higher
resolution and mass accuracy and eg tryptic peptides.
One major disadvantage of MALDI TOF is that it cannot perform
MS/MS of peptides other than in the postsource decay mode (PSD)
which is gives generally poor coverage.
Another configuration is the orthogonal TOF (o-TOF):
Source is orthogonal to mass analyzer. Ions are pushed toward the detector
by applying a voltage to the incoming ions.
l
TOF mass analyzer can be combined with other mass analyzers
such as TOF-TOF and Q-TOF instruments
TOF-TOF MS for (MS/MS)
Parent ion selection is done by ion gates deflecting the undesired ions
from the collision cell. The fragment are re-accelerated towards the detector source.
Katalin F. Medzihradszky, J. M. Campbell et al., Anal Chem 2000 72 pp 552 - 558
ABI/Sciex 4800 TOF/TOF (second generation)
Collision cell
Detector
linear
Detector of reflector
Timed ion selector
Source 2
Ion mirror
Decelleration stack optics prior
to collision cell enable the
kinetic energy ot the precursor
Ions entering the collision cell to
be tuned for controlled
fragmentation.
camera
laser
ABI 4700
ABI 4800
TOF/TOF Instruments are ideal for HTP peptide identification by MALDI
Extremely fast MS and MS/MS (10/sec) with excellent sensitivity
(low femtomole) and good mass accuracy (~15 ppm).
Collision energy can be very high and varied. High collision energy allow
for fragmentation of the side chains: TOF/TOF is the only MS/MS
instrument capable of distinguishing Leu and Ile.
Not cheap: ~700K!
Quadrupole mass analyzers
Device that separates ions in a quadrupole electric field based on
their m/z . The quadrupole electric field is created by a set of four parallel
rods on which both a DC and alternating voltage (RF) are applied.
By changing the applied field only some m/z will be transmitted
from one end of the quadrupole rods to the detector. For a given set of
voltage only a certain m/z range will be transmitted. To obtain the full
mass spectrum, perform scan for all m/z in their individual stability region.
Mass range: 10-4000 Da
Resolution: Operated at unit mass resolution up to ~2,000 Da
can be increased to ~4,000
Mass accuracy : ~0.1-0.2 Da
Scan speed: 5000Da/per second
Ions are lost
Mass filter; complete spectrum is obtained by scanning whole range
Mass range 50- 4,000 Da
1. DC only: no masses are
transmitted
4. Movement in 3 directions
2. RF ONLY: no separation,
all ions are transmittted
3. DC + RF only ions with narrow
mass range are transmitted
Quadrupole as Mass Filter:
Ideally hyperbolic rods
but are more difficult to produce
so use cylindrical rods
Rod potential Fo:
Fox = U – V cos wt
Foy = - U + V cos wt
U is the DC potential and V cos wt is the time dependent RF
voltage in which V is the amplitude, f = w/2p the radiofrequency,
and t, time. f is fixed at ~1MHz
Movement of ions is relatively complex; x,y,z directions
The ions emerging form the source are accelerated in at the entrance of the
quadrupole (z direction) over a potential of 5-20 v. (The resolution will be
negatively affected by higher velocity). All ions will be affected by the force
exerted by the fields in the x and y directions.
Consider the x plane:. Fox = U – V cos wt.
U is positive.
Large positive ions are less responsive to RF voltage
and will be repulsed towards the middle of the x plane. The low mass
positive ions respond faster to the RF voltage (less mass inertia).
Once every cycle the sum of the DC and RF voltage components will be
negative for a short time and the passing ions will experience an
attractive force. If the mass is low enough the ion will be accelerated
toward one of the x electrode and hit before it becomes positive again
(Lost!). This pair of rod act as high mass filter
Foy = - U + V cos wt
In the y direction pair of electrode will act as a low mass filter. The DC
potential U is negative and the high mass ions are more responsive to
the DC component (the RF is high and the potentials tends to average out).
The larger positive ions will be slowly dragged to the negatively
charged electrode.
The low mass will also experience an attractive force but will respond more to
positive RF potential forcing them more in the middle between the y electrodes.
Analogy with ball on top of a curve surface. It is unstable but by wiggling the
cylinder back in forth, the ball will not fall.
Ion Motion and Stability Diagram
The Motion of an ion traveling in a quadrupole field is described
by the Mathieu equation:
d2u + (au- 2qucos2x) u = 0
dx2
Where x = wt /2, and u represents x or y. The Mathieu parameters au and qu
are defined as
au = 8qchU
w2 r02m
(DC)
and
qu = 4qchV
(RF)
w2 r02m
Where r0 is half the distance between opposite rods, qch is the charge and m the
mass. Substituting for u for x and y gives au = ax = - ay and qu = qx = - qy
Only certain combinations of a and q gives stable solutions to the Mathieu eqn
that is, ions passing through the quadrupole. Moreover only the a/q combinations
that gives stable solutions for both the x and y directions will be useful.
Select for only one m/z. Higher resolution
but loose sensitivity
Larger m/z rang
Less resolution
Common to both x and y
Stability region I expanded
Change U and V at constant ratio a/q = 2U/V, while keeping w fixed. Since
a and q are proportional to U/(m/z) sn V/(m/z), for a certain setting of U and V
Only ions with a certain m/z range will be allowed trough the quadrupole.
Scan line vs resolution and intensity
Single Quad MS
Quadrupoles are very versatile and can be used in various configurations.
Can be operated in the SCAN mode or single ion monitoring SIM mode
which is a lot more sensitive if want to detect a single mass.
RF only quadrupoles can be used as ion guides: transmission of (almost)
all ions. Also hexapoles and octapoles with analogous functions.
One very popular configuration is a the triple quadrupole (Triple quad,
QqQ) for MS/MS experiments.
Consist of two sets of quadrupoles separated by a collision cell
(itself a quadrupole with RF only (q). Ions can be selected in the first
quadrupole and fragmented in the collision cell (q). The resulting
fragment ions are analyzed (separated) in the last quadrupole (Q3)
operated in the SCAN mode. This is called the fragment ion scan or
product ion scan.
Original way to perform peptide by MS/MS sequencing. Now this
better done with other more sensitive mass analyzer replacing Q3.
MS/MS with Triple Quadrupole Mass Spectrometer
Q1
Selection
Q2
Collision
Q3
Scan m/z
Detection
Data System
Doubly charged
precursor ion
Relative
intensity
700
50
m/z
Other experiments are also possible with triple quad:
2. Precursor ion scan:
Q3 is fixed a particular m/z. Q1 is scanned and the
transmitted ions are fragmented in Q2. This experiment can tell
what molecule (m/z) in the mixture contain a particular fragment
eg1 Q3 86 for immonium ion of Leu/Ile.
3. Neutral Loss Scan:
Q1 and Q3 are both scanned with a constant mass difference.
A peak in the spectrum is only recorded when the ions in Q1 loose
a neutral fragment of particular mass in Q2.
eg – H3PO4 (-98) of phosphopeptides
4. Mutiple reaction monitoring (MRM)
Ions are selected in Q1 and in Q3 obtain only a certain fragment of
a certain precursor. Useful for quantitation: less “chemical noise”
due to other species in the MS/MS:
MS/MS: modes of operation
Product Ion Scan
filter Precursor Ion
scan Product Ion
Neutral Loss Scan
scan Q1 and Q3 with
constant mass off-set
Precursor Ion Scan
scan Precursor Ion
filter Product Ion
Multiple Reaction
Monitoring (MRM)
filter Precursor Ion
filter Product Ion
Quadrupole Ion Traps (3D Ion Traps )
Similar principle to quadrupole but the geometry is different.
Consists of a ring electrode with hyperbolic surface
with end cap electrodes. Aperture in each end cap to allow ions and out.
Size of baseball, cheap to produce.
Wong and Cooks Current Separations
Movement of ions inside the 3D trap
Follow similar principle as in the linear quadrupole except that ions under ideal
conditions the ions would be trap for ever.
For = U – V cos wt
Foz= - U + V cos wt
The Mathieu paramaters for the cylindrical geometry are:
ar= -1 az =
2
8qchU
w2 (ro2 +2 z2o) m
qr = -1 qz = 4qchV
2
w2 (ro2 +2 z2o) m
Ions are injected in the trap from continuous (eg ESI) or pulse ion sources
(MALDI) guided by quadrupole mass filter.
The ions arrived at potential of 5-20V. During the injection the voltage is kept
constant so the ions are trapped and loose their energy by collision with
low pressure helium gas (1mTorr). The helium also helps to confine the ions
In the middle of the trap.
Mass analysis with 3 D ion trap ms
Endcaps electrodes are held at ground potential.
An RF potential is applied to the ring electrode which means
that the Mathieu potential a is equal to zero. (No DC current)
The trap is working on the q axis in the a/q stability diagram.
The ions lines up on the q axis with the lowest m/z at the highest q.
When the RF voltage is set low all ions are trapped (stored)
MS with 3D Ion Trap
Mass spectrum is acquired in the mass-selective instability scanning
mode by raising the RF voltage (DC = 0). Eventually the lowest m/z
ions will reach and cross the stability boundary and be ejected
through the small holes in the encap and detected.
With further increase in the RF potential higher m/z ions will be
ejected and a full mass spectrum can be obtained.
.
The ions are taped for a long time and several types of experiments
can be performed. One of the biggest advantage of ion trap MS
is that it is capable of multiple MS/MS experiments (MS)n .
MS/MS with 3D trap
MS/MS is done by ion isolation followed by fragmentation. Isolation
of ions is done by the application of a supplementary RF voltage
on the endcaps. The trapped ions will oscillate with different
frequencies according to their m/z.
To eject ions of certain m/z, a supplementary RF voltage with
corresponding frequency is applied (few to hundreds kHz). These ions
will be resonant with the oscillating potential and their oscillation
amplitude in the axial direction will increase and finally be ejected.
Another way to eject the ions is by applying a selected waveform
Fourier transform (SWIFT). Broad range frequency with a “notch” to
keep a pre-selected ion.
After ion selection, a supplementary voltage is applied low enough
to excite but not eject them. The higher energy ions will collide
with He gas and fragment (collision induced fragmentation, CID)
Then a mass selective instability scan is performed and eventually
all the fragment ions will be ejected and detected to give a full MS/MS
spectrum.
ADVANTAGES
Very fast scan 5000Da/sec
Excellent MS/MS capabilities
Inexpensive
Very sensitive (low femtomoles for peptides)
DISADVANTAGES
Low mass accuracy (100 ~ppm)
Poor dynamic range
Space charges effects at higher concentration
Low mass cut off (150-200) (no immonium ion!)
Linear Ion Traps (new design with higher capacity)
Add trapping plates to ends quadrupole
Tandem in Space: Triple Quads
Poor scanning sensitivity
Great for quant (MRM)
Very selective scans
Tandem-in-Time: Ion Traps
Very sensitive scanning
Only product ion scans
Only scanning
Solution: replace Q3 of triple quad with a linear ion trap by
adding trapping plates to end quadrupole
Trapping Forces in a Linear
Ion Trap
Radial Trapping RF Voltage
Axial
Trapping
DC
Voltage
Axial
Trapping
Exit Lens
Radial Trapping RF Voltage
Resonance Excitation
Linear vs. 3-D Ion Traps
• Trapping Efficiency:
• Linear ~10X better
• Extraction Efficiency
~5X Better sensitivity
• Linear ~2X worse
• Ion Capacity
• Linear ~45X better
Better immunity to
space charge
Enhanced Product Ion Scan
EPI
• RF/DC Q1
eV
Q2
frags.
Q3 linear trap
Advantages:
• No time required to isolate the precursor ion
• No loss for isolation of fragile precursor ions
• The ion trap is filled with only precursor and fragment ions
• Triple quad. fragmentation patterns
• No inherent low mass cut-off
Enhanced Product Ion
Scanning
Exit lens
N2 CAD Gas
Ion accumulation
Q0
Q1
Precursor ion
selection
1.
2.
3.
4.
5.
Q2
Q3
Fragmentation
Precursor ions selection in Q1
Fragmentation in Q2
Trap products in Q3
Mass scan
Concurrent trapping in Q0
linear ion trap
3x10-5 Torr
Linear Ion Trap
Movie from Finnigan WEB site
Hybrid Quadrupole Time-of-Flight Instrument (Q-TOF)
Initially designed for MS/MS of peptides after LC separation.
Replaces the scanning Q3 of triple quad with the more sensitive
and better resolution of the time-of-flight/reflectron.
Now can be used with MALDI sources including atmospheric pressure
MALDI (API MALDI).
Portions or slices of the incoming ions beam from the source are
orthogonally accelerated down the TOF tube (pusher).
Q-TOF (ABI/Sciex)
Qstar QqTOF System
Hybrid Quadrupole/Time-of-Flight (Q-TOF) MS
Q1
Selection
Q2
Collision
Pusher
Detector
Doubly charged
precursor ion
Relative
intensity
y9
y10
b8 b
9
50
m/z
700
TOF with reflectron
Q-TOF
Advantages
Good sensitivity for MS/MS sequencing of peptides
low femtomoles
Good resolution ~10,000-15,000 and mass accuracy
Disadvantages
Relatively slow duty cycle: ions have to reach the detector before
new ions are pushed
Can not perform neutral loss scan, MRM
Limited to MS/MS (cannot do MSn)
Fourier Transform Ion Cyclotron Resonance
Mass Spectrometry (FT ICR MS, FTMS)
General Principles
FTICR MS are a form of ion trap. Ions are trap by a magnetic field
not a quadruople. The stronger the magnet the better; 7-12 Tesla
magnets are commercially available.
The signals are measured as function of time and converted to
frequencies by a Fourier transformation (FT) (frequency domain)
Can be used with MALDI, electrospray and other sources
The vacuum is typically very low 10-8-10-10 Torr.
Provides extremely high resolution, but typically operated at
100,00-300,000, but up to 3,000,000 has been achieved.
A number of MS/MS experiments can be performed including MSn by SORI,
IRMPD, ECD, BIRD, and CAD with q FTICR MS
The principle of FT ICR MS is to force the ions into a periodic
motion that depends on m/z. Once injected in a magnetic field the
ions will have a circular trajectory.
For a circular motion:
F = ma = m v2/r
The magnetic field cause a Lorentz force: FL = q vB
The ion stabilizes on a trajectory resulting from the balance
of two forces
m v2/r = q vB, or qB = mv/r , v = (qB r)/m
The ions completes a 2p r circular trajectory with a frequency
n = v/ 2pr, substituting for v
n = qB/ 2p m
The angular w velocity is : w = 2p n = v/r = (q/m)B
As a result the frequency and angular velocity depends on the
(qB/m) ratio. However for a given ion the radius of the trajectory
increases with the velocity. If the radius becomes too larger than the
trapping cell the ion are expelled.
Cyclotron Motion
-
B
+
Static Magnetic
Field
Vxy
FL = qVxy x B
Y
B
X
Lorentz force (FL) is the
inward directed force that
causes the uniform
circular motion of an ion
in a magnetic field.
Magnetic field traps ions
in the x-y plane.
Frequency of Cyclotron Motion vs. Ion Mass
(Why we can put MS after ICR)
Vxy
Vxy
FL = qVxy x B
r
FC = m * accelerationC
B
X
ma = qvB
n=
v2
m
= qvB
r
mv
= qB
r
qB
FCyclotron =
2pm
centripetal acceleration a = v2/r
and the frequency of one cycle is n = v/2pr
The really important equation
Typical Cyclotron Frequencies
f = 1.535611x107*B
(m/z)
f in Hz; B in Tesla; m/z in Th
Cyclotron frequency is independent of ion kinetic energy
(radius, velocity). Cyclotron frequency is only a function
of an ion’s m/z.
Trapping Ions in a Bottle
Ions are trapped in x-y plane, but not along the magnetic field (z-axis)
+
B
Static Magnetic
Field
+
B
Static Magnetic
Field
Frequency of Trapping Motion
2
1.8
1.6
Potential (V)
1.4
1.2
B
1+
0.8
0.6
0.4
0.2
0
-2.4
-1.8
-1.2
-0.6
0
0.6
1.2
1.8
2.4
z-position (cm)
FTRAP =
1 2qVTrapα
2π
ma2
FTRAP = 7.2483x10
4
qVTrap
m
For our cubic cell (5.08 cm per side).
FTrap in Hz, VTrap in Volts, m in amu
If VTrap is constant, FTrap is inversely related to the square root of the
ion mass; tTrap is directly related to the square root of ion mass.
Detection of ion motions
Ions have very small radii (sub-mm) and the ion motion is not coherent.
Must apply an external RF electric field to increase the radius
of ion motion and to make motion more coherent.
Irradiating with an electromagnetic wave (excitation) that has the same
frequency as an ion allows resonance absorption of this wave. The
energy that is transferred to the ion increase its kinetic energy which will
cause an increase in the trajectory radius.
An “image” current will be induced by the ions circulating in the cell
wall perpendicular to the ion trajectory.
Ions of the same mass excited to the same energy will be on the same orbit
and rotate with the same frequency.
The RF can be used to excite the ions or to eject the ions. Can have
selected excitation for one particular ion (one frequency only) or excite
a wide range of m/z with broadband excitation to get a full ms spectrum
An inverse Fourier transform is used to calculate the excitation frequency. Starting
From the desired frequency spectrum, the corresponding the corresponding is
calculated and applied to the ICR cell. This is called SWIFT waveforms
(Stored Waveform Inverse Fourier Transform )
Excitation of Cyclotron Motion
r=
V ppT Excite
2dB
+
Useful for:
1. Excitation for
detection
2. Isolation (selective
ejection)
3. CID (increase KE)
B
Excitation of Cyclotron Motion for Detection
r=
V ppT Excite
2dB
1. Excitation radius is independent of m/z ratio
2. All ions of the same m/z ratio are excited coherently
Excite Electrodes
Detect Electrodes
FT
Detected signal (transient) for EI of carbon disulfide (m/z 76)
Took 64,000 data points at an acquisition rate of 5333.333 KHz – takes ~12 msec
From the transient, you can see one major component with a period of ~2 msec
which corresponds to ~500 KHz. The cyclotron frequency of m/z 76 is ~508 kHZ
Signal
200000.0
0
-200000.0
5.000
5.002
5.004
5.006
5.008
5.010
Time, msec
608816.0
Signal
365289.6
121763.2
-121763.2
-365289.6
-608816.0
0
2
4
6
Time, msec
8
10
12
Benefits of High Magnetic Field Strength
12 T
12 T