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-TOFQuadrupoleIon trapLinear ion trapQ-TOFFT ICR MSOrbitrap

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 timeb) adding (averaging) many individual scansc) 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

Formation of singly charged ionsSample is co-crystallized with matrix (solid)

Koichi Tanaka, Nobel Prize 2002

Absorb UV light, crystallize easily, sublimate easilyand transfer proton to analyte

MALDI-TOF…

E = qU = ez U = Ekin = ½ mv2

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

v = 2 ezUm

Since we the velocity is related to the time, we can relate the mass to the time: t = d/v

d

m2 ezU

t = d

2 eU t = m

z

Time to drift is proportional to square root of m/z

Smaller ions will arrive faster than heavier ions

MALDI Time-of-Flight (TOF)

Drift region (d)Source

Heavier ions arrived later

d

m2 ezU

t = d

2 eU t = m

z

MALDI-Time-of-Flight Mass Spectrometer

Mass range = 800-200,000

Sensitivity and accuracy decrease rapidly with size !

MALDI TOF (linear)

MALDI with Reflectron

Similar Ions may possess slightly different energies and arrive at different times = broad peaks ie poor resolution

Detector

Ion Mirror(reflectron)

Laser pulse

Laser pulse

Detector

Ions with more kineticenergy will penetrate more deeply

source

Refocussing

MALDI –Delayed Extraction

Earlier instruments use continuous extraction of ions, ie accelerating voltage was applied during and after laser pulse.

The resolving power is increased by a factor of 3-4 for linearMALDI and by a factor of 2-3 for MALDI with reflectron (MALDI-R)up to 10,000-20,000.

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.

This additional resolution is need for resolving more complexmixtures 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.

TOF mass analyzer can be combined with other mass analyzers such as TOF-TOF and Q-TOF instruments

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

Katalin F. Medzihradszky, J. M. Campbell et al., Anal Chem 2000 72 pp 552 - 558

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.

camera laser

Decelleration stack optics priorto collision cell enable the kinetic energy ot the precursorIons entering the collision cell tobe tuned for controlled fragmentation.

Timed ion selector

Detector of reflector Detector linear

Source 2 Ion mirror

ABI/Sciex 4800 TOF/TOF (second generation)

Collision cell

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 allowfor 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 ofvoltage only a certain m/z range will be transmitted. To obtain the fullmass spectrum, perform scan for all m/z in their individual stability region.

Mass range: 10-4000 DaResolution: Operated at unit mass resolution up to ~2,000 Da can be increased to ~4,000Mass accuracy : ~0.1-0.2 DaScan speed: 5000Da/per second

Mass filter; complete spectrum is obtained by scanning whole range

Ions are lost

Mass range 50- 4,000 Da

2. RF ONLY: no separation, all ions are transmittted

3. DC + RF only ions with narrow

mass range are transmitted

1. DC only: no masses aretransmitted

4. Movement in 3 directions

Quadrupole as Mass Filter:

Ideally hyperbolic rodsbut are more difficult to produceso use cylindrical rods

Rod potential x = U – V costy = - U + V cos t

U is the DC potential and V cos t is the time dependent RF voltage in which V is the amplitude, f = /2 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 thequadrupole (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:. x = U – V cost. 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

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.

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 tothe 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.

y = - U + V cos t

Ion Motion and Stability Diagram

The Motion of an ion traveling in a quadrupole field is described by the Mathieu equation:

d2u + (au- 2qucos2) u = 0d

Where tand u represents x or y. The Mathieu parameters au and qu

are defined as

au = 8qchUr0

2m

and qu = 4qchVr0

2m

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.

(DC) (RF)

Common to both x and yStability region I expanded

Change U and V at constant ratio a/q = 2U/V, while keeping 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.

Larger m/z rangeLess resolution

Select for only one m/z. Higher resolutionbut loose sensitivity

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 modewhich is a lot more sensitive if want to detect a single mass.

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.

RF only quadrupoles can be used as ion guides: transmission of (almost)all ions. Also hexapoles and octapoles with analogous functions.

Original way to perform peptide by MS/MS sequencing. Now this better done with other more sensitive mass analyzer replacing Q3.

Q1 Selection

Q2Collision

Q3 Scan m/z

m/z

Relative intensity

Data System

Detection

50700

Doubly charged precursor ion

MS/MS with Triple Quadrupole Mass Spectrometer

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 Ionscan Product Ion

Multiple ReactionMonitoring (MRM)

filter Precursor Ionfilter Product Ion

scan Precursor Ionfilter Product Ion

Precursor Ion Scan

scan Q1 and Q3 with constant mass off-set

Neutral Loss Scan

Quadrupole Ion Traps (3D Ion Traps )

Similar principle to quadrupole but the geometry is different.Consists of a ring electrode with hyperbolic surfacewith end cap electrodes. Aperture in each end cap to allow ions and out. Size of baseball, cheap to produce.

Wong and Cooks Current Separations

r = U – V costz= - U + V cos t

ar= -1 az = 8qchUro

2 +2 z2o) m

qr = -1 qz = 4qchVro

2 +2 z2o) m

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.

The Mathieu paramaters for the cylindrical geometry are:

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 keptconstant 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)

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 ejectedthrough 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 with 3D Ion Trap

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.

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.

MS/MS with 3D trap

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/secExcellent MS/MS capabilitiesInexpensiveVery sensitive (low femtomoles for peptides)

DISADVANTAGESLow mass accuracy (100 ~ppm)Poor dynamic rangeSpace charges effects at higher concentrationLow mass cut off (150-200) (no immonium ion!)

Linear Ion Traps (new design with higher capacity)

Tandem-in-Time: Ion TrapsVery sensitive scanningOnly product ion scansOnly scanning

Tandem in Space: Triple QuadsPoor scanning sensitivityGreat for quant (MRM)Very selective scans

Add trapping plates to ends quadrupole

Solution: replace Q3 of triple quad with a linear ion trap by adding trapping plates to end quadrupole

AxialTrapping

Exit Lens

Radial Trapping RF Voltage

Radial Trapping RF Voltage

AxialTrapping

DCVoltage

Resonance Excitation

Trapping Forces in a Linear Ion Trap

Linear vs. 3-D Ion Traps

• Trapping Efficiency:• Linear ~10X better

• Extraction Efficiency• Linear ~2X worse

• Ion Capacity• Linear ~45X better

~5X Better sensitivity

Better immunity to space charge

Enhanced Product Ion Scan

• RF/DC Q1 Q2 Q3 linear trapfrags.eV

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

EPI

Q0 Q1 Q2 Q3

Enhanced Product Ion Scanning

1. Precursor ions selection in Q12. Fragmentation in Q23. Trap products in Q34. Mass scan5. Concurrent trapping in Q0

N2 CAD Gas

linear ion trap3x10-5 Torr

Precursor ion selection

Ion accumulation

Fragmentation

Exit lens

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

Q2Collision

Q1 Selection Pusher

TOF with reflectron

Detector

Hybrid Quadrupole/Time-of-Flight (Q-TOF) MS

Relative intensity

50 700

Doubly charged precursor ion

b9

y10y9

b8

m/z

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

Provides extremely high resolution, but typically operated at 100,00-300,000, but up to 3,000,000 has been achieved.

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 Teslamagnets are commercially available.

The signals are measured as function of time and converted to frequencies by a Fourier transformation (FT) (frequency domain)

A number of MS/MS experiments can be performed including MSn by SORI, IRMPD, ECD, BIRD, and CAD with q FTICR MS

The vacuum is typically very low 10-8-10-10 Torr.

Can be used with MALDI, electrospray and other sources

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 balanceof two forces m v2/r = q vB, or qB = mv/r , v = (qB r)/m

The ions completes a 2 r circular trajectory with a frequency = v/ 2r, substituting for v

= qB/ 2m

The angular velocity is : 2 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

Static MagneticField

B+-

V xy

F = qV x BL xy

B

X

Y

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)

V xy

F = qV x BL xy

B

V xy

X

F = m * accelerationC C

r

centripetal acceleration a = v2/r

m2

qBF

qBr

mv

qvBr

vm

qvBma

Cyclotron

2

and the frequency of one cycle is = v/2r

The really important equation

Typical Cyclotron Frequencies

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 functionof an ion’s m/z.

f = 1.535611x107*B (m/z)

Trapping Ions in a Bottle

Static MagneticField

B+

+

Ions are trapped in x-y plane, but not along the magnetic field (z-axis)

Static MagneticField

B

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

-2.4 -1.8 -1.2 -0.6 0 0.6 1.2 1.8 2.4

z-position (cm)

Po

ten

tial

(V

)

Frequency of Trapping Motion

2

Trap

ma

α2qV

2π

1 FTRAP

m

qV7.2483x10 F

Trap4TRAP

For our cubic cell (5.08 cm per side). FTrap in Hz, VTrap in Volts, m in amu

+ B

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 cellwall perpendicular to the ion trajectory.

Ions of the same mass excited to the same energy will be on the same orbitand 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. StartingFrom 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

B

dB2

TVr

Excitepp

Useful for:

1. Excitation for detection

2. Isolation (selective ejection)

3. CID (increase KE)

+-

dB2

TVr

Excitepp

Excitation of Cyclotron Motion for Detection

1. Excitation radius is independent of m/z ratio

2. All ions of the same m/z ratio are excited coherently

FT

Detect Electrodes

Excite Electrodes

-608816.0

-365289.6

-121763.2

121763.2

365289.6

608816.0

-200000.0

200000.0

5.000 5.002 5.004 5.006 5.008 5.010

Tim e, msec

Sig

nal

0

Tim e, msec

0 2 4 6 8 10 12

Sig

nal

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 sec which corresponds to ~500 KHz. The cyclotron frequency of m/z 76 is ~508 kHZ

Benefits of High Magnetic Field Strength

12 T

12 T