Post on 09-Mar-2021
transcript
Chapter 81
Mass and charge
B1 .1 Historical review
1897
J. J. Thomson made the first measurement of the mass-to-charge ratio of el
ementary particle 'corpuscles', which later became known as electrons. This can
fairly be considered as the birth of mass spectrometry.
1918-1919
A. Dempster and F. Aston developed the first mass spectrographs. Photographic
plate was used as the array detector. The instruments were used for isotopic
relative abundance measurements.
1951
W. Pauli and H. Steinwedel described the development of a quadrupole mass
spectrometer. The application of superimposed radio-frequency and constant
potentials between four parallel rods acted as a mass separator in which only
ions within a particular mass range perform oscillations of constant amplitude
and are collected at the far end of the analyser.
1959
K. Biemann was the first to apply electron ionisation mass spectrometry to the
analysis of peptides. Later it was shown that for sequence determination, peptides
had to be derivatized prior to analysis by a direct probe.
1968-1970
M. Dole was the first to bring synthetic and natural polymers into the gas phase at
atmospheric pressure. This was done by spraying a sample solution from a small
tube into a strong electric field in the presence of a flow of warm nitrogen, to
assist desolvation. First experiments on lysozyme demonstrated the phenomenon
of multiple charging.
111
112 B Mass spectrometry
1974
D. Torgerson introduced plasma desorption mass spectrometry. This technique
uses 252Cf fission fragments to desorb large molecules from a target. It was the
first of the particle-induced desorption methods to demonstrate that gas-phase
molecular ions of proteins could be produced from a solid matrix.
1974
B. Mamyrin made the most important contribution to the development of time
of-flight (TOF) mass spectrometry. He constructed the so-called reflectron device,
which had been proposed by S. Alikanov in 1957. The reflectron essentially
improves mass resolution in the TOF mass spectrometer.
1978
N. Commisarow and A. Marshall adapted Fourier transform methods to ion
cyclotron resonance spectrometry and built the first Fourier transform mass
instrument. Since that time, interest in this technique increased exponentially,
as has the number of instruments.
1981
M. Barber discovered fast atom bombardment (FAB), a new ion source for
mass spectrometry. The mass spectrum of an underivatised undecapeptide, Met
Lys-bradykinin of M = 1318 was obtained by bombarding a small drop of glycerol
containing a few micrograms of the peptide with a beam of argon atoms of a few
kiloelectron-volts. The technique revolutionised mass spectrometry and opened
it to the biologist.
1984
R. Willoughby and, independently, M. Aleksandrov proposed the coupling of
liquid chromatography and mass spectrometry for analysing high-molecular
weight substances delivered by a liquid phase.
1988
J. Fenn and, independently, M. Yamashita were able to bring biological macro
molecules into the gas phase at atmospheric pressure. They proposed a new type
of ionisation technique called electro spray ionisation (ESI) to generate intact bio
logical molecular ions, by spraying a very dilute solution from the tip of a needle
across an electrostatic field gradient of a few kV. M. Karas and F. Hillencamp
and, independently, K. Tanaka developed a new ionisation technique called
matrix-assisted laser desorption-ionisation (MALDI). It was shown that pro
teins up to a molecular weight of 60 000 could be ionised if embedded in
a large molar excess of a UV-absorbing matrix and irradiated with a laser
beam. Taking advantage of high resolution, mass measurement accuracy, and
ion-trapping capabilities, MALDI provides not only molecular mass informa
tion but also structural information for various peptides and oligonucleotides.
B1 Mass and charge
K. Tanaka received the 2002 Nobel prize in Chemistry for his contribution to
mass spectrometry.
1992-1999
The molecular specificity and sensitivity ofMALDI-MS gave rise to a new tech
nology for direct mapping and imaging ofbiological macromolecule distributions
present in a single cell or in mammalian tissue. By rastering the ion beam across
a sample, and collecting a mass spectrum for each point from which ions are de
sorbed, it is possible to create mass-resolved images of molecular species across
a cell surface or in a piece of tissue.
2000 to present
Mass spectrometry has developed into an important analytical tool in the life sci
ences. Soft-ionisation techniques, such as FAB, ESI and MALDI, allow routine
mass measurements of proteins and nucleic acids with high resolution and accu
racy. Mass spectrometry has become one of the most powerful experimental tools
for the direct observation of gas-phase biological complexes, their assembly and
their disassembly in real time. Developments include the combination of mass
spectrometry with isotopic labelling, affinity labelling and genomic information.
It is clear that the rapid growth phase ofbioanalytical mass spectrometry has not
yet reached its peak. There is no doubt that in the next decade mass spectrom
etry will move at an extraordinary pace, extending from the world of structural
biology to that of medicine and therapeutics.
B1 .2 Introduction to biological applications
Since the 1930s, mass spectrometry (Comment Bl.1) has become an important
analytical tool in structural biology. This is a result of the ability to produce intact,
high-molecular-mass gas-phase ions of various biological macromolecules. Sev
eral ionisation techniques such as FAB, MALDI, and ESI revolutionised mass
spectrometry and opened it up to biology. New methods for ultrasensitive pro
tein characterisation based upon Fourier transform ion cyclotron resonance mass
spectrometry (FTIR-MS) have been developed, providing a detection limit of
approximately 30 zmol (30 x 10-21 mole) for proteins with molecular mass rang
ing from 8 to 20 kDa. Using this technique individual ions from polyethylene
glycol to DNA, with masses in excess of 108 Da can be isolated ( Comments B 1.2
andBl.3).
Comment B1.3 Molecular mass and molecular weight
Some confusion may arise when Mr is used to denote relative molecular mass. Mr is
a relative measure and has no units. However, Mr is equivalent in magnitude to M
and the latter does have units and for high-mass biological macromolecules the
dalton is usually used. Note that molecular weight (which is a force and not a mass)
is an incorrect term in this case.
Comment B1.1
The term 'mass
spectroscopy'
113
We would like to warn
the reader against the
term 'mass
spectroscopy'. The
term 'mass
spectroscopy' is not
correct because it
bears no relation to
real spectroscopic
techniques described
in Parts E, I and J. The
mass spectrum
depends mainly on the
stability of ions
produced and collected
during the experiment.
The stability of ions
strongly depends on
experimental
conditions and
therefore predicting of
a mass spectrum is
practically impossible.
Comment B1.2
Absolute and
relative masses
A mass spectrometer
does not measure
absolute mass, M. The
instrument needs to be
calibrated with
standard compounds,
whose M values are
known very accurately.
The carbon scale is
used most frequently
with 12c
= 12.000 ooo.
v
FB B outof page
Motion ofparticle
q+
B out ofpage
Increasing mass
+
Ion source
at 50% (FWHM)
at 50% (10% valley)
+1
116 B Mass spectrometry
Comment B1 .5 Monoisotopic mass
Most chemical elements have a variety of naturally occurring isotopes, each with a unique mass and natural abundance. The monoisotopic mass of an element refers specifically to the lightest stable isotope of the element. For example, there are two principal isotopes of carbon, 12C and 13 C, with masses of 12.000 000 and 13.003 355 and natural abundances of 98.9 % and 1.1 %, respectively. Similarly, there are two naturally occurring isotopes for nitrogen, 14N and 15N, with masses of 14.003 074(monoisotopic mass) and 15.000 109 and natural abundance of99.6% and 0.4%, respectively. A monoisotopic peak means that all the carbon atoms in the molecule are 12C, all the nitrogen atoms are 14N, all the oxygen atoms are 160, etc. The monoisotopic mass of the molecule is thus obtained by summing the monoisotopic masses of each element present.
Comment B1.6 Biologist's box: Measured mass
Measurements are made on a large, statistical ensemble of molecules and consist not only of species having just the lightest isotopes of the element present, but also of some percentage of species having one or more atoms of one of the heavier isotopes. The contribution of these heavier isotope peaks in the molecular ion cluster depends on the abundance-weighted sum of each element present. The theoretical probability of occurrence of these isotope clusters may be precisely calculated by solving the polynomial expression shown below:
where a is the percentage natural abundance of the light isotope, b is the percentage natural abundance of the heavy isotope, and m is the number of atoms of the element concerned in the molecule.
Calculations show that for small molecules such as n-butane (C4H10) there is a small but significant probability ( ~4%) that natural n-butane will have a molecule containing a 13C atom. The probability of there being two or three 13C atoms is negligible. For biological macromolecules containing several hundred carbon and nitrogen atoms the isotopic distribution pattern becomes extremely complicated. It can be calculated, however, with commercially available programs.
The main factor limiting accurate molecular mass determination for high-mass biological macromolecules is peak overlap. For MALDI the peaks correspond to [M + H]+, [M + Na]+, and [M + matrix]+.
High mass resolution is usually deemed to be a requirement for accurate mass measurements, but under appropriate circumstances ( sample ion completely separated from background ions), measurements with comparable accuracy may be made at low resolution (see Comments Bl.5-Bl.7).
2524 2525 2526 2527 2528 2529 2530 2531 2532 2533 2534 2535 2536 2537 2538 2539
2524 2525 2526 2527 2528 2529 2530 2531 2532 2533 2534 2535 2536 2537 2538 2539
2524 2525 2526 2527 2528 2529 2530 2531 2532 2533 2534 2535 2536 2537 2538 2539
2524 2525 2526 2527 2528 2529 2530 2531 2532 2533 2534 2535 2536 2537 2538 2539
2524 2525 2526 2527 2528 2529 2530 2531 2532 2533 2534 2535 2536 2537 2538 2539
2529.913Resolution = 25000Peak top mass = 2530.91Average mass = 2531.67
Monoisotopic mass
Resolution = 5000Peak top mass = 2530.91Average mass = 2531.67
Resolution = 1000Peak top mass = 2530.93Average mass = 2531.67
Resolution = 500Peak top mass = 2531.15Average mass = 2531.67
Resolution = 250Peak top mass = 2531.43Average mass = 2531.67
(a)
25560255552555025545 25565 25570 25575 25580 25585 25590 25595 25600 25605 25610 25615 25620
Monoisotopic mass
Resolution = 25000Peak top mass = 25579.04Average mass = 25579.58
Resolution = 5000Peak top mass = 25578.91Average mass = 25579.58
Resolution = 1000Peak top mass = 25579.32Average mass = 25579.58
Resolution = 500Peak top mass = 25579.48Average mass = 25579.58
(b)
B1 Mass and charge
accelerated in either an increasing negative gradient field or decreasing positive gradient field. Secondly, by adding an electron to form an anion. In this case the accelerating fields are exactly the opposite to what they were for cations. Thirdly, by removal or addition of protons. In this case, the mass of the resulting ion differs by ± 1 from the mass of the original neutral one. Below we describe the more common ways of producing ions in a mass spectrometer.
81 .5.2 Electron ionisation (El)
EI is the most widely used ionisation technique in mass spectrometry. EI is a relatively simple illustration of the general principles of ionisation under electron bombardment. The electron energy generated by a heated filament in the ion source is usually set to 70 e V ( Comment B 1.8). Upon impact with 70-e V electrons, the gaseous molecule may lose or capture one electron. The possible events that may occur are described below.
Covalent bonds are formed by the pairing of electrons. Ionisation resulting in a cation requires loss of an electron from one of these bonds, leaving a bond with a single unpaired electron. In this case events are
M (neutral) + e- - M*+ + 2e-
where M*+ means positively charged molecular ion. In the case of electron capture, an anion is formed by the addition of an unpaired electron and therefore
M (neutral)+ e- - M*-
where M*- denotes a negatively charged molecular ion. Such ions are relatively unstable under conditions of electron bombardment. They give a series of daughter ions, which are recorded as the mass.
81 .5.3 Field ionisation (Fl)
FI requires the sample to be introduced in the vapour state. The molecules are subjected to a high intense electric field, of the order of 107
-108 V/cm. The electric field strength required in FI is achieved by using a metal (Pt, W) tip emitter with a tip radius of 100-1000 nm, to which a voltage of about 5 kV is applied. Under such conditions the outer shell electrons are subject to large forces, sufficient to generate molecular cations.
81 .5.4 Fast atom bombardment (FA8)
In FAB mass spectrometry (FAB-MS) ionisation is produced by bombarding the sample surface with an atomic beam of Ar or Xe, accelerated to an energy of a few kiloelectron-volts. It is supposed that, in this case, a primary particle induces a collision cascade in a small volume of the sample.
Comment B1.8
Electron-volt (eV)
and joule (J)
119
The electron-volt (eV)
is a unit of energy
equal to the kinetic
energy a single
electron acquires
accelerating through a
potential difference of
IV
1 eV = 1.6 x 10-19
J
The joule (J) ist the
standard unit of energy
in Sl units. 1 J = 1
(McLafferty, 1993).
Xe
Solvent
A
C+
Fission fragment
/ detector
I Fission fragment I
Sample foil
Acceleralion grid
Excitation area (100 nm2)
B1 Mass and charge
I Desorbed ions I
� Fragment ions
I ©. Molecular ion
©.
Desorbed ions detector
time-of-flight (TOF) mass spectrometer is used (Section Bl.6.5). By measuring
the flight time of the secondary ions and knowing their energy and drift path,
it is straightforward to transform the ion TOF spectrum into a mass spectrum.
Figure Bl.4 shows the main features of the PD-MS technique with a TOF mass
analyser.
PD-MS has a reasonably good sensitivity with peptides and relatively small
proteins (7-20 kDa ). Typically, about 10 pmol material is necessary for a molec
ular mass determination. Mass resolution is about 1000.
81 .5.6 Laser desorption and matrix-assisted laser
desorption ionisation
In laser desorption ionisation (LDI), laser radiation is focused onto a small spot
with a very high power density that gives an extremely high rate of heating.
This leads to the formation of a localised laser 'plume' of evaporated molecular
species, either from adsorbed material or from the solid substrate itself. Direct
LDI ofintact biological molecules without using the matrix is limited to molecular
masses of about 1 kDa. The mass range limitation gave rise to the development
ofMALDI.
121
Fig. B1.4 The main
features of the PD-MS
technique with a 252Cf
source and TOF mass
analyser. (After Caprioli
and Suter, 1995.)
122
Fig. B1.5 Schematic
mechanism for MALDI
using lasers:
(a) absorption of radiation
by the matrix;
(bl dissociation of the
matrix, phase change to
supercompressed gas,
and transfer of charges to
sample molecules;
(c) expansion of the
matrix at supersonic
velocity, entrainment of
sample molecules in
expanding matrix plume,
and transfer of charge to
molecule.
B Mass spectrometry
(a) (b) (c)
Laser pulse
Analyte molecule
UV - absorbin!l matrix
The MALDI process differs from direct laser desorption because it utilises a
specific matrix material mixed with the sample. From this point of view, MALDI
is similar to FAB; the latter using liquid matrices to provide soft ionisation.
However, MALDI provides much softer ionisation than FAB, which allows the
analysis of large molecules up to 1000 kDa with minimum fragmentation.
The details of energy conversion and sample desorption and ionisation are still
not fully known. A general outline of the mechanism is presented in Fig. B 1 .5.
Energy from the laser beam is absorbed by the chromophor(ic) matrix, which
rapidly expands into the gas phase, carrying with it sample molecules. Ionisa
tion occurs by proton transfer between excited matrix molecules and sample
molecules, presumably in the solid phase, and also by collisions in the expanding
plume.
The matrix is the key component in the MALDI technique. The matrix func
tions as an energy 'sink' resulting in longer sample life. The material to be
analysed is mixed with an excess of matrix, which preferentially absorbs the
laser radiation. Commonly used matrix materials are aromatic compounds that
contain carboxylic acid functional groups. The aromatic ring of the matrix acts
as a chromophore for the absorption oflaser irradiation leading to the desorption
of matrix and sample molecules into the gas phase. The matrix not only increases
sample ion yield, but also prevents its extensive fragmentation.
Two types oflaser are most useful for laser desorption ofbiological materials:
the IR laser, which can couple efficiently with molecular vibrational modes,
and the UV laser, which can excite electronic modes in aromatic molecules.
Pulses of 100 ns or less duration are used in both wavelength ranges, because
longer exposure times would lead to thermal heating resulting in the pyrolytic
decomposition of biological molecules.
Because most laser sources are pulsed, TOF and Fourier transform ion
cyclotron resonance (FTR-ICR) mass spectrometers have been most widely used
withMALDI (SectionBl.6). Amass accuracy of ±0.01 % (±1 Da at a molecular
mass of 10 kDa) can be achieved under favourable conditions. If high-resolution
conditions are available, it is possible to resolve individual carbon isotope peaks,
for example (see Section B2.1 ). The MALDI technique is still under active devel
opment and improvements are occurring at rapid rate.
B1 Mass and charge
Nanoflow electro spray Free jet expansion ionisation in the ion source
0
Disassembly in the collision cell
B1 .5.7 Electrospray ionisation (ESI)
Mass analysis
1e-7- 1e-
10 mbar
ESI produces intact ions from sample molecules directly from solutions at atmo
spheric pressure. Ions are formed by applying a 1-5 kV voltage to a sample
solution emerging from a capillary tube, at a low flow rate (1-20 nl/min). The
high electric potential, which is applied between the tip of the capillary tube and
a counter-electrode located a short distance away causes the liquid at the tip of
the tube to be dispersed into a fine spray of charged droplets (Fig. Bl.6). The
solvent evaporates from the droplets as they move from the atmospheric pressure
of the ionisation region into the vacuum chamber containing the mass analyser.
The evaporation of the solvent is aided either by a counter-current flow of drying
gas or by heating the tube that transports the droplets from the ion source into
the vacuum of the mass analyser. The production of positive or negative ions is
determined by the polarity of the voltage applied to the capillary.
Comment B1.9 Number of attached protons
ln general, the maximum number of protons that attach to a peptide or protein under
ESI conditions correlates well with the total number of basic amino acids (Arg, Lys,
His) plus the N-terminal amino group, unless it is acylated. However, the
accessibility of these basic sites is an important factor. The distribution of charge
states thus depends on pH, temperature and any denaturating agent present in the
solution. This information can be used to probe conformational changes in the
protein.
For example, for bovine cytochrome c the most abundant ion has 10 positive
charges when electrospraying a solution at pH 5.2, but 16 charges at pH 2.6. A
similar effect is observed upon reduction of disulphide bonds. Hen egg white
lysozyme with four disulphide bonds shows a charge distribution centred at 12+ , but
upon reduction with DTT (dithiothreitol), a new cluster centred around 15+ appears
(see also Comments B2.5, B2.6 and B2.7).
123
Fig. B1.6 Schematic
representation of the
passage of ions from the
nanoflow electrospray
needle to the detector of
the mass spectrometer.
Protein solution, typically
1-2 µI of 5 µM
concentration, is placed in
a fine-drawn capillary of
internal diameter
approximately 10 µm.
A voltage of several
kilovolts is applied to the
gold-plated needle,
causing an electrospray
of fine droplets. The
positively charged
droplets are
electrostatically attracted,
dissolvated and focused
in the mass spectrometer
for detection. (After
Rostom, 1999.)
Sampleinjector
Ionisationchamber
Massanalyser
Iondetector
Datahandling
Massspectrum
Magnet
Ion source Detector
Ion source
+
+d
A
BSource slit
Magnet
Collector slit
ESA
Filament Electriclens
Top and bottomend cap electrodes
Ringelectrode
Electronmultiplier
Toamplifier
Electrongate
+
128
Fig. B1.13 Schematic
view of a MALDI mass
spectrometer based on an
ion trap.
B Mass spectrometry
Sample !Plate
Heated capillary tube and lens
Laser
Skimmer Quadrupole ion gulde
Octopole ion guide
Ion Trap
To Detector
B1.6.4 Ion cyclotron resonance mass spectrometry (ICR-MS)
As an ion-trapping technique, ion cyclotron resonance mass spectrometry (ICRMS) differs substantially from mass spectrometry that uses ion transmission to separate masses (Comment Bl.IO). In ICR, ions trapped in magnetic and de electric fields are detected when the frequency of an applied rf field comes into resonance with the cyclotron frequency (Comment Bl.11). The resonance frequency We is directly proportional to the strength of the magnetic field (typically 3-7 T) and inversely proportional to the mass-to-charge ratio, m/z, of the
10ns
Bz
Wc = m
Comment B1.10 Ion cyclotron principle
(B1.4)
In 1932, E. Lawrence and S. Livingstone demonstrated that a charged particle
moving perpendicular to a uniform magnetic field is constrained to a circular orbit
in which the angular frequency of its motion is independent of the particle's orbital
radius and is given by the cyclotron equation (Eq. (B1.4)). Lawrence showed that
cyclotron motion of a particle could be excited to a larger orbital radius by applying
a transverse alternating electric field whose frequency matched the cyclotron
frequency of the particle. The significance of Lawrence's discovery was that a
particle could be excited to very large kinetic energy by use of only modest electric
field strength. An alternating voltage of 1 kV would, after 1000 cyclotron cycles,
accelerate the particle to a kinetic energy of 1 Me V
Comment B1.11 Ion cyclotron frequencies
B1 Mass and charge
It follows from Eq. (B1.4), that ions of different m/z have unique cyclotron
frequencies. At a magnetic field strength of 6 T, an ion of m / z = 36 has a cyclotron
frequency of2.6 MHz, whereas an ion ofm/z 3600 has a cyclotron frequency of26
kHz. Equation (B 1.5) also shows that increasing the magnetic field linearly
increases the cyclotron frequencies of the ions, making high-mass ions easier to
detect over the environmental noise in the low-kilohertz region. Additional benefits
of increasing the magnetic field include an improvement in mass-resolving power
and the extension of the upper mass limit.
It should be noted that Eq. (B1.4) does not account for the presence of the electric
field produced by two trapping plates and can be considered as a first approximation.
Tesla (T)
The standard unit of magnetic flux density in the SI system
Torr
A unit of pressure, being that necessary to support a column of mercury 1 mm high
at 0 °Cat standard gravity
1 Torr= 133.322 Pa
Pascal (Pa)
The standard unit of pressure in the SI system.
1 Pa= 1 kg m-1 s-2
Once formed, ions in the ICR-MS analyser cell are constrained to move in circular
orbits of radius r
mv r=
zB (B1.5)
with the motion confined perpendicular to the magnetic field (xy plane) but not
restricted parallel to the magnetic field (z-axis) (Fig. Bl.14). Ion trapping along
the z-axis is accomplished by applying an electrostatic potential to the two plates
on the ends of the cell. The trapped ions can be in the cell for up to several hours,
provided that a high vacuum (1 o-s -10-9 Torr) is maintained to reduce the number
of destabilising collisions between the ions and residual neutral molecules.
After formation by an ionisation event, trapped ions of a given m / z have the
same cyclotron frequency but a random position in the cell. The net motion of the
ions under these conditions does not generate a signal on the receiver plates of
the ICR-MS cell because of their random location. To detect cyclotron motion,
129
Time-domainsignal
FT
Massspectrum
Transmitterplate
Trap plateB
Receiver plate
B1 Mass and charge
B1 .6.5 TOF mass spectrometer
Mass analysis in a TOF mass spectrometer is based on the principle that ions of
different m / z values have the same energy, but different velocities, after acceler
ation out of the ion source. It follows that the time required for each ion to pass
the drift tube is different for different ions: low-mass ions are quicker to reach
the detector than high-mass ions. From Eq. (Bl.I) we derive the expressions
for the velocity u of an ion of mass m and charge z
( )1/2 U
= 2z;acc
and for the time t, spent to cover a length L
( m )112 t- -- L
2z Vacc
(B1.6)
(B1.7)
Equation (Bl.7) shows that with an accelerating voltage of20 kV andL of 1 m,
a singly charged ion of mass 1 kDa has a velocity of about 6 x 104 mis and the
time spent traversing the drift tube is 1.4 x 10-5 s.
It is evident that for a TOF mass analyser the suitable ionisation techniques
are those by which ions are generated in a pulsed regime: using 252Cf fission
particles, a laser pulse, and introduction of ions from continuous ionization
sources (El, ES, FAB and so on) with pulsed deflection of an ion beam or
pulsed extraction from an ion source. The pulse gives the start signal for data
acquisition.
The TOF method can be advantageous compared with scanning technologies
because ofits 'unlimited' mass range, high transmission (most of the ions injected
into the analyser are detected), high speed (the experiment involves nearly simul
taneous detection of the mass spectrum on the microsecond time scale), and the
potential for high duty factors (percentage of ions formed that are detected). A
major drawback is the low mass resolving power. From Eq. (B 1. 7) it follows that
m / z is proportional to t2, which leads to the formula for resolution
m l tR=-=--
lim 2 lit
Standard linear TOF instruments typically have a resolution no greater than 1000.
A significant improvement of the resolution in the TOF method can be obtained
by using an electrostatic mirror or 'reflectron' and the orthogonal TOF mass
spectrometer (o-TOF-MS).
A reflectron TOF mass spectrometer is based on the fact that high-energy ions
penetrate deeper into the reflection electric field and, therefore, spend more time
there than low-energy ions. Because they must traverse a greater distance, the
more energetic ions arrive at the detector at the same time as the less energetic
one. With the reflectron, the resolution of the TOF mass spectrometer increases
up to 6000.
131
rf excite
Signalout
Fouriertransform
Fouriertransform
Time Frequency
B1 Mass and charge
in the cell. Because frequency can be measured precisely, the mass of an ion can
be determined to 1 part in 109 or better. It should be noted that resolution in
FTICR-MS is mass-dependent; ultrahigh resolution can be obtained at low mass.
The sensitivity ofFTICR is so high that the method has been successfully applied
to study individual multiply charged macro-ions.
B1 .6.7 Tandem mass spectrometry (MS-MS)
To obtain structural information by mass spectrometry the molecule must undergo
fragmentation of one or more bonds in such a manner that ions are formed, the
m/z ratio of which can be related to the structure. We recall that 'soft' ionisation
methods, such as FAB, MALDI and ESI, generate single molecular ions that
contain insufficient excess energy to fragment. However, by converting the kinetic
energy of the ion into vibrational energy, fragmentation can be achieved. This
can be done in MS-MS using a special collision cell.
The most common MS-MS experiment is the product ion scan. In the exper
iment, ions of a given m / z value are selected with the first mass spectrometer
(MS 1, Fig. Bl.16). The selected ions are passed into the collision cell (CC),
typically filled with helium, argon or xenon. The ions are activated by collision,
and induced to fragment. The product ions are then analysed with the second
mass spectrometer (MS 2), which is set to scan over an appropriate mass range.
Since it takes only 1-2 min to record the spectrum, one can then set MS 1 for the
next precursor ion and obtain its collision spectrum, and so on.
There are two main types of instrument that allow MS-MS experiments. The
first is made of two mass spectrometers assembled in tandem. Two mass analysing
quadrupoles, or two magnetic analyser instruments or hybrids containing one
magnetic and one quadrupole spectrometer are representative cases. From this
standpoint coupling of a magnetic and an electric sector can be considered as MS
MS (double-focusing MS, Section Bl.6.1). The second type of MS-MS instru
ment consists of analysers capable of storing ions: the ICR (Section Bl.6.5) and
the quadrupole ion trap (Section B 1.6.3) mass spectrometers. These devices allow
the selection of particular ions by ejection ofall others from the trap. The selected
ions are then excited and caused to fragment during a selected time period, and
the ion fragments can be observed with a mass spectrometer. The process may be
P1 P4-
F,
P2 F:1
P3
{"] F3
{ P1, P3, P2'} MS 1 � @g F1, F2, F3, MS 2 F4 �, P5 F4, F5, F5
P5 F5
F5-
133
Fig. B1.16 Principle of
tandem mass
spectrometry. MS 1 and
MS 2 are the first and the
second mass
spectrometer
respectively. CC is the
collision cell. A mixture of
five peptides is scanned
to produce the spectrum
of the five (M + HJ+ ions
(P1 -P5 ). After the scan
only one selected ion (P 4)
passes into collision cell.
The fragments (F,-F6)
produced upon
collision-induced
decomposition of the
precursor ion (part of
which remains intact) are
then mass analysed by
scanning MS 2 to record
the product ion spectrum.
(After Biemann, 1992.)
134
Fig. B1.17 Lay-out of the
triple quadrupole system.
QI and QII are the first and
second quadrupole
systems, respectively. The
third quadrupole q, is
used as the collision cell.
S, source; D, detector; rf,
radio frequency. Such a
geometry is named
01 q02• {After Gordon,
2000.)
B Mass spectrometry
rf only q (collision cell)
QI
repeated to observe fragments of fragments, over several generations. The instru
ments exploit a sequence of events in time.
An alternative approach is to use the triple quadrupole design (Fig. Bl.17,
and Section B 1. 5 .2), which, although much cheaper, suffers from poor sensitivity
and mass limitation. The first quadrupole, QI, is used as a mass spectrometer, a
selected peak being injected into the collision cell (CC), and the decomposition
products are analysed in the second quadrupole QII.
Finally, there are also 'hybrid' instruments, which are so-named because they
combine the use of magnetic sectors, quadrupoles and TOF instruments in linear
and orthogonal projections.
B1.7 Checklist of key ideas
• A mass spectrometer does not measure absolute mass. The instrument needs to be
calibrated with standard compounds, whose mass values are known very accurately.
• The ESI technique produces intact ions from samples directly from solutions at atmos
pheric pressure by spraying a very dilute solution from the tip of a needle across an
electrostatic field gradient of a few kilovolts.
• A unique feature ofESI process is the formation of multiply-charged molecular species.
ESI is the most gentle ionisation method yielding no molecular fragmentation in practice.
• The MALDI technique produces intact ions from the sample mixed with specific matrix
material, which preferentially absorbs the laser radiation.
Suggestions for further reading
Historical review
B1 Mass and charge
Griffiths, L W. (1997). J. J. Thomson - the centenary of his discovery of the electron and his
invention of mass spectrometry. Rapid Commun. Mass Spectr., 11, 2-16.
Comisarow, M. B., and Marshall, A.G. (1996). The early development of Fourier transform
ion cyclotron resonance (FT-ICR) spectroscopy. J. Mass Spectr., 31, 581-5.
Ionisation techniques
Smith, D.R., Loo, J. A., Loo, R.R. 0., Busman, M., and Udseth, H. R. (1991). Principles and
practice of electrospray ionization - mass spectrometry for large polypeptides and
proteins. Mass Spectr. Rev., 10, 359-451.
Muddiman, D. C., Gusev, A. I., and Hercules, D. M. (1995). Application of secondary ion and
matrix-assisted laser desorption-ionization time-of-flight mass spectrometry for the
quantitative analysis of biological molecules. Mass Spectr. Rev., 14, 383-429.
Gordon, D. B. (2000). Mass spectrometric techniques. In Principles and Techniques of
Practical Biochemistry, Chapter 11, eds. K. Wilson and J. Walker. Cambridge: Cambridge
University Press.
Instrumentation and innovative techniques
Caprioli, R. M., and Suter, M. J.-F. Mass spectrometry. Chapter 4 in Introduction to
Biophysical Methods for Protein and Nucleic Research, Academic Press.
Amster, I. J. (1996). Fourier transform mass spectrometry. J. Mass Spectr., 31, 1325-1337.
Hofmann, E. (1996). Tandem mass spectrometry: a primer. J. Mass Spectr., 31, 129-37.
Dienes, T., Pastor, J. S., et al. (1996). Fourier transform mass spectrometry - advancing years
(1992-mid 1996). Mass Spectr. Rev., 15, 163-211.
Guilhaus, M., Mlynski, V. and Selbi, D. (1997). Perfect timing: time-of-flight mass
spectrometry. Rapid Commun. Mass Spectr., 11, 951-962.
Belov, M. E., Gorshkov, M. V., Udeseth, H. R., Anderson, G. A. and Smith, R. D. (2000).
Zeptomole-sensititivity electrospray ionization - Fourier transform ion cyclotron
resonance mass spectrometry proteins. Anal. Chem., 72, 2271-2279.
135