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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




(a)

25560255552555025545 25565 25570 25575 25580 25585 25590 25595 25600 25605 25610 25615 25620

Monoisotopic mass





(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.

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