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Organic mass spectrometry at the beginning of the 21st century
Article in Journal of Analytical Chemistry · December 2008

DOI: 10.1134/S1061934808120022
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ISSN 1061-9348, Journal of Analytical Chemistry, 2008, Vol. 63, No. 12, pp. 1128–1154. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © A.T. Lebedev, V.G. Zaikin, 2008, published in Zhurnal Analiticheskoi Khimii, 2008, Vol. 63, No. 12, pp. 1236–1264.
REVIEWS
Organic Mass Spectrometry at the Beginning

of the 21st Century
A. T. Lebedeva and V. G. Zaikinb
aDepartment of Chemistry, Moscow State University, Moscow, 119992 Russia
b Topchiev Institute of Petrochemical Synthesis of the Russian Academy of Sciences,
pr. Lenina 29, Moscow, 119991 Russia
Received May 25, 2006
Abstract—Mass spectrometry in the last 15–20 years has become one of the first methods of the qualitative
analysis and quantitative determination of various substances, from isotopes of chemical elements to synthetic
polymers, proteins, and nucleic acids. This method allows analysts to work with both individual compounds
and most complex mixtures that contain hundreds and thousands of compounds. It is the best in terms of the
sensitivity, information content, and rapidity. This review covers the present-day achievements of mass spec-
trometry, including ionization and ion separation methods and different versions of its application in various
fields of science and human activity.
DOI: 10.1134/S1061934808120022
If we consider the year 1901, when German physi- ment of the analytical performance, and the reduction
cist W. Kaufmann created the first prototype parabolic in the cost and miniaturization of instruments. In
mass spectrograph for studying “cathode rays,” as the almost all of the above characteristics, mass spectrom-
date of birth of mass spectrometry, this method has etry now surpasses the overwhelming majority of the
recently celebrated its centenary. The 2002 Nobel Prize other methods of analysis.
in chemistry awarded to two mass spectrometrists, John We can hardly find a field of knowledge where mass
B. Fenn and Koichi Tanaka, was found to be symbolic. spectrometry is not used. Nowadays, it is most effi-
It is evident that this award was in no way dated to the ciently used in different branches of physics and partic-
jubilee, but was the recognition of the revolutionary ularly chemistry, from inorganic to bioorganic chemis-
changes in the mass spectrometry during the last try, biochemistry (proteomics, etc.), biophysics and
decade of the 20th century was marked by, in particular, medical chemistry (pharmacology, toxicology, and
because of the contribution of the above scientists. The doping control), geochemistry, and petrochemistry. We
most important result of these changes was the onrush cannot overestimate the success of mass spectrometry
of mass spectrometry into studies of thermally labile, in solving many applied problems, including industrial
nonvolatile, highly polar, and high-molecular-weight ones. These include clinical medicine, food industry,
compounds, which could not previously been studied geology, space research, forensic science, environmen-
by direct mass spectrometry. However, the rapid pene- tal protection, hygiene and sanitary, archeology, control
tration of mass spectrometry into various life sciences of industrial processes, defense, industrial separation
has became particularly important; this opened up the and the investigation of radioactive isotopes, antiterror-
possibility of investigating various biological polymers ist activity, and many other fields. Let us note the main
(proteins and peptides, nucleic acids, and carbohy- advantages of mass spectrometry distinguishing it from
drates) and even entire microorganisms, which were other research and analytical methods:
previously practically inaccessible not only to mass (i) The method allows the determination of the
spectrometry but to other methods as well. This success molecular weight of a compound, that is, its major indi-
was supported by the creation of new unique techniques vidual characteristic. Knowing the exact weight found
of ionization, ion separation and detection, and the with the use of high-resolution mass spectrometry, one
wide introduction of computers. Simultaneously, new can determine the elemental composition of com-
instruments were developed and traditional equipment pounds without their separation from mixtures, not
was considerably sophisticated, so that the basic char- resorting to traditional methods of elemental analysis.
acteristics of the method (sensitivity, rapidity, resolu-
tion, reproducibility, and automation level) were (ii) Present-day mass spectrometers ensure the
improved. Nowadays, the deeper and deeper penetra- determination of molecular weights of superheavy mol-
tion of mass spectrometry into cell life studies necessi- ecules. The record today is 110000000 Da.
tates the elaboration of new versions of mass spectrom- (iii) The set of fragment ions formed upon the
etry, its combinations with other methods, the improve- decomposition of the initial molecules provides infor-
1128
ORGANIC MASS SPECTROMETRY AT THE BEGINNING OF THE 21ST CENTURY 1129
I, % 105
100
90
80
70 77
60
50
40 51
30
20 148
10
0
20 30 40 50 60 70 80 90 100 110 120 130 140 150
m/z
Fig. 1. Electron ionization mass spectrum of butyrophenone.
mation about the structure of the studied substance. To and 77 almost unequivocally point to the presence of
identify and determine the compound structure, one can the ë6H5CO- fragment in the structure, which is also
use both the computer libraries of mass spectra and supported by the presence of a peak with m/z = 51 and
“manually” interpret the spectra taking into account the the absence of an ion peak with m/z = 91 [C6H5C H 2 ] + .
known fragmentation laws. The remaining 43 mass units can be assigned only to
(iv) Mass spectrometry is characterized by an the propyl or isopropyl group. The ion peak with
extraordinary sensitivity and, in many cases, can be m/z = 120 testifies to the abstraction of an ethylene
used in the quantitative analysis. Studies on standard molecule from a molecular ion, which is possible only
equipment require 10–12 g or 10–14 M of an individual for the propyl substituent. Therefore, Fig. 1 presents the
compound. The attained sensitivity records are as low spectrum of butyrophenone.
as 10–18 g or 10–21 M, i.e., analysts deal with several hun-
dreds of analyte molecules in a test sample. Relatively simple compounds are now easily identi-
(v) Mass spectrometry offers a unique possibility of fied by their mass spectra using computers, which are
directly analyzing the most complex mixtures of normal constituents of modern mass spectrometers.
organic compounds (to several thousand components) The last versions of commercial computer libraries
using combinations of a gas or liquid chromatography, contain several hundred thousands of mass spectra,
capillary electrophoresis, or their multidimensional which allow the identification of the studied compound
versions with mass spectrometry in the real-time mode. within several seconds. The NIST/EPA/NIH mass spec-
Methods of tandem mass spectrometry in the analysis tral library is most efficiently used in the electron ion-
of mixtures in some cases do not require the use of ization mass spectrometry; the next NIST05 version
additional separation techniques. was issued in 2005. The library contains 190825 spec-
(vi) Taking into account the most important struc- tra for 163198 compounds. In addition, it contains
tural and quantitative information gained from mass 120786 published and estimated gas-chromatographic
spectra and the automation of the method, it provides indices for 25728 compounds and also 5191 MS/MS
the highest speed of analysis. For example, time-of- spectra for 1920 primary ions. For the sake of conve-
flight analyzers with a registration speed of up to nience, the library also includes software for analyzing
500 spectra/s ensure the qualitative and quantitative mass spectra, an isotope calculator, an interpreter of
determination of 150 priority organic pollutants for mass spectra, and also an improved AMDIS program
8-10 min. for deconvolution and GC/MS data processing.
The interpretation of the data of mass spectrometry As noted above, very important events occurred in
is often easier than that of other spectrochemical meth- mass spectrometry in the last 15–20 years, when so-
ods. In many cases, standard chemical knowledge is called “mild” methods of the transfer of difficultly vol-
sufficient for determining the structure of compounds. atile, polar, thermally unstable, and high-molecular-
An example is provided by the spectrum of the electron weight organic compounds into gaseous ions were
ionization shown in Fig. 1. The weight of the heaviest developed and their active application was started. At
ion registered by the mass spectrometer is 148 Da. This the same time, traditional ionization methods, such as
is the molecular weight of the compound. High-resolu- electron ionization, chemical ionization, and photoion-
tion mass spectrometry gives its exact weight, ization in different versions, have still been used by the
148.0888 Da, which corresponds to the element com- researchers. The scope of this review does not allow us
position ë10ç12é. The small amount of peak fragment to consider in detail the entire diversity of mass spectro-
ions is indicative of their relatively stability and the metric methods and their application. We will only dis-
ease of formation, which is usually characteristic for cuss some of the most interesting and promising results
aromatic compounds. Intense ion peaks with m/z 105 obtained recently.
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

1130 LEBEDEV, ZAIKIN
(‡) (b) Counter electrode Separators
Test
Chromatograph
sample
Matrix Capillary
Analyzer
Fig. 2. Laser pulse interaction with a sample in MALDI: (a) N2 To the pump
before laser pulse and (b) after laser pulse.
PRESENT-DAY METHODS Fig. 3. A conceptual sketch of the electrospray method.

OF MASS SPECTROMETRY
Mass spectrometry is based on molecular ioniza- organic matrix, by short laser impulses (Fig. 2). The
tion, usually accompanied by their decomposition, and matrix is selected so that its molecules actively absorb
the determination of the whole set of ions formed photons emitted by a UV or, more rarely, an IR laser. A
through their separation by the mass-to-charge ratio dense high-temperature plasma is created above the
m/z. In addition to an ion source and an ion analyzer, sample surface, in which analyte molecules are present
mass spectrometers are equipped with systems of ion along with matrix molecules and ions. Although the
detection and the trapping of data processing. Tens of ionization mechanism itself has not been studied in
methods of molecule transformation into gaseous ion- detail, it is clear that the absorption of photon energy is
ized compounds based on different physical principles accompanied by the protonation, cationization, or
are known along with several types of mass analyzers. deprotonation of substance molecules with the forma-
The implementation of different combinations of such tion of positive and negative gaseous ions. These ions
instruments gives a wide variety of mass spectrometric possess low internal energies and almost do not decom-
techniques, each type of which is of particular effi- pose; therefore, MALDI spectra are characterized by
ciency in solving specific analytical and research prob- intense peaks of such ions used for the determination of
lems. molecular weights.
Ionization methods. The most widespread ioniza- Note that the character of the mass spectra in
tion methods and their characteristics are summarized MALDI strongly depends on many parameters, such as
in Table 1. It does not include well-known but virtually the matrix nature, the quantitative ratio of the matrix to
replaced methods of field ionization and desorption, the sample, the wavelength, the pulse duration and the
plasma desorption, thermal spray ionization, electric power of laser radiation, the method of sample applica-
hydrodynamic ionization, and also rarely used or only tion, and the presence of impurities. Therefore, the
being developed methods of photoionization, photoat- determination or study of each type of compound
mospheric pressure chemical ionization, multiphoton requires the optimum selection of these parameters.
mass spectrometry, etc. The table also does not contain Electrospray ionization (ESI), is based on another
methods used to study primarily inorganic compounds physical principle; it is classified with atmospheric
and materials: spark sources, element analyzers, induc- pressure ionization methods. This method was simulta-
tively coupled plasma, and thermal ionization. We will neously developed by J.B. Fenn [5], the 2002 Nobel
not consider in detail the principles of electron and Prize winner, and the group of L.N. Gall’ [6], who
chemical ionization, fast atom bombardment and sec- named it the method of ion extraction from solutions
ondary ion (FAB and SIMS), because they have been under atmospheric pressure (Russian abbreviation
considered many times, also in recent monographs ERIAD). Figure 3 shows the flowchart of the ESI
[1, 2]. We will consider in sufficient detail the newest method. A flow from a liquid chromatograph is directed
and most often used methods based on desorption prin- into a needle 0.1 mm in diameter to which a high volt-
ciples and atmospheric pressure ionization. The main age of about 6 kV is applied. At the exit of the needle in
success in life sciences in the last decade is due to these the ion source, an aerosol of charged drops with a high
methods. surface charge is obtained. These drops move to the
The desorption ionization methods are FAB, SIMS, counter electrode with the Earth potential. The pressure
and also MALDI. The last method was proposed by is decreased in the same direction; note that the pres-
F. Hillenkamp and M. Karas in [3] and K. Tanaka in [4], sure in the entire region before the counter electrode is
who won the 2002 Nobel Prize in chemistry. The maintained at the level of atmospheric pressure. In
MALDI method consists in the irradiation of a sample, moving to the entrance of the first separator, the drops
which is a solid solution of the studied compound in an decrease in size because of solvent evaporation. When

Table 1. Characteristics of the most widely used ionization methods
Ionization Fields
Mass range Sensitivity, mol Special advantages Disadvantages
method of application
Electron ionization up to 2000 10–15 Analysis of gas- Good reproducibility; Intensive fragmenta-
(IE) eous and highly availability of voluminous tion because of the
volatile substances computer databases; con- strong interaction
in quite different venient combination with with electrons; pos-
areas gas chromatography; com- sibility of studying
pact instruments; fragmen- only substances that
tation assists structural can be transferred to
analysis the gas phase with-
out decomposition,
the possibility of
thermal decomposi-
tion
Chemical ioniza- The same 10–13–10–15 The same Possibility of determining Possibility of study-
tion (CI) molecular weight; solving ing only substances
structural and often stere- that can be trans-
ochemical problems; com- ferred to the gas
bination with GC; model- phase without de-
ing of catalytic processes in composition
the presence of Lewis and
Broensted acids in the gas
phase
Atmospheric pres- The same 10–13–10–15 Analysis of low- to Convenient interface for Poor reproducibility
sure chemical ion- medium-polarity combining liquid chroma- of the spectra
ization (APCI) compounds in mix- tography and capillary
tures; monitoring electrophoresis with mass
industrial process- spectrometry; possibility of
es, ecology analyzing low- and medi-
um-polarity compounds
Fast atom bom- up to 10000 10–9 Analysis of oligo- Possibility of analyzing Possibility of over-
bardment (FAB) meric compounds low- and medium-polarity lapping with the ma-
and ionic compounds, ra- trix spectrum; poor
pidity, ease of cationization sensitivity, necessity
of sample dissolu-
tion in the matrix,
problems with non-
polar compounds
Secondary ion- <103 (quadrupole), Increases by four Studies of polymer Can be used as a “chemical Sample spraying
mass spectrometry 104 (magnetic ana- orders of magni- surfaces, catalytic microscope” for studying
(SIMS) lyzer), >103 (time- tude in going from systems, biomole- biological materials; Stud-
of-flight analyzer) a quadrupole to cules on the sur- ies of surface and deeper
time-of-flight ana- face; Construction material layers
lyzer of mass spectrome-
try images
Matrix-assisted la- Unlimited 10−12–10−15 Analysis of mainly Mild ionization; analysis of Insufficient resolu-
ser desorption/ion- to 10−21 biological and syn- bio- and synthetic poly- tion in the traditional
ization thetic high-molec- mers, ionic compounds; combination with
(MALDI) ular compounds application of low (PSD)- time-of-flight ana-
and high-energy lyzers; overlapping
(TOF/TOF) impact activa- of matrix peaks with
tion for gaining structural the spectrum of the
information; is not affected analyzed compound;
by the presence of salts; limited possibilities
possibility of mixture anal- in quantitative deter-
ysis minations; no on-
line combination
with HPLC

Table 1. (Contd.)
Ionization Fields of appli-
Mass range Sensitivity, mol Special advantages Disadvantages
method cation
Electrospray 200000 10–12–10–17 Analysis of po- The mildest ionization; analy- Stringent requirements on
ionization (ESI) and more lar compounds, sis of highly polar compounds the purity of solvents; bad
(in regis- biopolymers, and biopolymers; formation reproducibility of mass
tering and their com- of multiply charged ions; use spectra; strong effect of the
multiply plexes of impact activation for struc- cone potential and liquid
charged tural determination; conve- phases on the character of
ions) nient interface for combining mass spectra; necessity of
with HPLC and capillary salt removal
electrophoresis; possibility of
revealing noncovalent inter-
actions; absence of matrix
ions
they reach a critical size, at which the surface tension than by masses, so that, for example, an ion with the
cannot withstand the Coulomb repulsion forces (Ray- mass 100000 Da and a charge of 100 will be registered
leigh limit), the drop “blows up” to form smaller drop- as a singly charged ion with the mass of 1000. Unfortu-
lets. This process is repeated. As a result, we obtain nately, the formation of multiply charged ions, or, more
microdrops containing only one charged particle, precisely, multiply protonated or deprotonated mole-
which comes to the gas phase after the evaporation of cules, is typical mainly for proteins and oligopeptides
residual solvent molecules. An alternative mechanism that consist of amino acids with basic or acidic func-
implies the release of a charged molecule from the sur- tional groups in the side chain. The registered record in
face of the same charged drop. Gaseous ions of unsol- the mass determination by this method was
vated molecules of the studied substance in both cases 110000 kDa [11]. In that work, ESI was an ion source
pass through the separator and arrive at the analyzer [7]. for a Fourier-transform ion cyclotron resonance
The gas flow (usually nitrogen), which is fed (FT ICR) spectrometer, and the corresponding molec-
between the counter electrode and the first separator ular weight of the T4 bacteriophag RNA could be deter-
coaxially to the capillary (sputtering gas), improves the mined by registering multiply protonated molecules
dispersion of the liquid flow and the conditions for ion with charges from 28000 to 35000.
desolvation. This modification of the method, named The ions formed under the conditions of mild ion-
ion spray, also allows the analyst to increase the flow ization by electrospray possess low internal energies,
rate of the liquid in the device up to 200 µL/min in com- they virtually do not undergo fragmentation, and their
parison to 1–40 µL/min in the conventional version. mass spectra are often sets of the peaks due to polypro-
The additional modification of the interface [8] opens tonated molecules with different charges (more pre-
up the possibility of working with flows from usual cisely, [M + nH]n+). The fragmentation of these ions can
chromatography columns (to 2 mL/min). In addition to be initiated using tandem mass spectrometry. A typical
attempts at increasing the flow rate, analysts worked at spectrum of a biopolymer is presented in Fig. 4. By
the reduction of the flow of liquid. The method named measuring the value of m/z for these multiply charged
nanospray [9] was developed as the result of these ions, one can easily calculate the molecular weight of
works. This modification of electrospray deals with the compound.
flows with flow rates of several nL/min, requiring a Let us mention one more ionization method, atmo-
smaller amount of the sample for analysis, and makes it spheric pressure chemical ionization (APCI). It con-
possible to increase the efficiency of the analyte ion for- sists in the ionization of analyte molecules by their
mation by almost two orders of magnitude. Thus, the interaction with plasma ions formed under the action a
electrospray ionization source has appeared to be an corona discharge. Similarly to ESI, this method forms
excellent interface for combining high-performance a basis for the interface between the liquid chromato-
liquid chromatography with mass spectrometry. graph and mass spectrometer. Many LC/MS systems
In addition to the high sensitivity and the possibility include replaceable interfaces capable of registering
of working with thermally labile and nonvolatile com- mass spectra in the ESI or APCI mode. Note that the
pounds, the electrospray allows the detection and deter- last method imposes fewer requirements on sample
mination of high-molecular-weight compounds with properties (hydrophobicity, polar functional groups)
molecular weights of millions of Dalton [10]. This is and is of particular interest for industrial control.
accomplished because of the formation of multiply A disadvantage of mass spectrometry is the neces-
charged ions as a result of ESI. The mass analyzer sep- sity of sample injection into a vacuum system. In 2004,
arates ions by the their mass-to-charge ratios rather a new method of mass spectrometric analysis was first

I, arb. units
728.1
8 773.5
6 687.6
825.0
4
651.5
2 883.8
619.0
589.7 951.8
0
500 550 600 650 700 750 800 850 900 950 m/z
Fig. 4. Mass spectrum of cytochrome C obtained by the electrospray method.
HV power source Atmospheric-pressure

inlet of the mass spectrometer
Solvent V
N2
Sputtering gas flow
Desorbed ions
Water spray
Freely moving sample

is placed in air
Fig. 5. A conceptual sketch of the method of desorption electrospray ionization (DESI).
reported, in which any surface is bombarded with a human finger was described in [12]. The method opens
flow of charged electrosprayed solvent drops directly in up new horizons for the use of mass spectrometry in
air [12]. This method was named desorption electro- pharmacology, medicine, and ecology, both under sta-
spray ionization (DESI). The microdrops hitting the tionary and field conditions. Forensic examinations and
surface desorb ions of different organic compounds antiterrorist applications of this method are highly
(from low-molecular to proteins) form due to the elec- important. Luggage screening at airports for traces of
trostatic and pneumatic interactions (Fig. 5). The result- chemical and biological agents or drugs can easily be
ing gas-phase ions are sucked into the mass spectrome- carried out using stationary devices. Prototypes of por-
ter by an atmospheric interface of a mass spectrometer table instruments with a weight of about 10 kg for work
and analyzed in the MS or MS/MS mode. An advantage in the field have been constructed [13].
of the method is the absence of the sample preparation. Mass analyzers. The characteristics and possibili-
Any surface (metal, plastic, paper) can be used as a ties of mass spectrometers depend not only on the ion-
sample substrate. One can analyze samples directly ization method, but also on the type of the mass ana-
from the ground and also organs and plant and animal lyzer. The main types of mass analyzers and some of
tissues. For example, an experiment on the determina- their properties are summarized in Table 2. We will not
tion of medical preparations in a human body by direct- consider them in detail, because their operation princi-
ing charged drops of an aqueous–alcoholic mixture at a ples were considered in many monographs. Let us only

Table 2. Characteristics of the main types of mass analyzers (m/z analyzers)
Analyzer type Resolution; m/z range Scanning rate Principle of ion separation
Magnetic and sec- up to 2000 or to 80000 up to 20000 0.1–1 s/decade Deviation of the ion beam due to
tor electrostatic (at double focusing) magnetic or electric moment
Linear quadrupole up to 2000 up to 4000 ds/decade Separation of an ion beam by the
stability of the trajectories in a
linear radio-frequency quadru-
pole field
Linear and three- up to 2000 up to 2000 ds/decade The same, with the possibility of
dimensional ion retention and carrying out op-
quadrupole trap erations with ions
Time-of-flight up to 30000 or more unlimited The spectrum is regis- Separation of ion beam pulses by
tered for milliseconds the time of flight
Ion cyclotron reso- More than 105–106 up to 10 kDa High Ion separation by the cyclotron
nance frequency; ion retention and op-
eration with ions
note some of the important achievements in their were not so efficient. Makarov has proposed a device
improvement and the development of new types. that interfaced a classical linear quadrupole LTQ trap
If we consider analyzers used nowadays, we should from Thermo Finnigan with an analyzer of a new type,
note a substantial increase in the number of time-of- named orbitrap analyzer, through a quadrupole mem-
flight systems among them. This is due to the fact that ory trap (C-trap). This additional quadrupole trap
classical scanning analyzers (magnetic sector, quadru- serves as the accumulation and cooling of ions before
pole) can register only 1 to 10 full spectra per second, their injection into the orbitrap analyzer. The path of the
which is insufficient for analyzing rapidly changing ion motion in the device is shown in Fig. 6.
substance flows arriving at the mass spectrometer, for
example, in GC/MS (see more below). Commercial To analyze ions, a symmetric static electric field of
time-of-flight mass analyzers can register up to a special shape is created in the orbitrap analyzer
500 spectra per second. However, their most important between the inner and outer electrodes. The ions com-
advantage is the virtually unlimited upper detected ing to the field begin moving along stable cyclic trajec-
mass. tories around the central electrode and simultaneously
oscillating along its axis. The ion is detected by the
The wide introduction of reflectron proposed by image current at the outer electrodes. The procedure of
B.A. Mamyrin [14] in its numerous newest modifica- spectrum recording is finished by Fourier transforma-
tions, in particular, the curved-field one, as well as the tions, through which the frequency spectrum is trans-
combination of continuous ionization sources with the formed into the classical mass spectrum. The device
analyzer (orthogonal acceleration, M. Guilhaus [15] efficiently operated in the LC/MS mode. Its resolution
and A.F. Dodonov [16]), significantly improved the at a data selection rate of 1 spectrum per second is
parameters of time-of-flight mass spectrometers (reso- 60000. The maximum resolution exceeds 100000. The
lution, sensitivity, reproducibility, data selection rate, accuracy of mass measurement is below 2 ppm with an
and the possibility of working in tandem mass spec- internal standard and less than 5 ppm with an external
trometry). In particular, a multireflective time-of-flight standard. In the MS/MS mode, the device can record 3
mass spectrometer with an ion path length of up to spectra per second at a high resolution. Taking into
800 m and a resolution of 200000 was created at the account the absence of liquid helium-cooled supercon-
Institute of Analytical Instrument Making of the Rus- ducting magnets, the new mass spectrometer will
sian Academy of Sciences in 2005 [17]. This opened up undoubtedly become highly competitive to the today’s
the possibility of using such instruments in various most expensive mass spectrometric method, Fourier
applied research in biology, medicine, ecology, etc.
transform ICR mass spectrometry.
In 2005, Thermo Scientific presented an essentially
new type of mass spectrometric analyzer. The device In fact, present-day Fourier transform ICR mass
was created by the group headed by A. Makarov [18]. spectrometers are rather cumbersome, because they
The high resolution and accuracy of measuring the include superconducting magnets with a field strength
masses of electrostatic traps were always favorable for of 7, 9, and 12 T, which are also responsible for the high
combinations with pulse ion sources. However, in using cost of the instruments and their maintenance. One of
continuous ion sources with wide mass ranges, a high the ways to solve these problems is the development of
sensitivity, and wide dynamic range, electrostatic traps permanent magnets with longitudinal magnetic fields

Ions are collected in an ion trap, pushed out axially, and trapped in a C-trap
where they shrink to a small volume and are sent to the orbitrap
Linear trap ë-trap
Rotating around the central electrode

and vibrating axially, the ions create
an image current on the outer halves
of the orbitrap; the current is detected Orbitrap
by a differential multiplier
Ions of each mass generate

a corresponding sinusoidal wave
Fig. 6. The principle of the LTQ Orbitrap analyzer.
for ICR mass spectrometers with atmospheric ioniza- (n is usually below 10). CIDs are usually subdivided
tion sources [19]. into low- and high-energy ones. Low-energy collisions
Tandem and combined versions of mass spec- (below 100 eV) are usually implemented in ion traps,
trometry. Tandem mass spectrometry. As noted quadrupole systems, and ICR. High-energy collisions
above, many ionization methods used nowadays (3–25 keV) are typical for instruments with sector or
(MALDI, ESI, APCI) relate to mild methods, because two time-of-flight analyzers.
primary ions formed from the initial molecules possess Electron-capture dissociation (ECD) [20] should be
insignificant internal energies and virtually do not related to relatively new and highly efficient versions of
undergo further decomposition. To obtain the data on tandem mass spectrometry. This method can be used in
the molecular structure, analysts use induced dissocia- electrospray instruments and is based on the interaction
tion of these primary ions, which forms the basis of tan- of multiply charged (polyprotonated) ions with slow
dem mass spectrometry or MS/MS (åS2). This is most electrons (energy below 0.2 eV). The cross section of
often attained using collision cells filled with a noble this reaction considerably increases with an increase in
gas. While colliding with gas atoms, the primary ions of the ion charge:
the studied substance extracted from an ion beam by an [M + nH]n+ + e– [M + nH](n – 1)+.
appropriate method gain internal energy and dissociate
into fragments (collision-induced dissociation, CID); The arising radical center initiates a specific fragmenta-
the spectrum of these fragments is registered by any tion of molecules. This method is particularly widely
mass analyzer. used for the determination of a link sequence in
biopolymers by Fourier-transform ICR with an electro-
Several versions of tandem mass spectrometry are spray ion source [21].
known. In the instruments with magnetic and electro- We should note one more method, infrared mul-
static analyzers, collision cells are installed between the tiphoton dissociation (IRMPD) which is based on the
analyzers. The ions separated by the first analyzer dissociation of activated absorbed photons [21]:
undergo ion-molecular reactions in the cell, and the
m 1 + xhν m 2 + n.
products of their decomposition are registered as a + +
mass spectrum. Mass spectrometers with three quadru-

pole analyzers in which the central one is a collision The energy of photons can be varied. The method is
cell are widely used. MALDI mass spectrometry with suitable for use with ICR mass spectrometry, in which
time-of-flight analyzers involves the so-called post- ions can be retained under the conditions of photon
source decay (PSD) of metastable ions that left the excitation for a long time.
source, which also relates to tandem mass spectrometry Combination of mass spectrometry with other
methods. Because ion traps and ICR cells can retain methods. Mass spectrometers can be conveniently
ions for a long time, ion-molecular reactions can be combined with other instruments providing real-time
conducted in them, and the activating ions can be cho- analysis, that is, in the on-line mode. The combination
sen at different stages of decomposition by implement- of mass spectrometry with gas chromatography
ing the conditions of tandem mass spectrometry åSn (GC/MS) is known most widely; it is effectively used

Dodecane
width 150 ms
7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 8.1
Time, s
10 spectra/s 250 spectra/s
Fig. 7. The shape of dodecane signal in the total ion-current chromatogram at a registration rate of 10 and 250 spectra per second.
everywhere where the qualitative and quantitative anal- In addition to an increase in the reliability of the
ysis of complex organic mixtures is necessary for deter- qualitative and quantitative analysis, the acceleration of
mining trace amounts of particular organic compounds in the registration of mass spectra gives good-quality
large volumes of liquids, in particular, biological fluids. mass spectra even in the case of unresolved chromato-
The present-day determination of organic com- graphic peaks. The computer processing of the spectra
pounds in mixtures requires increasing rates. The limit- with the construction of the mass chromatograms for
ing step in GC/MS is the rate of passing the mixture each m/z value followed by the determination of the
components through the chromatography column. This maxima of the corresponding peaks and the construc-
process can be accelerated using shorter columns or by tion of a reconstructed chromatogram and recon-
increasing the temperature gradient. However, mixture structed mass spectra opens up the possibility of math-
separation in this case worsens. To solve this problem, ematically resolving the chromatographic and mass
one can increase the rate of scanning the mass spec- spectrometric coelution problems. Figure 8 illustrates
trum. The effect of the scanning rate on the reliability the possibilities of the deconvolution (computer pro-
of the substance determination is shown in Fig. 7. It can cessing of the results) in the identification of the mix-
be seen that, at the time width of the chromatographic ture components elutes as the overlapping chromato-
peak of 150 ms, the scanning of one spectrum even graphic zones. The human eye can distinguish in the
within 0.1 s will give absolutely incorrect results for the presented segment of the chromatogram seven or eight
quantitative determination (peak area). In this case, the peaks. After computer processing, we could find in this
identification of a component can become problematic, segment 21 individual compounds [22].
because only one mass spectrum with considerable The mathematical processing occupies an increas-
peak intensity discrimination will be obtained in the ingly significant place in mass spectrometric analyses.
region of high masses. Both these problems can easily For example, Fig. 9 (bottom) presents a reconstructed
be solved in registering 250 spectra per second. This chromatogram differing from the initial one by the sep-
example illustrates considerable advantages of the aration of almost all peaks near the zero line and the
time-of-flight mass analyzers providing the registration absence of the chromatographic “hump.” Note that the
of up to 500 spectra per second in comparison to the deconvolution of the chromatographic data, the deter-
sector and quadrupole analyzers allowing the recording mination of retention indexes, and the automatic iden-
of no more than 1–10 spectra per second. tification of complex mixture components, in addition

1 3 5 7 9 11 13
t, s
Fig. 8. Computer processing of a 13-s chromatogram section (above) allows the detection of 21 individual compounds (below) [22].
to software products built-in to any instrument, can also with the analysis of similar samples, this gain in time is
be done using the AMDIS program included in the undoubtedly significant.
complete set of the NIST05 mass spectral database.
One more direction of the development of GC/MS
Because of the substantial increase in the sensitivity in the last decade was the appearance of the first com-
of the instruments, the use of the method of mass frag- mercial mass spectrometers based on the so-called two-
mentography (selected ion monitoring) has consider- dimensional gas chromatography (GC/GC/MS). In
ably reduced. In this version, the full mass spectrum is such instruments, two capillary columns are connected
not registered, and the substance is detected by the time in a series through a special cryofocusing device; the
of elution from the chromatography column by regis- first of them (long) contains a nonpolar and the second
tering only the intensity of the chosen characteristic (short) one, a polar stationary phase. This approach
ion; therefore, the large volume of additional informa- allows the separation of the most complex compound
tion on the sample is lost. However, mass fragmentog- mixtures not only by boiling temperatures, but also by
raphy has still been used under the conditions of high- polarity. As an illustration, Fig. 10 presents the chro-
resolution mass spectrometry. This method is of partic- matograms of a dichloromethane extract from an aque-
ular importance, for example, for the determination of ous solution of diesel fuel before and after its chlorina-
superecotoxicants (polychlorinated dibenzodioxins and tion with sodium hypochlorite. The analysts found in
dibenzofurans), when ultratrace amounts of com- the samples more than 2000 individual organic com-
pounds (femtograms per liter) must be detected. pounds, and the figures demonstrate that saturated
hydrocarbons do not participate the reaction, alkylben-
Note that the above approaches have led to the intro- zenes are consumed by one-half to give numerous chlo-
duction of fast chromatography methods into experi- rinated congeners of chloroalkylbenzene, and alkyl-
ments by chromatography–mass spectrometry; in this naphthalenes enter the reaction almost completely to
case, mixture analysis, for example, the determination form mono- and polychlorinated alkylnaphthalenes
of hundreds of organic pollutants in environmental [23].
samples takes 6–8 min instead of the usual 50–60 min
without a deterioration of the results of the qualitative Another combined method widely used nowadays is
and quantitative determination. For laboratories dealing liquid chromatography/mass spectrometry (LC/MS).

1.8e+007
1.6e+007
1.4e+007
1.2e+007
1e+007
8e+006
6e+006
TIC
1.2e+007
1e+007
8e+006
6e+006
4e+006
2e+006
0
800 1000 1200 1400 1600 1800 Time, s
6756 9759 12762 15765 18768 21771 Spectrum number
TIC
Fig. 9. Primary (above) and reconstructed (below) total ion current chromatograms.
The introduction of interfaces based on electrospray dard high-performance liquid chromatography. One
and atmospheric pressure chemical ionization (see can see the improvement of all three parameters simul-
above) has allowed researchers to solve very complex taneously, although this is not essential in the quantita-
problems which arise while combining these two meth- tive aspect as in Fig. 11, at a change in one of the
ods. Today, LC (especially reversed-phase) in combina- parameters.
tion with mass spectrometry has become a powerful
tool for qualitative and quantitative research in biolog- Among the combined methods we should mention
ical chemistry. the combination of mass spectrometry with various
decomposition methods that allow the analyst to per-
As in the case of GC/MS, the problems of increasing form analyses in the real-time mode. The combination
the rapidity and sensitivity in this case are highly of mass spectrometry with pyrolysis has received the
important. One of the ways to increase the productivity widest acceptance. The pyrolitic system in this case can
and efficiency of the method was the use of stationary
be connected directly to a mass spectrometer (pyrolitic
phase particles below 2 µm in size and an ultrahigh
pressure for pumping the eluant (up to 15000 psi). This mass spectrometry) or to a GC/MS system (pyrolitic
gave the method of an ultraperformance liquid chroma- GC/MS). These methods are most efficient in studies of
tography (UPLC). A combination of UPLC with a different polymers, especially synthetic ones, and pro-
time-of-flight mass spectrometer has led to a significant vide the determination of their microstructure, spatial
shortening of the analysis and an increase in its sensi- structure, composition, and various possible impurities
tivity and resolution. Figure 11 illustrates this statement and additives; the study of the mechanism and kinetics
for the case when it was necessary to improve one of the of thermal processes; and many other things [24]. Such
specified parameters while the other parameters must combined systems, along with pyrolizers, can contain
remain the same. Figure 12 allows the reader to esti- various reaction systems, in particular, those based on
mate the total advantage of UPLC–MS over the stan- heterogeneous catalysts.

Alkane
(‡)
Alkylbenzenes
Alkylnaphthalenes
0.935573
650
1.99869
1650
2650
2.99688
3650
4650
3.99668
Chlorinated
alkylbenzenes
(b)
Chlorinated
alkylnaphthalenes
0.935573
650
1.99869
1650
2650
2.99688
3650
3.99668 4650
Fig. 10. Three-dimensional total ion-current chromatograms of a dichloromethane extract from a sample of diesel fuel (a) before
and (b) after chlorination [23].
Mass spectrometry of isotope relations based on the USE OF MASS SPECTROMETRY

precise measurement of the ratios of stable isotopes IN BIOORGANIC CHEMISTRY,
13ë/12C, 15N/14N, 18O/16O, 34S/32S, and 2H/1H in different BIOCHEMISTRY, AND BIOPHYSICS
organic and inorganic objects has recently also became Considerable efforts and funds were contributed to
widely used. Corresponding instruments usually proteomic and metabolomic studies; the analysis of
include a certain system for transforming studied sub- nucleic acids and nucleotides, sugars, and other bio-
stances into gases suitable for measurements by mass molecules and biopolymers. The greatest recent
achievements of mass spectrometry have been in the
spectrometry. Instruments including a special combus- analysis of various proteins and oligopeptides.
tion chamber between the gas chromatograph and the
mass spectrometer (this system is known as GC–IR–MS) Proteins have quite diverse functions in the cell: pro-
tection, transport, catalysis of enzymatic reactions, par-
are the most efficient. In this case, the isotope ratio can be ticipation in the cell’s alarm and communication pro-
determined for each mixture component successively cesses, etc. From the beginning of the 1990s, because
eluted from the gas-chromatography column [25]. of the creation of new mass spectrometric instruments,

I, arb. units the application of mass spectrometry to the analysis of

0.25 Rate: 9 times greater proteins continuously increased. Today, this method
has become virtually the primary method of such
research, and it actually has no alternative. As a result,
a new scientific area named proteomics [26] appeared 8
to 9 years ago; its main task is the large-scale analysis
of proteins by the unambiguous identification and the
precise determination of the concentration and time
0 behavior of the maximum number of proteins in a bio-
Time, min
logical system (the whole organism, tissue, cell, intrac-
ellular structure). Although the success of such research
I, arb. units was favored by the completion of the genome decoding,
Sensitivity: 3 times greater mass spectrometry has become an irreplaceable tool for
0.030
0.025 protein identification, and its increasing use in pro-
0.020 teomic research is due to the possibility of determining
0.015
0.010 post-translation modifications, the quantitative aspects
0.005 of the synthesis of various proteins by living organisms
0 depending on external conditions, and the structure of
Time, min multiprotein complexes. Note that proteomic research
deal with much more complex objects compared to the
genome ones and additional approaches are required
I, arb. units for the successful application of mass spectrometry to
Resolution: 1.7 times better
0.10 such research. Mass spectrometry in such research is
0.08 normally used for the determination of the primary
0.06 structure of proteins (amino acid sequence), post-trans-
0.04 lation modifications, conformational transformations, and
0.02 intra- and intermolecular cross links or noncovalent inter-
0 actions; the identification of protein biomarkers; the quan-
titative determination of particular proteins, etc.
0 5 10 15 20 25 30 35 40 45 50 55 60
Time, min The use of mass spectrometry for the identification
of proteins is based on several principles. One of them,
Fig. 11. Comparison of the possibilities of UPLC–MS and so-called peptide mass fingerprinting, is based on the
HPLC–MS. separation of a mixture of proteins by two-dimensional
I, arb. units
0.05
0.04
0.03
0.02
0.01 5.0 µm
0
0 2 4 6 8 10 12 15
Time, min
I, arb. units
0.05
0.04 60% more rapid
30% more sensitive
0.03 70% better resolution
0.02
0.01 1.7 µm
0
0 1 2 3 4 5 6 7
Time, min
Fig. 12. Cumulative advantages of UPLC/MS (below) over HPLC/MS (above).

polyacrylamide gel electrophoresis, their proteolysis, x3 y3 z3 x2 y2 z2 x1 y1 z1

the measurement of the molecular weights of the pep-
tides obtained by MALDI, and a comparison of the R1 O R3 O H+
results obtained with the data in the theoretical peptide H H
libraries compiled on the basis of databases of amino N N
acid sequences of the known proteins. To use this H2N N OH
method, one should know the weights of only several H
peptides. O R2 O R4
One more approach named accurate mass tags pro- a1 b1 c1 a2 b2 c2 a3 b3 c3
vides the identification of proteins on the basis of the
exact masses of single proteolytic peptides measured
by high-resolution Fourier transform ICR mass spec- Fig. 13. The accepted nomenclature and the origin of the
trometry. most typical ions formed upon the decomposition of proto-
nated protein and peptide molecules and used for determin-
One more mass spectrometric method includes a ing the sequence of amino acid residues.
version of tandem mass spectrometry (low- or high-
energy collisional activation, metastable post-source
decay). Mass spectra are registered for the correspond- ferentiate the isobar lysine and glutamine residues in
ing protonated or cationized peptides; they correspond such a peptide. Mass spectrometric approaches for the
to the statistical break of amide bonds in peptides. Pro- differentiation of the isomeric leucine and isoleucine
teins are identified by such mass spectra using special- residues is also available.
ized computer software, for example, SEQUEST or Although many publications report the successful
MASCOT. exclusive use of mass spectrometry for solving similar
This is usually preceded by protein decomposition problems, this in most cases is not true. The reliability
by a chemical or, more often, an enzymatic method to and reproducibility of such analyses today are far from
obtain small peptides, the amino acid sequence of perfect. New computer software makes sequence deter-
which is determined by mild ionization tandem mass mination by mass spectra easier, but do not cardinally
spectrometry. A sufficiently effective method for deter- change the situation. Therefore, new techniques and
mining the amino acid sequence of proteins is based on principles are constantly developed on the basis of mass
a comparison of the partial sequence determined exper- spectrometry, which allow the analyst to obtain inde-
imentally with those in protein libraries created on the pendent data sets.
basis of genome sequences. These works can be auto- One of the techniques is based on the use of comple-
mated using various software (SEQUEST, ProFound, mentary tandem methods. In particular, the classical
Mascot, MSFit) and databases (dbEST, NCBlnr, OWL, MS/MS version (collision activation) for determining
Swiss-Prot), available on the Internet. However, note the primary structure peptides obtained after the trypsi-
that the genome databases can be used only for organ- nolysis of proteins can be successfully augmented by
isms whose genome sequences are known. MS/MS spectra due to the electron capture dissociation
In the general case, especially in studying new pro- (ECD) or electron detachment. If classical collision
teins and peptides, peptidomimetics, proteins contain- activation leads to the breaking of peptide bonds with
ing “inconvenient” amino acids inhibiting the processes the formation b and y series fragment ions, electron
of the ion formation suitable for determining capture results in the break of the N–Cα bond with the
sequences, etc., the problem of the de novo determina- formation of Ò and z series fragment ions, and electron
tion of the primary structure arises, when the corre- detachment, causes the break of the ëα–ë bond with the
sponding spectra are not present in the databases. Then, formation of ‡ and ı series fragment ions [2]. The com-
the spectra are interpreted using the laws of cation or bination of these independently found sequences
anion decomposition in tandem mass spectrometers allows a considerable improvement in the reliability of
revealed for peptides and proteins. Complementary proteomic research. Using the dissociation of high-
ions of b and y types formed upon the amide bond break energy ions resulting from the electron capture, one can
and charge localization at the N- or C-terminal frag- solve the most difficult problem in the mass spectrom-
ment, respectively, should be considered most informa- etry of the separate determination isomeric leucine and
tive. One or another ion can form depending on the pep- isoleucine in peptide sequences [27].
tide character. The possible set of the basic diagnostic ions As an example, Fig. 15 presents MS/MS spectra of
formed from protonated oligopeptides and used to deter- a protonated FFPAIFR heptapeptide registered in the
mine the amino acid sequences is shown in Fig. 13. CID (top) and ECD (bottom) modes. In the first case,
As an example, Fig. 14 presents a MS/MS spectrum we observe four Û-series fragments caused by the
of a protonated peptide, which allows the determination abstraction of the F residue (m/z 750) from the proto-
of the amino acid sequence in the chain by splitting the nated molecule, then the F residue (m/z 603), P residue
amide bonds from both N- and C-termini. Using high- (m/z 506), and A residue (m/z 435). In the second case,
resolution mass spectrometry, one can successfully dif- three z-series fragments are shown: the release of the

V V P A I V G H F(NH2)
(B)
I, % 1232
100 1331
(MH+)
2069
1612
1499 2052
1133
1711
1074 1905
1503
1616 1768 1899

1161 1428
1359 1446 1786
1260
0
1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
(Y + 2)
S V V S G L (LG) L (LG)
S H
I, % (B)
100 739
359 458 838

302
909 937
996
0
100 200 300 400 500 600 700 800 900 1000
m/z
(Y + 2)
G V (PAI) V V H
Fig. 14. Mass spectrum of a (GL)L(GL)LGSVVSHVVPAIVGHF-NH2 peptide (here and below, A is alanine, R is arginine, N is
asparagine, D is aspartic acid, C is cysteine, Q is glutamine, G is glycine, H is histidine, I is isoleycine, L is leucine, K is lysine, M
is methionine, F is phenylalanine, P is proline, S is serine, T is threonine, W is thryptophan, Y is tyrosine, and V is valine).
whole FFP fragment (m/z 490) from the protonated An example is presented in Fig. 16, in which one
molecule and, then, the A fragment (m/z 419) and I frag- can see that the corresponding spectrum MS/MS con-
ment (m/z 306). It is clear that the information obtained tains fragment ions formed upon the break of peptide
by these two independent methods supplement each bonds inside the disulfide bridge. Unfortunately, com-
other and allows one to determine the sequence of binations of positive and negative ion spectra today are
amino acid residues in the peptide. described in a few works, whereas it can potentially be
successfully used in high-performance automated
Good results could be obtained by simultaneously research.
using collision activation spectra with the registration
of positive and negative ions [28]. In particular, under One more popular approach to the de novo sequence
the conditions of registering positive ions, the presence determination is the use of derivatization. A number of
of a disulfide bridge in the peptide structure inhibits derivatization agents interacting with various func-
bond breaks necessary for gaining information about tional groups of peptides, most often, with the terminal
the amino acid sequence on the corresponding place in amino group or the ε-amino group of lysine, were pro-
the chain. Therefore, the widespread approach in this posed. Reagents bearing a fixed charge, for example, in
case is the preliminary preparative chemical decompo- quaternary ammonium or the phosphonium group
sition of the S–S bond. Using the spectra of negative present the most interest among them. The presence of
ions obtained in the collision activation, one can often such a charge in the derivatized peptide leads to the
decode the sequence without the preliminary decompo- break of bonds along the chain and to the formation of
sition of the bridge [29]. ions used for the sequence determination. Derivatiza-

295.14 435.27 506.31 603.36
A
1.5
1.4
1.3
1.2
1.1
1.0
0.9 576.32
0.8
0.7
0.6
0.5
0.4
0.3 611.35
726.57 750.43
0.2 300.68 686.46
0.1
0
300 350 400 450 500 550 600 650 700 750 800
m/z
A 897.50
7.5
7.0
6.5
6.0 306.17
5.5
5.0
4.5
4.0
3.5
677.37
3.0 390.21 490.29 723.39
2.5 419.25 446.28
2.0 576.32 630.35
1.5 349.17
450.30 735.41 882.48
1.0 838.47
705.38
0.5
0
350 400 450 500 550 600 650 700 750 800 900 950
m/z
Fig. 15. MS/MS spectra of a FFPAIFR heptapeptide: upper, CID; lower, ECD.
tion accompanied by the introduction of the sulpho- glycosylation serine and threonine residues, and also
nium group at the N-terminus of the peptide molecule methionine oxidation, formylation, acetylation, ubiqui-
has won the highest popularity [30]. An important fact tylation, the formation or removal disulfide bonds, etc.
is that, in collision-activated dissociation, such deriva- Considerable success was reached in developing mass
tives are formed only by y-series ions, which is very spectrometric methods for the detection, determination
convenient for determining the sequence both “manu- of phosphorylation and glycosylation places, and the
ally” and using the simplest software (Fig. 17 [31]). quantitative determination of the depth of these pro-
The results of determining the amino acid sequence cesses. Derivatization by the introduction of a sulfonat-
of peptides using such sulfonating agents were so ing group mentioned above has appeared convenient
impressive that Amersham Biosciences has started the for the determination of phosphorylation places in pro-
production of a special reagent kit (EttanTM CAFTM teins. An interesting approach was offered for deter-
MALDI Sequencing Kit) including a sulfonating agent mining the location of post-translation ubiquitinylation
(3-sulfopropionic acid succinimidyl ester) and a of proteins [32]. Ubiquitin is a 76-member polypeptide;
reagent for the transformation of the lysine residue into it contains an asparagyl–glycyl–glycine fragment at the
the guanidine one. C-terminus and the carboxyl group of the last glycine is
The protein after its synthesis by a ribosome can bound to the ε-amino group of lysine. The trypsinolysis
function both in the initial and the modified state. We of such a conjugate is accompanied by the hydrolysis
know dozens of such post-translation modifications, the asparagyl-glycine amide bonds with the retention of the
most typical of which are phosphorylation and variable glycyl–glycine fragment in the peptide structure. The

FLPLLAGLAANFLPKIFCKITRKC-OH
(AAN) (GL) (654)
(α)
A
100
γN11 –(H2S2 + CO2 + MeCHO)
1534
δC18
50 1841
2517
(M-H)–
1437
2671
1435 1693 1823 1863
0
1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 24002500 2600 2700
(KIF) (β)
R G I K C* F (PKI) L F (α)
σN11
A 982
100
1078
692
709
50 1177
382 623
839 1225
169 325 495 1290
0
100 200 300 400 500 600 700 800 900 1000 11001200 1300
(β)
(LAAN) F (LP)
–(H2S2 + CO2 + MeCHO)]–

[(M-H)– (α)
*
F-L-P-L-L-A-G-L-A-A-N-F-L-P-K-I-F-C-K-I-G-R-K(NHCHCH2)
(β)
Fig. 16. A negative ion collisionally induced dissociation MS/MS spectrum of a peptide with a disulfide bridge at the C-terminus.
L G Q H –184 Da
I, % y11
100
R L L N E N L V E Q P
80
60 y15
y4 y5 y7 y
8
y6 y9
40
y2 y13
y1 y3 y10 y14
20
100 340 580 820 1060 1300 y12
m/z # # # *
0 400 800 1200 1600 2000

m/z
Fig. 17. Spectrum of the ion products formed from the protonated HQGLPQEVLNENLLR peptide under MALDI–MS/MS conditions
after the preliminary guanidization of lysine and the sulfonation of the N-terminus with 2-sulfobenzoic acid cyclic anhydride [31].

P1
1747.9
(–1 tag)
I, % P2
1804.8
100 (–2 tags)
tag-GG GG
1590.0
90
1691.7
80 tag-LKFAGKQLEDGR
y11y10 y7y6
70 y11
1532.8
1632.1
60
1476.9
50 MH+
KGG
1846.9
2019.6
40 KGG yy11'
1419.8
30 y6 y8 y
y5 9 y10
y3 y4
1016.6
y7
717.1
y2
1087.9
1234.7
20
589.4
476.4
347.4
960.0
232.2
10
0
200 400 600 800 1000 1200 1400 1600 1800 2000
m/z
Fig. 18. (MALDI–MS/MS) Mass spectrum of ion products forming from a protonated peptide bearing two sulfonated fragments at
the glycine residue [32].
last fragment is processed by a sulfonating agent and SO3Na

SO3Na
the peaks of the y ions are observed in the MALDI–
O O
MS/MS mass spectrum (Fig. 18). The mass difference O O
between the y6/y7 and y10/y11 ions correspond to the N N
mass of two glycine residues and the sulfonating agent, O (CH2)n O
which proves the position of ubiquitine in the initial O O
peptide. Disodium salts
of di(sulfosuccinimidyl)dicarboxylic acids
Mild ionization mass spectrometry has relatively
recently been used for determining the high-order +
structure of proteins, tertiary (macrochain conforma- NH2Cl–
tions), quaternary (noncovalently bound protein–pro- H3C O
tein, protein–peptide, protein–DNA, etc. complexes) O CH3
[33]. This is done after the preliminary intramolecular NH2 Cl–
+
or intermolecular cross linking of the approaching pro-
tein sites by the so-called cross linkers. The last agents Dimethyladipimidate
are bifunctional compounds with the terminal (usually
We should pay attention to one more approach used
two) reactive groups specifically reacting with the func-
to determine conformations and the intramolecular
tional groups of amino acids (most often the ε-amino mobility of proteins by mass spectrometry, namely,
group of lysine or the sufohydryl group of cystein). H/D exchange [34]. The experiments are usually per-
Thus, cross-linked proteins then undergo proteolysis formed using electrospray mass spectrometry. In the
and the peptides containing cross links are identified by simplest case, the protein is placed in D2O and the rate
mass spectrometry among the peptides formed. of deuterium exchange, which depends on the accessi-
Because the length of the linker chain is usually known, bility of a solvent approaching the exchanging center,
it determines the distance between the approaching the ease of the hydrogen bond formation, primary struc-
amino acids in the chain, between the two chains in the ture, temperature, and pH, is controlled by mass spec-
corresponding conformations or in complexes. Analy- trometry. By varying the last two parameters, one can
sis is usually performed by MALDI or, more rarely, by follow the rate of H/D exchange and, hence, the
ESI mass spectrometry. The most efficient analyzer is a changes in the spatial structure of the protein. For
Fourier-transform ICR. The most typical reagents for example, it is quite clear that denaturated and noncoag-
cross linking by amine groups are alkanedicarboxylic ulated proteins will possess higher exchange rates than
acid disuccinimide cross linking and also imidates. The partially or completely coagulated ones. The study of
use of a sulfonated succinimide ester derivative is of protein conformations by controlling the rate of proto-
particular convenience because they react with proteins nation and the formation of multiply charged ions will
under physiological water conditions: be based on the same principles.

I, arb. units become one more remarkable application of mass spec-

6000 (‡) trometry to solving proteomics problems. The devel-
16
O Digest
5000 oped approaches are based mainly on the introduction
of an isotope label. In principle, all of these methods are
4000 similar to the method of isotope dilution, widely used
3000 in quantitative mass spectrometry. In the simplest case,
attempts were made to use an internal standard, which
2000 was a peptide analog labeled with stable isotopes, for
1000 the quantitative determination of proteins and peptides.
However, this approach is poorly suitable, because the
0 internal standard is a labeled analog of one of the pep-
m/z tides, whereas the number of proteins or peptides to be
I, arb. units (b) quantitatively determined in the test mixture, there can
6000
95 % 18O Digest 18
O2 be several tens of thousands, and all of them can mani-
5000 fest themselves with different sensitivities.
4000 For the global quantitative analysis of the whole set
of proteins in the proteome, analysts proposed methods
3000 for determining the proteins at the comparative (rela-
2000 18 O1 tive) level rather than at the absolute level [see, for
example, 35]. All of these methods are based on the use
1000 of the mass difference between the proteins or peptides
0 from the proteomes to be compared and the introduc-
I, arb. units (c) m/z tion of “light” and “heavy” isotopes. The convenience
6000 of this strategy is in the fact that it allows the introduc-
50 % 16O Digest + 50 % 18O Digest tion of specific (isotope) markers into two populations
5000 16O2 of proteins, which are then united and proteolytically
4000 18
O2
hydrolyzed, and the peptides formed will be separated
and analyzed by mass spectrometry.
3000 18
O1
The known approaches of this type are subdivided
2000 into three groups:
1000 (i) Metabolic in vivo label introduction by
0 (a) cell cultivation in the presence of a culture
920 925 930 935 940 medium enriched by stable isotopes;
m/z
(b) cell cultivation in a medium containing amino
Fig. 19. (MALDI–QqTOF) mass spectra of positive ions
acids labeled by stable isotopes;
from the protonated YLYEIAR peptide obtained by proteol- (ii) The introduction of 18O isotopes during the
ysis in (a) H216O, (b) H218O, and (c) peptide mixtures course of or after proteolysis in water enriched with
obtained by proteolysis in light and heavy water [36]. 18O;
(iii) Chemical in vitro modification of proteins or

Ion mobility mass spectrometry is used to study pro- peptides using reagents bearing stable isotopes and
tein conformations more rarely. In this case, the ions interacting with N- or C-terminal functional groups or
formed in an ion source of a mass spectrometer move in groups in the side chain of some amino acid residues.
a gaseous medium, and the open structures possess a
lower mobility than the closed ones. Calculations of The general principle of the in vivo introduction of
theoretically possible spatial structures are used for the an isotope label involves the following stages: the sep-
more reliable determination of conformations. arate cultivation of cells in an unlabeled and labeled
medium under different conditions, joining equal
Note that all possible complexes constructed of sev- amounts of proteins from the proteomes to be com-
eral proteins and also of other biomolecules in some pared, proteolysis of the united sample, and the analysis
cases are studied by electrospray ionization with ICR of peptides by LC–MS/MS.
mass analyzers. However, this method most often The third method involves the simultaneous proteol-
detects just the formation of such noncovalently bound ysis of the proteins to be compared in light and heavy
biomolecules rather than the character of bonding. water, joining hydrolyzates, the separation of peptides,
The creation of efficient methods for the quantita- and their analysis by mass spectrometry. As an exam-
tive determination of proteins in the proteome has ple, Fig. 19 presents the mass spectra used to determine

the quantitative difference in the expression of the com- functional groups in proteins and peptides are
pared proteins [36]. rather widespread. One of the most widespread and
Although the above methods of quantitative pro- industrially accessible reagents for such a modifica-
teomics relate to global strategies affecting all pro- tion is the so-called ICATTM reagent kit, which is
teins in the proteome, the methods of introducing offered as an unlabeled reagent or its D8- or 13ë9
an isotope label by the modification of specific analogs:
HN NH
X X
X X H
O O N
S O I
X X
O X X O
Biotin Linker (heavy X = D; Thiol-specific
light X = H) reactive
group
A characteristic feature of these reagents is the pres- (gene). Many of them are biomarkers of an endogenic
ence of iodacetamide groups reacting with the sulfohy- origin formed as a result of metabolism. The other
dryl group of cystein, a linker that can be unlabeled or objects of analysis are xenobiotics formed as a result of
contain a deuterium or a 13C label, and also biotin nec- the exogenic metabolism after the introduction of a
essary for mixture enrichment with peptides by affine medicine or under the action of environmental factors
chromatography. The strategy of the analysis involves [37]. Generally, a combination of metabolome, pro-
the separate alkylation of sulfohydryl groups of proteome, and genome analyses would be most desirable
teins from two compared cells by an unlabeled or for the diagnostics.
labeled ICAT reagent, joining their equal amounts, and The most widespread object of analysis is urine, but
the proteolysis and analysis of the peptide of the mix- blood plasma is also quite often used. In urine analysis
ture by microcolumn GC combined with electrospray by GC/MS, organic acids are investigated most often
mass spectrometry. By the peak ratio of the labeled and [38]. LC/MS is most convenient for determining amino
unlabeled peptides, the analyst can assess the relative acids and constructing their profiles. The most impor-
amount of proteins in the proteome. tant advantage of this method is the possibility of deter-
USE OF MASS SPECTROMETRY mining the quantitative profile of quite diverse acids as
IN CLINICAL MEDICINE the most important biomarkers for the recognition of
particular diseases in one analysis. Note that, because
The creation of desktop compact mass spectrome- of the highest sensitivity of the mass spectrometric
ters, the introduction of mass spectral databases, and detection, one can analyze not only urine or blood
the simplification of experimental and data processing plasma extracts, but also spots on filter paper.
methods, favored the intensive penetration of the mass
spectrometry into clinical laboratories. Although clini- The introduction of MALDI mass spectrometry
cal analyses can be performed by GC/MS and LC/MS opened up the possibility of detecting and determining
and also their tandem versions, the first method is used protein or peptide biomarker profiles in cells, cell
most widely. lysates, etc. A particularly important direction is the
revelation and measurement of protein biomarker pro-
Metabolic disturbances due to particular diseases
files in tumor cells (oncoproteomics) [39].
lead to changes in the concentrations of certain sub-
stances often named biomarkers. The estimation of We should also mention successful diagnostic appli-
these changes forms the basis for diagnosing corre- cations of isotope ratio mass spectrometry in medicine.
sponding diseases. The most widespread objects of This method can be used, for example, for the noninva-
analysis in such studies are rather low-molecular fatty sive diagnostics of Helicobacter pylori (13ë-urease res-
acids, other organic acids, amino acids, monosaccha- piratory test) by the ratio of 13C and 12C isotopes in the
rides, prostaglandins, bile acids, various steroids and exhaled carbon dioxide; it is important that it is possible
their conjugates responsible for the metabolome. How- both to perform screening examinations of the popula-
ever, it also becomes necessary to determine some ele- tion and reveal the carriers of the dangerous infection in
ments and also various high-molecular peptide and pro- a timely manner and to control the efficiency of the
tein metabolites (proteomes) and oligonucleotides therapy used [40].

Note that, despite the considerable potential of mass spectrum registration. Studying reaction mechanisms is
spectrometry in clinical analysis, the majority of such one of the corner problems of organic chemistry. The
analyses are now performed in specialized laboratories majority of such reactions are conducted in solutions,
instead of in clinical laboratories. However, the works where their mechanism is significantly affected by the
on the automation and miniaturization of mass spectro- solvent nature, temperature, the presence of impurities,
metric instruments are considered, and mass spectrom- etc. The variation of one of the parameters can result in
etry will increasingly penetrate into hospital and clini- considerable changes in the reaction system or even in
cal laboratories. the change of the reaction direction. Modeling such
chemical processes in the gas phase of the mass spec-
trometer allows one to carry out research in the absence
USE OF MASS SPECTROMETRY of the effects of the solvent, counterions, and intermo-
IN PHARMACOLOGY, TOXICOLOGY, lecular interactions and, hence, provides an opportunity
AND DOPING CONTROL to investigate the behavior of the system under “ideal”
GC/MS is an exclusively important method for solv- conditions. A comparison of the data of the gas-phase
ing various problems in pharmacology, toxicology, and experiments with solution chemistry opens up the pos-
doping control, although in recent years LC/MS has sibility of revealing the role of the specified effects and
been used for this purpose more and more intensively. of selecting the optimum conditions for the required
Medicinal substances and their metabolites are most process. In addition, mass spectrometry is a reliable
often quantitatively determined by the method of iso- method for generating and studying fully isolated or
tope dilution. partially solvated ions and, therefore, occupies an inter-
Mass spectrometric systems with membrane injec- mediate place in physical organic chemistry between
tion are very convenient for studying the metabolism of experimental solution chemistry and theoretical calcu-
medicinal substances, because membrane injectors lations.
allow the extraction of organic substances from aque- By now, a representative set of the known organic
ous solutions and their admittance into the ion source of reactions in the gas phase has been studied using mass
the mass spectrometer. Blood flow systems of labora- spectrometry. The majority of these gas-phase pro-
tory animals can also be included in such a system; this cesses exhibit a clear correlation with reactions in the
allows the observation of a change in the level of a condensed phase [41]. Therefore, attempts were made
medicinal substance in a living organism in the real- at predicting chemical reactions on the basis of studies
time mode. of the reactivity of ions in the gas phase of the mass
The wide introduction of methods of tandem mass spectrometer. Thus, even classical electron ionization
spectrometry, the monitoring of specified reactions, and (EI) yields ionized particles whose analogs form as
high-resolution mass spectrometry in addition to the intermediates in solution chemistry. Using EI, chemists
classical monitoring of specified ions has made possi- can predict the reactions of thermolysis, photolysis and,
ble a considerable improvement in the sensitivity of the to a certain extent, acid-catalyzed reactions. Using the
determination and the reliability of the results. whole arsenal of present-day mass spectrometric meth-
ods, chemists can obtain positive and negative even-
electron ions identical to those formed in solution under
MASS SPECTROMETRY the conditions of acid and base catalysis, respectively.
IN ORGANIC CHEMISTRY The attraction of high-resolution mass spectrometry,
Mass spectrometry has remained one of the primary tandem mass spectrometry, and also isotope-labeled
analytical tools in organic chemistry. GC/MS methods analogs open up the possibility of reliably following the
with electron and chemical ionization are highly effi- directions of the transformation of the initial com-
cient in the qualitative and quantitative analysis of reac- pounds and the structures of the formed ions [41].
tion mixtures; this is aided by the availability of highly
efficient and voluminous mass spectral databases.
Using high-resolution mass spectrometry, one can MASS SPECTROMETRY OF
determine the elemental composition of each mixture SYNTHETIC POLYMERS
component. Hardly volatile, thermally unstable, and Almost from the appearance of organic mass spec-
highly polar compounds are studied using LC/MS trometry, this method was used to study polymers and
methods. Note that, in recent years, designers work on polymeric materials. However, the low volatility of
the development of LC/MS systems providing the reg- macromolecules and their large molecular weights,
istration of electron ionization spectra using the avail- falling outside the mass range measured by the first
able large mass spectral databases with automatic com- mass spectrometers practically excluded the possibility
pound identification. of their direct analysis. Therefore, many mass spectro-
One of the successfully developing independent metric approaches to studying macromolecules, used
directions in organic mass spectrometry is revealing until now, involved their preliminary decomposition to
correlations between real chemical processes and small fragments, often in the on-line mode (pyrolysis,
molecular decomposition under the conditions of mass hydrolysis, ozonolysis, etc.); the fragments were then

I, arb. units
–1625.4
–1581.4
–1689.4
–1537.4
–1713.4
–1493.3
–1757.4
2000
–1449.3
–1801.4
–1845.4
–1405.3
1500
–1361.3
–1889.4
–1317.3
–1933.4
1000
–1641.3
–1977.4
–1273.3
–1229.3
–2021.3
–1597.3
–1553.3
–1685.3
–1509.3
–1729.3
–2085.4
–1185.3
–1773.3
–1465.3
–1817.3
500
–1421.3
–2109.3
–1861.3
–1141.3
–1377.3
–1905.3
–1949.3
–2153.3
–1097.3
–1333.3
0
1000 1200 1400 1600 1800 2000 2200
m/z
Fig. 20. MALDI spectrum of a sample of polyethylene glycol.
analyzed by mass spectrometry (pyrolitic mass spec- separation of polymers into fractions by gel permission
trometry and GC/MS, thermogravimetry/mass spec- chromatography is very helpful in the quantitative stud-
trometry, etc.). ies of polymers by MALDI mass spectrometry.
The breakthrough in the direct analysis of intact One more application of the newest versions of
polymers took place because of the creation of desorp- mass spectrometry is the study of mechanisms and the
tion ionization methods and, in particular, MALDI and kinetics of polymerization processes, and also various
SIMS, and also ESI, capable of ionizing macromole- types of polymer transformations (thermal, hydrolytic,
cules without their decomposition. As a result, it biological, mechanical, and other).
became possible to determine the average molecular In the analysis of industrial plastics, it is highly
weights and the molecular weight distribution, the important to identify various additives (softeners; anti-
composition of copolymers, the nature of the terminal oxidants; antiozonizing agents, filling oils, cross-link-
groups, and the sequence of the monomer links; to ing and vulcanizing agents; heat, light, and radiation
identify polymers; and to study their mixtures [42]. As stabilizers; and others), and also residual, low-molecu-
an example, Fig. 20 shows the mass spectrum of a sam- lar oligomers, and solvents. The basic method in their
ple polyethylene glycol, from which one can determine qualitative and quantitative determination is mass spec-
many of the above parameters. trometry in different versions.
Both new and old mass spectrometric methods are In the processing of polymeric materials, the chem-
sometimes rather informative in polymer studies and ical structure of their surface is of principal importance.
can sometimes compete even with nuclear magnetic In the general case, it can differ from the bulk structure,
resonance spectroscopy. We know examples of the effi- and even molecular weight distribution at the surfaces
cient applications of pyrolitic GC/MS to determining can be different. Various additives can tend to migrate
the character of the binding monomer links; the identi- to the surface at different rates and, hence, change its
fication and localization of the branching sites and properties. The chemical structure of the polymer sur-
cross-linking groups; the differentiation of alternating, face predetermines the ways of its subsequent modifi-
statistical, and block copolymers; the analysis of the cation for preparing materials with the specified prop-
distribution by the lengths of the monomer unit erties. Secondary ion-mass spectrometry [43] plays an
sequences in copolymers at the qualitative or quantita- increasing role in solving such problems.
tive level; and the stereoregularity. MALDI and ESI
mass spectrometry proved to be a convenient tool in
determining the nature of terminal groups and identify- USE OF MASS SPECTROMETRY IN ECOLOGY,
ing cyclic structures; to a lesser degree, it is now used HYGIENE, AND SANITARY
to determine other elements of the microstructure, Starting from the 1970s, mass spectrometry has won
whereas we should expect the appearance of new the positions of the most reliable, highly sensitive, and
methodical works in this direction. The preliminary the most informative method for monitoring environ-

2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
5 10 15 20 25 30 Time
10000
24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 Time
10000
24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 Time
10000
24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 Time
Fig. 21. Total ion current chromatogram of a dichloromethane extract of seal fat and mass chromatogram ions m/z = 326, 360, and
394 typical for pentachloro-, hexachloro-, and heptachlorobiphenyls, respectively [2].
mental pollutants [44]. The possibilities of the method plete separation and the result in the appearance of a
in the GC/MS mode are much superior to those of clas- chromatographic “hump.”
sical gas chromatography. As an example, Fig. 21 pre-
sents the most complicated chromatogram of a sample Nevertheless, mass chromatography consisting in
of the seal fat (top figure). Several thousands of individ- the computer processing of the results with construct-
ual organic compounds in the sample prevent its com- ing chromatograms by ion currents characteristic for

6678 6833
100
80 7081
60 B. amthracis, Vollum
40
20
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time, min
100 5683, 6825

80
60 B. cereus, 6464 6713
40
20
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Time, min
6827
100
80
60 B. thuringiensis, 19267 6695
40 7081
20
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Time, min
Fig. 22. (LC/MS) chromatograms of three microorganism stain lysates and molecular weights of the main components [47].
particular compounds opens up the possibility of qual- has appeared highly efficient in applied studies. For
itatively and quantitatively determining dangerous example, its use for revealing the origin of environmen-
ecotoxicants present in trace amounts. In particular, tal pollution with mineral oil was found to be the most
Fig. 21 shows the ion current mass chromatograms for reliable in comparison to IR spectrometry, GC–MS,
the ions m/z = 326, 360, and 394, characteristic for rel- and nuclear magnetic resonance [45].
atively widespread chlorinated ecotoxicants, pen- Among the problems of environmental protection is
tachloro-, hexachloro-, and heptachlorobiphenyl, the determination of microorganisms, particularly
respectively. It can be seen that all peaks near the zero pathogenic or toxin producing ones, in various ecosys-
line are resolved, which allows the reliable determina- tems. The application of MALDI mass spectrometry
tion of the number of compounds in the sample, even in providing the desorption/ionization of protein biomar-
the absence of corresponding chromatographic peaks in kers from intact viruses, bacteria, spores, and fungi
the initial total ion current chromatogram. opens up the widest possibility [46].
Among the most widespread ecotoxicants that must
be identified and quantitatively determined in different
media, we should mention various chlorinated organic USE OF MASS SPECTROMETRY IN MILITARY
compounds, polynuclear aromatic compounds, phtha- DEFENSE AND ANTITERRORIST ACTIVITY
lates, organophosphorous compounds, various pesti- Being a high-sensitivity and selective method, mass
cides, phenols, etc. This is in many cases done using spectrometry and its combinations are widely used in
GC/MS in different versions; at the same time, LC/MS the analysis of chemical weapons and decomposition
with the atmospheric pressure chemical ionization products in monitoring the processes of weapon
(APCI) or ESI interface is very efficient for the detec- destruction. In recent years, in view of the revitalization
tion and determination of residual pesticides. A large of antiterrorist activity, considerable efforts are made at
body of basic research has been performed on the selec- working out the methods for the rapid identification of
tion of the most convenient method for determining causative agents of diseases: bacteria, viruses, and
superecotoxicants (polychlorinated dibenzodioxins and other pathogenic organisms. Because the correspond-
dibenzofurans) in water, soil, and biological objects. ing biomarkers are different, just the direct MALDI
The most reliable and efficient method proven to be a analysis of protein profiles in the cells of various organ-
combination of GC with high-resolution mass spec- isms allow for their identification. Figure 22 presents a
trometry. chromatogram of cell lysate from three relative bacte-
The number of ecological research studies using ria, including the antrax strain [47]. Using the molecu-
isotope mass spectrometry is increasing. This method lar masses of the basic components elutes as the chro-

Frozen MS images
section
Matrix
application
MALDI TOF
mass spectrometer
4000 6000 8000 10000

Laser m/z
Fig. 23. A mass spectrometry image of the compound distribution in the mouse brain [48].
matographic peaks, one can reliably distinguish the structural studies, and quantitative determination of
samples. bioorganic molecules, but also for the investigation of
Rapid methods for the detection of explosives, poi- individual cells and even tissues. The method of mass
sons, toxins, and military strains of microorganisms by spectrometry images (imaging) is extremely promising
mass spectrometry have been developed and introduced for biology, chemistry, and medicine. It consists in the
into practice in connection with antiterrorist activity. visualization of the distribution of the molecules of
Taking into account the rapidity and reliability of deter- interest in plants and animal tissues. In this method, one
mination, such analyses are right hands in today’s can follow, for example, the distribution of biomole-
unquiet world. cules or medicinal substances and their metabolites in
an organism. Figure 23 presents the conceptual sketch
The use of isotope ratio mass spectrometry in anti- of the experiment [48]. A thin frozen tissue section is
terrorist activity seems principally possible. With this placed on a metal substrate treated with a solution of
method, one can judge the psychoemotional condition the substance, used as an MALDI matrix. The applied
of a person by the isotope composition of the exhaled sample is dried and put into a mass spectrometer. A
substances [40]. laser impulse gradually scans the whole surface of the
sample registering thousands of mass spectra. The sub-
USE OF MASS SPECTROMETRY IN FORENSIC sequent computer processing with constructing images
EXAMINATION AND ARBITRAMENT of characteristic ions of the compounds of interest gives
a map of this compound distribution along the whole
Mass spectrometry in different versions is a very sample surface. The signal intensity in the spectrum,
important method in forensic examination. which correlated to the amount of a particular sub-
Mass spectrometry can also be irreplaceable in solv- stance at the specified point, in the final map is dis-
ing some arbitrament problems. For example, the prob- played by a certain color intensity. Because each indi-
lem of revealing the origin of oil pollution in surface vidual mass spectrum exhibits ion peaks for quite dif-
waters and the coastal line often appears. GC/MS with ferent compounds, this method allows the researcher to
constructing total ion current or separate ion chromato- control the distribution of almost all of these substances
grams can give fingerprints suitable for oil identifica- in one experiment. In particular, if a medical prepara-
tion. For many years, steranes and triterpanes were used tion is assumed to interact with a protein, in one exper-
as reliable oil markers in such analyses. With the devel- iment we can gain information about the distribution of
opment of isotope ratio mass spectrometry, this method the protein, the preparation, its metabolites, and the
has become very convenient for determining the author modified protein along the sample surface. The smaller
of oil spills [45]. the laser spot and the finer the irradiation grid, the
higher the resolution of the mass spectrometry images
obtained. Unfortunately, this method has not yet been
METHOD OF MASS SPECTROMETRY IMAGES used in Russia.
The application of desorption ionization has opened A similar approach in a simpler version is used to
up new possibilities not only for the identification, construct various protein profiles in tissue sections.

This approach is also based on MALDI which allows mass spectrometry. All this forms the basis for the
for the detection of proteins by their molecular weights development of highly rapid and informative methods
and the comparison of their expressions depending on for the qualitative and quantitative determination of
the tissue origin [49]. quite diverse low- and high-molecular organic com-
The development of so-called molecular scanners pounds in various matrixes.
[50] started in the early 1990s should be considered
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Organic mass spectrometry at the beginning of the 21st century

Article in Journal of Analytical Chemistry · December 2008

Albert T Lebedev Vladimir Zaikin

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Organic Mass Spectrometry at the Beginning

Fig. 1. Electron ionization mass spectrum of butyrophenone.

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

(‡) (b) Counter electrode Separators

PRESENT-DAY METHODS Fig. 3. A conceptual sketch of the electrospray method.

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

Table 1. Characteristics of the most widely used ionization methods

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

Fig. 4. Mass spectrum of cytochrome C obtained by the electrospray method.

HV power source Atmospheric-pressure

Freely moving sample

Fig. 5. A conceptual sketch of the method of desorption electrospray ionization (DESI).

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

Table 2. Characteristics of the main types of mass analyzers (m/z analyzers)

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

Rotating around the central electrode

Ions of each mass generate

Fig. 6. The principle of the LTQ Orbitrap analyzer.

mass spectrum. Mass spectrometers with three quadru-

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

10 spectra/s 250 spectra/s

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

Mass spectrometry of isotope relations based on the USE OF MASS SPECTROMETRY

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

I, arb. units the application of mass spectrometry to the analysis of

Fig. 12. Cumulative advantages of UPLC/MS (below) over HPLC/MS (above).

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

polyacrylamide gel electrophoresis, their proteolysis, x3 y3 z3 x2 y2 z2 x1 y1 z1

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

1616 1768 1899

359 458 838

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

–(H2S2 + CO2 + MeCHO)]–

0 400 800 1200 1600 2000

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

last fragment is processed by a sulfonating agent and SO3Na

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

I, arb. units become one more remarkable application of mass spec-

(iii) Chemical in vitro modification of proteins or

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

Fig. 20. MALDI spectrum of a sample of polyethylene glycol.

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

100 5683, 6825

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4000 6000 8000 10000

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 63 No. 12 2008

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