1
MASARYK UNIVERSITY
Faculty of Science
Ondřej PEŠ
MULTIDETECTION PLATFORM FOR MICROCOLUMN
SEPARATIONS
Dissertation thesis
Supervisor: assoc. prof. Mgr. Jan Preisler, Ph.D. Brno, 2010
2
Bibliographic identification
Author’s name: Ondřej Peš
Thesis name: Multidetection platform for microcolumn
separations
Study program: PřF D-CH4 ANAL
Study domain: Analytical chemistry
Supervisor: Assoc. prof. Mgr. Jan Preisler, Ph.D.
Year of defense: 2010
Keywords: capillary electrophoresis, liquid chromatography, mass
spectrometry, laser-induced fluorescence, fractionation,
detection
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© Ondřej Peš, Masaryk University, 2010
4
Declaration:
The work submitted in this dissertation is the result of my own investigation, except
where otherwise stated.
5
Acknowledgments:
I would like to thank my supervisor Jan Preisler for conscientious, systematic and
encouraging guidance in the entire course of the doctoral study. Next, I greatly
acknowledge all co-authors of publications presented hereinafter, as well as all my
colleagues in the division of analytical chemistry.
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We do not know whether Homer truly existed; however, we do know he was sightless
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Abstract
Multidetection platform setup allowing recording separation progress on a
suitable target is the currently requested solution for the majority of applications in
analytical chemistry. Unlike other systems coupled off-line, the development of the
platform setup inheres in the multi detection assessment which has started to be
accepted merely as a necessity being able to ensure multiple species detection and
identification in a complex sample mixture.
The presented work submits a survey on the platform setup which comes out
from an innovative vacuum deposition interface in 2002. The re-designed, laboratorybuilt
device enables a microcolumn separation record to be collected on a target in a
form of sub-microliter fractions. These fractions then may undergo detection either
for to be localized on the target (solid-state laser-induced fluorescence), or to be
identified (on-target reactions, mass spectrometry).
9
Abstrakt
Platforma, která by umožnila zaznamenání průběhu separace a vícenásobnou
detekci, je často vyžadována na poli současného vědeckého výzkumu a aplikacích.
Narozdíl od ostatních off-line systémů je multidetekční platforma založena právě na
detekci pomocí několika analytických technik, které mohou výrazně přispět k
identifikaci a kvantifikaci látek v komplexních směsích.
Platforma obsažená v této práci vychází z rozhraní pro vakuové nanášení z
roku 2002 a umožňuje mikrofrakcionaci na vhodný substrát s následnou detekcí
pomocí laserem indukované fluorescence z pevné fáze, příp. identifikaci a
kvantifikaci pomocí specifických reakcí a hmotnostní spektrometrie.
10
Table of Contents
1. PREFACE .............................................................................................. 11
2. THEORY................................................................................................ 13
2.1. HYPHENATED TECHNIQUES ................................................................... 13
2.2. ON-LINE VS. OFF-LINE.......................................................................... 14
2.3. SEPARATION ...................................................................................... 15
2.3.1. Liquid chromatography ................................................................................15
2.3.2. Capillary electrophoresis .............................................................................17
2.4. DETECTION ........................................................................................ 20
2.4.1. Luminescence...............................................................................................23
2.4.2. Time-resolved fluorescence ..........................................................................25
2.5. MASS SPECTROMETRY .......................................................................... 26
2.5.1. Matrix-assisted laser desorption/ionization...................................................26
2.5.2. Electrospray ionization ................................................................................29
2.5.3. Laser ablation inductively-coupled plasma....................................................30
3. EXPERIMENTAL ................................................................................... 33
3.1. PLATFORM SETUP – DEPOSITION ............................................................ 33
3.1.1. Sub-atmospheric chamber.............................................................................33
3.1.2. Effluent deposition .......................................................................................34
3.2. PLATFORM SETUP – DETECTION ............................................................. 37
3.2.1. (Time-resolved) laser-induced native fluorescence ........................................37
3.2.2. Matrix-assisted laser desorption/ionization MS .............................................38
3.2.3. Laser ablation inductively-coupled plasma MS..............................................39
4. RESULTS AND DISCUSSION.................................................................. 40
4.1. MULTIDETECTION PLATFORM ................................................................ 40
4.1.1. Effluent deposition .......................................................................................40
4.1.2. Laser-induced native fluorescence ................................................................41
4.1.3. Time-resolved laser-induced native fluorescence ...........................................42
4.1.4. Matrix-assisted laser desorption/ionization MS .............................................44
4.1.5. Substrate-assisted laser desorption inductively-coupled plasma MS ...............45
4.2. OPERATIONAL SOFTWARE DEVELOPMENT ................................................ 47
5. CONCLUSION........................................................................................ 49
6. LIST OF PUBLICATIONS AND PRESENTATIONS .................................. 50
6.1. FULL PAPERS ...................................................................................... 50
6.2. EXTENDED ABSTRACTS ........................................................................ 50
6.3. ORAL PRESENTATION ........................................................................... 50
6.4. POSTER PRESENTATIONS....................................................................... 50
6.5. CONTRIBUTIONS NOT ENCLOSED WITHIN THE THESIS ................................. 51
7. SELECTED PUBLICATIONS AND PRESENTATIONS .............................. 53
8. ATTACHMENTS .................................................................................... 74
8.1. CURRICULUM VITAE ............................................................................ 74
9. LIST OF ABBREVIATIONS .................................................................... 76
10. REFERENCES ........................................................................................ 78
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1. Preface
In order to identify and characterize target compounds in complex mixtures,
exploitation of methods comprising one or more separation techniques and one or
more sophisticated detection modes, such as spectrophotometry, laser-induced
luminescence, soft and/or hard ionization mass spectrometry or on-target reactions,
becomes more conventional in modern science. In proteomics, countless studies rely
on two-dimensional gel electrophoresis (GE) followed by excision of the target spots
from the gel. The proteins contained within the gel piece are subjected to protease
digestion, and mass spectrometry (MS) and/or MS/MS is used to identify the peptides,
thereupon the original protein. Other approaches employ liquid chromatography (LC)
of peptides, produced by proteolytical digestion of one-dimensional GE bands, on-line
coupled to tandem MS. The wild card in the field of protein separation and
identification may be bestowed to microcolumn separation techniques coupled either
on-line or off-line to MS.
Employment of microcolumn LC and, on-line coupled, MS have been
successfully accepted in further scientific areas, e.g. genomics, metallomics,
metabolomics, pharmacokinetics, combinatorial chemistry or polymer analysis.
Microcolumn separation techniques, such as capillary liquid chromatography
(cLC, µLC) or capillary electrophoresis (CE), belong among the most powerful
analytical tools in separation science. The advantages of these techniques include high
separation efficiency, low limits of detection, minute sample size requirements, low
sample consumption and economical operating costs.
In order to study multiple species in a sample, more than one type of detection
is preferable. Detectors are typically located in series one after one, on a position- or a
timescale. The timescale approach may shown to be beneficial, especially in the case
of a platform arrangement consisting of a target platform capable to serve as a tool for
effluent collection, as well as analyte detection. In that case, the detection is
performed from a solid-state sample, which predetermines it to be a spectral and/or a
desorption technique.
This work should enable a reader to gain a thorough view into the off-line
platform layout for multifaceted access to multidisciplinary science with the primary
interest in proteomics and applications.
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Tasks ensuring stable long-term performance include:
• experimental setup development and investigation for
o effluent deposition
o solid-state fluorescence detection
o solid-state time-resolved fluorescence detection
o MS detection
§ MALDI MS
§ SALD ICP MS
• operational software development
The theoretical part depicts separation techniques and detection modes, which
can be productively employed as a segment of the multidetection setup. The
experimental part faces the effective multidetection system development, as well as
coordination of the discrete, partial steps required for effective optimization and
applicability. Contribution of mine, in particular, involved CE separation with effluent
collection, fluorescence setup and detection, soft ionization MS analysis, software
development and testing, or data processing, evaluation, and interpretation.
Additionally, a review, gathering current possibilities and approaches in
microcolumn separations coupled to MS, was compiled as the thesis work piece.
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2. Theory
2.1. Hyphenated techniques
Hyphenated techniques refer to a combination of two or more instrumental
methods being able to provide more power when analyzing compounds from a
complex mixture.1-5
Generally, it includes a combination of a chromatographic or
electromigration separation technique and a sophisticated detection mode such as MS,
Fourier transform infrared spectroscopy (FTIR) or nuclear magnetic resonance
(NMR). The connection is maintained by a suitable interface allowing two options for
linkage. An on-line interface predetermines the detection to be performed
simultaneously as the separation proceeds, hence providing immediate data collection
and easier and more straightforward coupling options. An off-line interface relies on
an ability to record the separation process in a form of sample fractions, which are
then transferred to the detection compartment. Principally, it allows a user to perform
several types of detection and benefits from a possibility of complete decoupling of
the methods involved, which allows independent optimization of each.
Mass spectrometry belongs to a set of absolute methods being able to obtain
qualitative and quantitative information about a wide range of analytes. However,
mass spectrometry may suffer from a lack of selectivity, i.e. not being able to provide
the required results when applied to complex sample mixtures without prior
separation. To reduce sample complexity, coupling MS to one or more available
separation techniques, in either an on-line or an off-line arrangement, is then
unavoidable. As the on-line approach appears to be universal, thus widespread6-11
,
challenges resulting from time scales imposed by analyzing complex mixtures on-line
seem to be a major driving force for technological advances in the field of off-line
coupling. The majority of mass spectrometers coupled off-line include matrix-assisted
laser desorption/ionization12
(MALDI) or laser desorption/ionization (LDI) as an
ionization source, especially due to nature of the separation record consisting of
dried-droplets. Nevertheless, the sample introduction techniques may further include
desorption/ionization on silicon13
(DIOS), material-enhanced laser
desorption/ionization14
(MELDI), desorption electrospray ionization15
(DESI), nano-
14
assisted laser desorption/ionization16
(NALDI), laser ablation17
(LA), or the recently
presented surface-assisted laser desorption inductively-coupled plasma18
(SALD ICP).
As MALDI MS is amongst the foremost ionization techniques at the present time,
most applications involving off-line coupling pertain to the proteomic area. The
principles and applications, however, can be extended to the other desorption
techniques and analyte types.
2.2. On-line vs. off-line
Due to the inherent nature of liquid column separations, spraying techniques,
such as electrospray ionization (ESI) or atmospheric-pressure chemical ionization
(APCI), offer, as an on-line detector, easier and more straightforward utilization.
While being predominating techniques, they still possess shortcomings originating
from the time constraints of MS measurements. Particularly, there is a short time
available for the analysis of all components in highly resolved peaks, i.e. by the time
MS/MS of a selected compound is being carried out; co-eluting or closely eluting
compounds may have been skipped. In some cases, the entire separation has to be
repeated in order to complete data acquisition on missed analytes.
Ideally, the capabilities of both separation and detection instrumentation
should not be affected by being linked. This may be achieved merely by an off-line
approach, which provides an option of complete decoupling of the involved
techniques, thus enabling separate optimization of each. Nonetheless, if a publication
interest in on-line and off-line interfacing of microcolumn separations to MS were
compared, papers dealing with off-line coupling would be less frequent, presumably
due to a requirement of a relatively complex deposition interface, as well as early
expansion of MS/MS-capable mass spectrometers equipped with ESI. Moreover, the
key difference between time-dependent and result-driven analyses19
is constantly
being equalized by increasing power of data acquisition and processing hardware.
Although on-line coupling prevails, the off-line approach is capable of
combining the advantages of column (ruggedness) and gel (archiving) separations.
The ability to store the eluted fractions and expose them to another (spectrometric)
technique has appeared to be rewarding, e.g. in metallomics, where both species,
metal and bio-ligand, are expected to be determined together.18,20
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In combination with microcolumn separation, the off-line approach offers
improved sample throughput and cost-effective exploitation of the consequent
detection method. In other words, the duration of an on-line hyphenated technique
analysis is given by the time in which the entire liquid separation is accomplished. In
the off-line arrangement, a complete separation, which may take hours, can be stored
on a target and analyzed repeatedly, under various conditions, in minutes.
Furthermore, the availability of commercial spotters allowing various modes
of fraction collection considerably expands the application development.21
2.3. Separation
Generally, separation methods, which may be considered as a combination of
separation and detection, embrace chromatography and electrophoresis, and their
associated subforms in a planar or column arrangement. In many analyses, the
compounds of interest are found as portion of a complex mixture and the role of the
separation technique is to separate the components of that mixture to allow their
identification and/or quantitative determination.22
2.3.1. Liquid chromatography
The history of modern column chromatography can be traced to the turn of the
20th
century when the Russian botanist Mikhail Tswett (1872–1919) used a column
packed with a stationary phase of calcium carbonate to separate colored pigments
from plant extracts. There was little interest of the technique until 1931 when the
chromatography was re-established as an analytical technique for chemical
separations.
The basis of modern chromatography, formed by the process of extracting the
solutes back and forth between fresh portions of two phases was developed by Craig
in the 1940s.23
Initial work by Martin and Synge24
established the importance of
liquid–liquid partition chromatography and directed the advance in a theory for
chromatographic separations.25
Chromatographic separations are accomplished by constantly passing one
sample-free mobile phase over a second sample-free phase that remains stationary or
fixed. The sample injected, or placed, into the mobile phase moves with the mobile
phase while the sample’s components partition themselves between the mobile and
16
stationary phases. Components whose distribution ratio favors the stationary phase
require a longer time to advance through the chromatographic system.25
Although plain column chromatography, introduced by Tswett, is nowadays
used in large-scale preparative work, the main focus is on high-performance liquid
chromatography (HPLC). In HPLC, a liquid sample, or a solid sample dissolved in a
suitable solvent, is passed through a chromatographic column by a liquid mobile
phase. Separation is determined by interactions between solute and stationary phase
which include liquid–solid adsorption, liquid–liquid partitioning, ion exchange and
size exclusion, and between solute and mobile phase. In each case, however, the basic
instrumentation is principally the same. A schematic diagram of a typical HPLC
instrument is shown in Fig. 1.
The most commonly used columns for HPLC are constructed from stainless
steel with inner diameters ranging from 2.1 mm to 4.6 mm, and lengths from
approximately 30 mm to 300 mm. These columns are packed with 3–10 µm porous
silica particles that may be spherically or irregularly shaped. Typical flow rates are 0.5
– 5 ml.min-1
. In order to collect microfractions, it is necessary to reach the low flow
rate compatibility (several microliters per minute), by using a microcolumn LC or
splitting the effluent from the column in a suitable ratio, e.g. 100:1.
Smaller column dimensions and less solvent use in microcolumn separations
result in enhanced efficiency with the sample being diluted to a lesser extent and
producing larger signals at the detector. These columns are made from fused silica
capillaries with internal diameters of 50–200 µm and lengths of up to several meters.
Microcolumns may be packed with 3–5-µm particles or exist as open-tubular
microcolumns containing no packing material, with internal diameters of 1–50 µm
and lengths of approximately 1 m.
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Fig. 1: Schematic representation of a high-performance
liquid chromatograph. Adapted from (26).
2.3.2. Capillary electrophoresis
A theory for the mechanism of electrophoresis and the role of electroosmosis
was outlined by Hittorf, Helmholtz and Kohlrauch in the end of 19th
century. In 1930,
Tiselius published a work concerning blood plasma proteins separation in free
solution. He also demonstrated that the electrophoretic mobility of proteins might be
related to their molecular weight.
Electrophoresis belongs to a class of separation techniques in which analytes
are separated based on their ability to move through a conductive medium in response
to an applied electric field. In the absence of other effects, cations migrate toward
negatively charged cathode, and anions migrate toward the positively charged anode.
18
Ions with a higher charge-to-size ratio migrate at a faster rate than ions with lower
charge and/or larger ions. Differences in their migration rate enable the separation of
analytes in complex mixtures.25
In capillary electrophoresis the conducting buffer is maintained within a
capillary tube whose inner diameter is typically 25–100 µm. Samples are placed into
one end of the capillary tube filled by a buffered solution by hydrodynamic or
electrokinetic injection. When an electric field is applied to the capillary tube, the
sample’s components migrate as the result of two types of mobility: electrophoretic
mobility and electroosmotic mobility. Electrophoretic mobility is the solute’s
response to the applied electric field. Cations move toward the negatively charged
cathode, anions move toward the positively charged anode, and neutral species, which
do not respond to the electric field, remain stationary. The other contribution to a
sample migration is electroosmotic flow, which occurs when the buffer solution
moves through the capillary in response to the applied electric field. As the sample
migrates through the capillary, its components separate and elute from the column at
different times.25
The velocity with which a solute moves in response to the applied electric
field is called its electrophoretic velocity, νep and it is defined as
Ev epep µ= (1)
where µep is the solute electrophoretic mobility, and E is the magnitude of the applied
electric field. A solute electrophoretic mobility is defined as
r
q
ep
πη
µ
6
= (2)
where q is the solute charge, η is the buffer solvent viscosity, and r is the solute’s
radius.
The fundamental instrumentation for capillary electrophoresis is shown in Fig. 2 , and
includes a power supply for applying the electric field, anode and cathode
compartments, which contain reservoirs of the buffer solution, a sample vial
containing the sample, the capillary tube, and a detector.
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Fig. 2 : Schematic representation of a capillary electrophoresis with UV-Vis detection.
Adapted from (27).
Both cLC and CE belong to microcolumn separation techniques representing
actual analytical tools in separation science. The advantages of these techniques
include low limits of detection, high separation efficiency, high reproducibility, low
sample requirements and consumption, economical operating costs. Different
detection modes allow both LC and CE to establish the most powerful tools for the
identification and quantification of unknown substances in complex matrices.
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2.4. Detection
Improved levels of confidence in identification and structure elucidation can
be achieved by the use of multiple detection strategies. Although detection with a
variety of detectors can be achieved via multiple analysis of the sample on systems
equipped with different detectors, there has been requirement and a growing interest
in systems or setups which enable the use of multiple detectors coupled to a single
analytical separation system.28
Detection of analytes in microcolumn techniques might be performed using
several types of detectors. Generally, the detection techniques can be classified as:29
• bulk property or differential measurement
• sample specific
• mobile phase/electrolyte modification
• hyphenated techniques
A bulk property detector can be considered as a universal detector since it
measures a property which is common to all compounds. The detector responds to a
change in this property allowing a differential measurement between the separation
medium containing the sample and that without the sample, e.g. refractive index or
light scattering detector. The bulk property detector has the ability to detect all
compounds, which may be both an advantage and a disadvantage since all sample
components that are eluted from the column will generate a detector signal. This
means that additional chromatographic selectivity may be requested to make up for
the lack of detection selectivity.
Sample-specific detectors respond to characteristic which is unique to the
sample, or at least is not conventional to all analytes. The most commonly used
sample-specific detectors rely on the ability of an analyte to absorb ultraviolet (UV) or
visible light (UV-Vis), fluoresce (fluorescence), conduct electricity (conductivity), or
react under specific conditions (electrochemical).
The mobile-phase modification detectors change the mobile phase after the
column to produce a change in the analyte properties. Such changes include a specific
liquid-phase chemical reaction with the analyte (reaction detectors), a gas-phase
21
reaction (e.g. corona discharge, mass spectrometric detectors), or creation of analyte
particles suspended in a gas phase (e.g. evaporative light-scattering detectors).
Hyphenated techniques refer to the coupling of an independent analytical
instrument to the separation system, as mentioned in chapter 2.1
Although the UV-Vis and fluorescence detectors lack the option to provide
structural information, thus failing at identification of unknown compounds, both
belong to the most widespread types of detection. This may be explained by the
relatively low cost, simple interface design and decent selectivity. Detection is done
on-column before the solutes elute from the capillary tube and additional band
broadening occurs.
Since absorbance is directly proportional to light path length, the small
diameter of the capillary tubing results in signals that are smaller than those obtained
in narrow-bore LC. Several approaches have been used to increase the path length,
including a Z-shaped sample cell, a bubble cell, or multiple reflections. Fluorescence
detection, particularly when using a laser as an excitation source, is capable of
obtaining much lower limits of detection. Since the laser provides an intense source of
radiation that can be focused to a narrow spot, detection limits are ultimate, capable of
“single molecule” detection. Detection limits (LOD) along with other important
characteristics are mentioned in Table 1.
Flow rates in microcolumns techniques are much lower and thus compatible
with MS detection. The power of MS lies in the fact that the mass spectra of many
compounds are acquired with sufficient specificity to allow their identification with a
high degree of confidence. In other words, if the analyte of interest exists as part of a
mixture the mass spectrum obtained will contain ions from all of the present
compounds and identification with any degree of certainty is made much more
difficult, if not impossible. Subsequently, the combination of the separation ability to
allow unmixed compounds to be introduced into the MS with the identification
capability is clearly advantageous.22
22
Table 1: Characteristics of selected detectors for microcolumn separation techniques.
Detection LOD (mol.l-1
) Advantage Disadvantage
all purpose low selectivity
photometric 10-5
–10-7
spectral characteristics interference
all compounds non-selective
refractometric 10-5
–10-7
low-cost
selectivity fluorophore requirement
lamp fluorescence 10-7
– 10-9
spectral characteristics
ultimate sensitivity demanding
laser-induced fluorescence 10-13
– 10-15
selectivity
sensitivity
electroactivity
requirementamperometric/coulometric 10-7
– 10-10
selectivity
all purpose
electroactivity
requirementconductivity 10-6
– 10-8
contactless option
selectivity demanding
mass spectrometric 10-7
– 10-9
qualitative and
quantitative
selectivity labeling requirement
radiochemical 10-10
– 10-12
sensitivity risky environment
structural characteristics demanding
nuclear magnetic resonance 10-3
insensitive
indirect 10-4
–10-6
versatile
less sensitive than direct
detection
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2.4.1. Luminescence
Luminescence represents an optical phenomenon allowing a molecule to
absorb an energy quantum in a form of a photon and emit it in a form of a photon with
longer wavelength, i.e. lower energy. The energy absorbed may originate from a
different source which classifies the luminescence into categories as
photoluminescence, chemiluminescence, bioluminescence and electroluminescence.
The principle of absorption and emission of light by a fluorophore are
typically presented by a Jablonski energy diagram (Fig. 3), named in honor of the
Polish physicist Alexander Jablonski.30
The nature of the excited state allows several phenomenons with a varying
degree of probability to take place depending on an energy relaxation pathway from
the excited singlet state. The electron energy may be converted into heat by internal
and/or external collisions and the molecule loses the energy by a non-radiate way.
Else, the electron may drop to any vibrational state of the ground singlet state. If
relaxation from the excited state is accompanied by emission of a photon, the process
is formally known as fluorescence and the duration of such emission takes 10-9
to 10-7
s. Else the electron may undergo an intersystem crossing in which is converted to an
excited triplet state and, if not converted to heat by internal and/or external collisions,
is irradiated as light in a form of phosphorescence. As the crossing to the triplet state
includes a change in the electron spin, it generally takes longer for the electron to be
permitted re-entering the ground singlet state; thus time-taken by phosphorescence
ranges from 10-3
s to several days.30
The substances responsible for the fluorescence phenomenon are called
fluorophores and can be generally classified as intrinsic (native) or extrinsic. While
intrinsic fluorophores are those that occur naturally, extrinsic fluorophores are added
to the sample in a form of a covalently or non-covalently bound label either to enable
fluorescence, or to change the spectral properties of the sample.
Although many biologically important compounds provide native
fluorescence, e.g. the aromatic amino acids, the reduced form of nicotinamide adenine
dinucleotide (NADH), flavins, quinine, green-fluorescent protein, derivatives of
pyridoxyl and chlorophyll, etc., the proteomic character of the samples in the focal
24
point of the multidetection setup refers further text only to fluorescence of peptides
and proteins.
Fig. 3: Jablonski energy diagram. Adapted from (31)
Generally, native fluorescence of peptides and proteins is based on
fluorescence of three amino acids, tryptophan (W), tyrosine (Y) and phenylalanine
(F), which is excited by a light source emitting in UV region. Contribution of these
amino acids to the fluorescence yields is not evenly balanced and, as indicated by
chemical structures, the insole groups of tryptophan provide the highest fluorescence
yield. Tyrosine has a quantum yield similar to tryptophan, however, its fluorescence
emission is often quenched, which may be due to its interaction with the polypeptide
chain or energy transfer to tryptophan. Phenylalanine fluorescence can be observed
only when the peptide or protein sample lacks both tryptophan and tyrosine
molecules.30
Subsequently, for sensitive native fluorescence detection, the total amount of
tryptophan molecules in the protein sample is indeed essential.
Eventually, proteins are often requested to be labeled with fluorophores
providing longer excitation and emission wavelengths than the aromatic amino acids,
which would enable the labeled protein to be studied in the presence of other
unlabeled proteins. Nevertheless, the challenges originating from quantities
25
fluorescence labeling of peptides, and especially proteins, significantly outbalance the
practical use in the multidetection setup.32,33
2.4.2. Time-resolved fluorescence
The fluorescence lifetime is an average time that a molecule remains in an
excited state prior to returning to the ground state. Time-resolved fluorescence lays in
measurement the fluorescence intensity as a function of time which results in a
spectrum showing a decay of fluorescence intensity in a tight time window. It is
worthy to note that time-resolved measurements can efficiently filter-out the
undesirable scatter light, thus increase the signal-to-noise ratio, and furthermore,
provide more information about the fluorophore than is available from the steady-state
data.
Predominantly, three approaches are applied to obtain a time-resolved
spectrum. In time-domain or pulse fluorometry, the fluorophore is excited with a
pulse of light. The width of the pulse is made as short as possible, and is preferably
much shorter than the decay time of the target molecule.30
The alternative method of measuring the decay time is the frequency-domain
or phase-modulation method. In this case the sample is excited with intensitymodulated
light. Fluorescent sample excited by this light makes the emission respond
at the same modulation frequency. The fluorophore lifetime causes the emission to be
delayed in time observable as a shift, which can be used to calculate the decay time.
This shift is also accompanied by a decrease in the emission peak height relative to
the modulated excitation.30
The third technique to record fluorescence decay is based on the detection of
low-level light signals with pico- or femtosecond time resolution, commonly known
as time-correlated single photon counting (TCSPC). Photon counting instruments are
equipped with high repetition rate picosecond or femtosecond laser light sources, and
high acquisition rate microchannel plates. The method exploits the fact that low level,
high repetition rate signals provide such low light intensity that the probability of
detecting one photon in one signal period is much less than one.30
The fluorophore
lifetime is then estimated from a waveform histogram of all collected photons.
26
2.5. Mass spectrometry
The origins of mass spectrometry lie in the work done in the Cavendish
Laboratory in Cambridge by J. J. Thomson and his colleagues at the start of the
twentieth century on electrical discharges in gases.34
The mass spectrometer consists essentially of a source, which produces a beam
of ions, an analyzer which separates the beam according to the m/z ratio and a
collector which determines the fraction of the total ion current carried by each of the
ions.
For application context and further character of the work, only MALDI, ESI,
and (LA) ICP MS are brought in details.
2.5.1. Matrix-assisted laser desorption/ionization
MALDI time-of-flight (TOF) MS belongs into a group of analytical techniques
used for most peptidomics and proteomics analyses. Additionally, it may be employed
in metabolomics, pharmacokinetics, polymer analysis, and imaging MS.
MALDI, as an ionization process, was largely developed in the laboratory of
Franz Hillenkamp at the University of Münster, Germany.12,35,36
A similar method
was developed almost simultaneously by Koichi Tanaka et al. at Shimadzu Research
Laboratories in Kyoto, Japan.37
Hillenkamp’s method, which has been widely used,
works by embedding the sample in an organic matrix to facilitate desorption and
ionization of the sample upon irradiation by a pulsed UV laser. Tanaka reported the
use of finely dispersed metal powder in a glycerol matrix for the same purpose.
The sample to be analyzed is mixed with a chemical matrix, which typically
contains a small organic molecule containing a desirable chromophore absorbing light
at a specific wavelength. Common matrix compounds for UV-MALDI include αcyano-4-hydroxycinnamic
acid (CHC), 2,5-dihydroxybenzoic acid (DHB), and 3,5dimethoxy-4-hydroxycinnamic
acid (SA). The mixture of sample and matrix is
spotted onto a small plate and allowed to evaporate in air. The evaporation of residual
water or other solvent from the sample allows the formation of a crystal lattice into
which the peptide sample is integrated. The sample holder is then introduced into the
high vacuum of the ion source through an interlock. The pulse of a laser is focused
onto the sample to initiate desorption and ionization; a small part of the laser pulse is
27
split off to trigger the data acquisition. The matrix preferentially absorbs photons from
the beam and becomes excited. This excess energy is transferred to the sample, which
is then ejected from the target surface into the gas phase. This ionization process
produces both positive and negative ions, depending on the nature of the sample. For
peptides and proteins, the positive ions are usually the species of interest. The positive
ions are formed by accepting a proton as they are ejected from the matrix. A peptide
molecule tends to pick up a single proton, therefore most of the resulting peptide ions
are singly charged.38
The ions formed are all accelerated to the same kinetic energy and are mass
separated usually in a field-free drift tube. Detection is usually done by ion-to-electron
conversion using a multichannel plate detector, whose signal is read by a digital
oscilloscope. The modest mass resolution of linear TOF instruments can be greatly
improved using appropriate ion optics, delayed extraction, and a reflectron TOF mass
analyzer (ion mirror, see Fig. 4), which prolongs the ions flight trajectory in order to
correct for the flight times of ions having slightly different kinetic energies.
The advantage of the TOF instrument, in addition to its simplicity, is its fast
scanning capability and for this reason it is increasingly being encountered in LC–MS
instrumentation, particularly when fast analysis or high chromatographic resolution is
involved.22
There are other variants of MALDI mass spectrometers. Other mass analyzers
such as Fourier transform ion cyclotron resonance (FTICR) MS, quadrupole - based
instruments, sector-field mass spectrometers, or hybrid instruments can be used. The
TOF mass analyzer, however, is by far the most frequently used, because it is
naturally disposed for ions produced by a short laser pulse.
28
laserprism
acceleration
voltage
ion
optics
vacuum
pumping
reflectron
detector
linear
detector
reflectron
m1/z1m2/z1 >
lens
laser pulse
analyte molecules
matrix molecules
sample carrier
laserprism
acceleration
voltage
ion
optics
vacuum
pumping
reflectron
detector
linear
detector
reflectron
m1/z1m2/z1 >
lens laserprism
acceleration
voltage
ion
optics
vacuum
pumping
reflectron
detector
linear
detector
reflectron
m1/z1m2/z1 >
lens
laser pulse
analyte molecules
matrix molecules
sample carrier
laser pulse
analyte molecules
matrix molecules
sample carrier
Fig. 4: Schematic representation of a MALDI TOF mass spectrometer with a detail of
the MALDI ionization process. Adapted and modified from (39).
There are several peculiarities about the MALDI process that deserve a deeper
understanding of the mechanism for ion formation:38
• the yield of matrix ions in the absence of added cationization agents is
relatively low (~10-4
);
• MALDI is known to produce predominantly singly charged ions;
• mutual signal suppression when analyzing mixtures. For instance, a
basic peptide in a mixture of other, less basic peptides, completely
dominates the MALDI mass spectrum;
• MALDI works at both UV and IR wavelengths, yielding similar
spectra;
29
• a limited selection of “suitable” matrices exists for a given analytical
problem with the choice being largely empirical.
2.5.2. Electrospray ionization
In many mass spectrometric applications, the sample intended for analysis is
present as a solution in a solvent; moreover, the solution is often an effluent from a
(micro-) column separation technique. In any case, for a solution flowing into the
front end of a MS, the bulk of the solvent has to be removed without losing the
sample. If the solvent were not removed, then its vaporization in the vacuum of a
mass spectrometer would produce a large increase in pressure and made the MS
analysis impossible. Electrospray belongs to the methods, which enable the
differential solvent removal.
The sample solution is passed along a short conductive capillary tip with a
high positive or negative electric potential applied, typically 3–5 kV. When the
solution reaches the end of the capillary, the electric field causes it to be immediately
vaporized into a jet or spray of small charged droplets of a sample solution. Unlike
other methods of a liquid sample introduction, electrospray frequently produces
multicharged ions, which grants an option to measure analytes with large masses,
such as peptides and proteins, even with a mass analyzer with a relative low mass-tocharge
(m/z) ratio limit. Furthermore, the ions produced from macromolecules
overcome the tendency to fragment when ionized.40
The development of electrospray ionization (ESI) for the analysis of biological
macromolecules was rewarded the Nobel Prize in Chemistry to John Bennett Fenn in
2002.41
30
2.5.3. Laser ablation inductively-coupled plasma
ICP belongs into a category of hard ionization, element-specific techniques
and provides features such as multielemental capability, respectable precision, a wide
linear dynamic range, simple spectra, low detection limits and the ability to do rapid
isotopic analysis. ICP MS has found wide application to the analysis of a variety of
samples, e.g. in biology, geology, environmental chemistry, archeology, or forensic
science.28
In an Ar ICP, the degree of ionization of 52 elements is expected to be more
than 90 %. Only three elements (He, F and Ne) with a first ionization potential greater
than that of Ar would not be ionized and cannot therefore be determined by Ar ICP
MS. Correspondingly, the highest degree of double ionization is 10% and occurs only
for a few elements. The ICP is therefore an efficient elemental ion source for MS
since the majority of elements in the periodic table are singly ionized.28
However, it
has a number of limitations, such as matrix effects, or the need to keep the solute
concentration low to avoid clogging.
Fig. 5 shows a schematic diagram of an ICP MS instrument. The ICP is a
high-temperature (5000 – 10000 K) electrodeless discharge that is maintained in
argon flowing within a torch. Basically, the samples may be introduced into ICP as
liquids via nebulizers, which present the advantages of control over homogeneity and
ease of calibration, or as solids, which is advantageous at insoluble or solution
incompatible (archeological, forensic, art) samples.
31
Fig. 5: Schematic representation of a conventional nebulizer ICP MS instrument.
Reprinted from (28).
The nebulizer converts the sample solution into an aerosol that is transported
through a spray chamber where large droplets condense and drain out. The fine
aerosol is injected into the core of the plasma, and undergoes several sequential
processes as it moves deeper into the plasma: desolvation, vaporization, atomization
and ionization.
For solid-state samples introduction, laser ablation (LA) has been introduced
in 1985 by Gray et al.17
A short-pulsed high-power laser beam is focused onto a
sample surface resulting in an instantaneous conversion of a finite volume of the solid
sample into its vapor phase constituents which may be directly analyzed or flushed
into the ICP torch. Even as the primary role of LA is to be engaged in bulk analysis, it
is striking that the possibility of employing LA for desorption of deposited samples
has not been considered yet.
Separation and detection of ions generated by an ICP ion source are typically
performed by a low-resolution quadrupole, a TOF tube in an orthogonal setup, or by a
high-resolution double-focusing electrostatic and magnetic sector-field analyzer.
While the quadrupole-based and the sector-field instruments allow a sequential
measurement, TOF provides simultaneous detection of all ICP-produced ions at once.
32
Ions passing through a sector-field instrument experience a magnetic and
electric force, by which the ion beam is focused, corrected for aberrations in shape,
and, after reaching a collector, the individual m/z ratios are measured. The width and
shape of the ion beam is controlled by a series of slits located between the ion source
and the collector.40
In a quadrupole instrument, only the electric field is used to separate ions
according to their m/z. Ions pass along the central axis of four parallel, equidistant
rods with direct and alternating current (DC and AC, respectively) voltage applied to
them. Depending on both intensity and frequency of the electric field, only ions of one
selected m/z can pass through the rod assembly while all others are deflected to strike
the rods. By changing the AC/DC parameters, different m/z can be directed through
the system to produce a mass spectrum.40
33
3. Experimental
3.1. Platform setup – deposition
3.1.1. Sub-atmospheric chamber
In 2002, a vacuum deposition interface has been designed by Karger and
coworkers.42
The sub-atmospheric assembly, arisen from the vacuum deposition
interface, consisted of a rigid, circle-shaped chamber with a target plate being located
on a stepped-controlled linear translation xy-stage (Standa, Vilnius, Lithuania) in
order to act as an effluent microfraction collector, see Fig. 6, and consequently as a
dried-droplet sample carrier; see Fig. 8. The xy-stage travel range was 10 cm with 2.5
µm resolution in both axes.
LC or CE separation deposition either in a form of a continuous streak or
discrete fractions was enabled by a z-probe comprising of a deposition capillary and
another stepped-controlled linear stage with a 2-cm travel range. The deposition
process might have been monitored by an externally mounted black and white camera.
After a slight chamber setup modification, the target plate with a separation
record could have been detected by laser-induced (time-resolved) fluorescence; or
forwarded for MALDI MS, LA ICP MS or SALD ICP MS. Prior to MS detection, ontarget
reactions, such as enzymatic digestion might have been performed as well.43
To provide a linkage between separation and deposition capillary, the oncolumn
detector in Fig. 2 was replaced by a grounded liquid junction. It maintained
high separation efficiency and sample utilization due to the self-focusing effect and
lack of pressure-induced flow in comparison with on-line nebulizer-like interfaces. In
other words, the junction allowed the electrophoretically separated analyte to be
imprisoned in the middle of the laminar flow of the deposition capillary, thus keeping
the analyte zone width unaffected.
34
Fig. 6: Schematic representation of a sub-atmospheric deposition chamber with a
detail of a grounded liquid junction (left) and a deposition capillary probe (right)
when readied for CE effluent deposition.
3.1.2. Effluent deposition
Deposition of liquid effluent obtained as a result of a separation record or a
model sample testing was performed via capillary tubes of various lengths (10 – 40
cm), outer (100, 360 µm) and inner (30, 50, 75, 100 µm) diameters (O.D. and I.D.,
respectively). It is worthy to note that stable deposition depends on many variables,
such as effluent flow rate, temperature and viscosity, concentration of organic
solvents in effluent, substrate surface, surface tension, or environment pressure. The
volumetric flow rate was calculated according to Hagen–Poiseuille's law, which
stands for a volumetric flow of an incompressible fluid through a long circular tube,
and is defined as
l
pR
t
V
η
π
8
4
∆
= (3)
35
where R is the tube radius, ∆p is the pressure differences between the ends of the tube,
η is the dynamic fluid viscosity, and l is the tube length.
The pressure difference between ends of the deposition capillary was held at
80 kPa by generating a lower (20 kPa) pressure inside the chamber using a membrane
pump (KNF Neuberger, Freiburg, Germany). The lower pressure inside has shown to
be advantageous when depositing liquids with relatively high vapor tension allowing
the droplets to evaporate faster thus providing tinier dried-droplets ideally reaching
less than 100 µm in diameter.
As previously mentioned, the laboratory-built chamber was equipped with an
xy-translation stage ensuring horizontal movement and with a z-probe embracing the
deposition capillary. In respect to several ways of deposition of the liquid onto a target
surface,21
this experimental setup has allowed contact deposition without the capillary
stroking the target, see Fig. 7. This significantly decreased the possibility to introduce
a memory effect between consecutive fractions. The lowest volume, which could have
been reproducibly deposited was 6 nl and relied on a use of 30 µm I.D. capillary. The
lower limit for the droplet size was essentially given by the speed of the three-axis
ensemble of stages to perform the entire deposition cycle, and the intention not to
employ capillaries with I.D. lower than 30 µm to avoid clogging problems.
The process of contact deposition of a single droplet is described in Fig. 7.
Aqueous solution of 10 µM Cr(III) species is spotted onto a polyethylene
terephthalate glycol (PETG) plate as 100 nl droplets.
36
Fig. 7: A 360 µm O.D., 50 µm I.D. capillary contact deposition frame capture. Frame
1) and 2), droplet creation while the xy-stage is moving onto a target position; frame
3), the capillary has started to move downwards, droplet volume ~ 100 nl; frame 4),
proceeding contact deposition of the liquid onto the hydrophobic surface; frame 5) the
capillary has started to move upwards; frame 6) a new droplet is immmediately
growing and the xy-stage is moving onto a next position.
1) 2) 3)
4) 5) 6)
37
3.2. Platform setup – detection
3.2.1. (Time-resolved) laser-induced native fluorescence
In order to enable fluorescence collection in solid-state deposited fractions, the
sub-atmospheric chamber has been slightly modified. The core of the chamber, i.e.
xy-translation stage seizing the target plate remained, and an opaque tube was
mounted in a z direction, as seen in Fig. 8. The tube contained a reflecting microscope
objective (Ealing, Rocklin, CA), a pair of long-pass filters (Omega Optical,
Brattleboro, VT), a 1-mm slit, a pair of band-pass filters (Omega Optical, Brattleboro,
VT), and a photomultiplier tube (PMT) (Hamamatsu, Hamamatsu City, Japan). Laser
beam from a diode-pumped solid-state Nd:YAG (frequency quadrupled, 266 nm)
laser (Laser Compact, Moscow, Russia) was focused on a spot beneath the objective
via a concave mirror (focal length = 10 cm, diameter = 2.5 cm). Electric current,
generated by photoelectric effect, was acquired and processed by the 6035E data
acquisition board (NI, Austin, TX) for steady-state fluorescence or by the digital
oscilloscope WaveRunner 6050 (LeCroy, New York, NY). Sampling rate = 5
Gsample.s-1
and rise time = 750 ps allowed for time-domain measurements in
nanosecond resolution. Two long-pass filters with transmission characteristics T269
~5%, T275 ~50% and T295 ~5%, T298 ~50%, respectively, were used to filter out the
undesirable laser radiation, and the pair of band-pass filters with maximal
transmission 31% in 330 – 360 nm, and 65% in 380 – 625 nm, respectively, were
chosen to collect the majority of the native tryptophan fluorescence.
To guarantee several points per peak, as well as resolution of the fluorescence
signal acquired from a dried-droplet fraction, with the size being ~2 mm on a
stainless-steel target, the xy-stage was set to perform a linear scan trajectory along the
spot at speed of 1 mm.s-1
with 1 – 20 of points-per-fraction. The data acquisition time
from PMT ranged from 50 to 1000 ms. A value of 10 points per a fraction, 100-ms
steady-state and 500-ms time-resolved, fluorescence collection time-per-point was set
to all acquisitions to keep a balance between peak resolution and a decent acquisition
speed required for the entire target scanning.
38
Fig. 8: Schematic representation of a platform setup for solid-state (time-resolved)
fluorescence collection.
3.2.2. Matrix-assisted laser desorption/ionization MS
In order to detect and identify fractions with a MALDI MS, the MALDI target
served as the deposition plate. Commercial sample plates accessible for effluent
collection included a 96- and 384-well plate, or a smooth, well-free one.
MALDI MS data were acquired by Axima CFR (Kratos Analytical,
Manchester, UK) equipped with a nitrogen laser emitting at 337 nm (Laser Science,
Franklin, MA), a programmable xy-stage and a flight tube with reflectron. High
vacuum (~10-5
Pa) inside the instrument was generated by a couple of pumps. A builtin
black and white camera system provided sample imaging and laser targeting.
39
3.2.3. Laser ablation inductively-coupled plasma MS
Samples intended for LA ICP MS were deposited and collected onto
standalone (5.0 × 2.7 cm) sample targets, which fitted into the ablation cell.
Instrumentation for LA ICP MS consisted of a laser ablation system (model
UP 213, New Wave, Fremont, CA) and an ICP MS spectrometer (model Agilent 7500
CE, Japan). The commercial Q-switched Nd:YAG laser ablation system worked at the
5th
harmonic frequency, 213 nm. The ablation device was equipped with a
programmable xyz-stage to move the sample along a programmed trajectory during
ablation/desorption. Visual target inspection as well as photographic documentation
was accomplished by means of a built-in microscope/CCD-camera system. A sample
target with the deposited fractions was inserted into the SuperCell (New Wave,
Fremont, CA) and scanned by the laser beam, which was focused onto the sample
surface through a quartz window. The ablation cell was flushed with a carrier gas
(helium), which transported the laser-induced aerosol into the ICP torch, at a flow rate
1 l.min-1
. A sample gas flow of argon was admixed to the helium flow subsequent to
the laser ablation cell resulting in the total gas flow being 1.6 l.min-1
. To reduce
possible polyatomic interference, a helium collision cell was used (gas flow 3 ml.min-
1
) and natural isotope ratio abundance was monitored. Signals of 52, 53, 63 and 65
a.m.u. were set for detection of Cr or Cu, respectively.
40
4. Results and discussion
4.1. Multidetection platform
4.1.1. Effluent deposition (I, II, III, V, VIII, IX, X)
The dried-droplet size, shape, and appearance depended on the solution
constitution, hydrophobic/hydrophilic character of the sample plate, and the shape of
the deposition capillary tip. This presumably influenced sample behavior during the
deposition process.
It is obvious that different surfaces on which the liquid has been deposited
provide different dried-droplet diameters, as can be seen in Fig. 9. Two 100 nl
droplets were deposited onto a glass sheet and PETG surface. Dissimilar
hydrophobicity of target surfaces resulted in a different shape and size of the
developed dried-droplets. While evenly spaced ~100 µm diameter dots were formed
on the relatively hydrophobic surface of the PETG sheet, the spots on the glass sheet
were spaced more randomly and spot diameter was ~500 µm due to the more
hydrophilic nature of glass surface.
Fig. 9: A 100 nl droplet of 10 µM CrIII
deposited onto A) PETG and B) glass. The
dried droplet diameter is 3 to 4 times larger in the case of more hydrophilic glass
surface.
300 µm300 µm
A) B)
41
4.1.2. Laser-induced native fluorescence (IV, VI, VII, IX)
Solid-state fluorescence detection assumed that the native fluorescence of
tryptophan (λex = 295 nm, λem = 353 nm) in peptides and proteins may be considered
as an analogy to protein staining in gel electrophoresis. Furthermore, the located
fractions may undergo consequent on-target reactions, such as enzymatic digestion for
peptide mass fingerprinting.
As many compounds absorb at excitation wavelengths of trpytophan, several
challenges have been brought to the solid-state fluorescence detection of peptides and
proteins. The undesirable scatter light produced by dust particles or chemical residues
significantly decreased the signal-to-noise ratio. Major attention has then been paid to
the MALDI target cleaning techniques to ensure a complete oxidation of organic
residues, selection of buffers, which could recompense the irregularities of the
MALDI target, and tuning the optical system by suitable filters and slits. Organic
residues on the target that would have been excited at 266 nm were removed by
specific, time- and labor-consuming cleaning procedures, which involved ethanol,
methanol, acetonitrile, hexane, potassium permanganate, or strong colorless oxidizing
agents (hydrogen peroxide, hypochlorite acid). The best results were given by
employing a 5 min H2O2 cleaning procedure followed by 30-s treatment in ethanolic
ultrasonic bath. The fluorescence background signal decreased 3.5 times in
comparison to standard cleaning only with organic solvents.
Even as the background signal was lowered, an idea to compensate the
background signal using a spin-coating procedure was tested. MALDI matrices, CHC
and DHB, were as 6 mM solutions spin-coated onto a MALDI target. Although both
matrices absorb in UV region, deposition of peptides and proteins onto a target, which
would have been readied in advance for MALDI analysis, seemed advantageous.
Unfortunately, concentration of spin-coated matrix was extremely high as the
deposition capillary immediately started to clog up as the matrix crystals dissolved in
the built-up droplet. There was no way in decreasing the concentration since then the
matrix excess would not suffice for efficient matrix-assisted ionization. Other
solutions, i.e. phosphate or acetate buffer, citric acid, and nitrocellulose dissolved in
acetonitrile, did not cause any clogging problems; however, their contribution to the
signal-to-noise ratio increment was controversial.
42
Furthermore, the protein samples encountered strong photobleaching, which
resulted in a >50 % decrease in intensity when re-reading the same scan trajectory.
This was solved by setting the xy-translation stage to perform a fine offset shift (~10
µm) between two consecutive scans.
Fig. 10: Solid-state native fluorescence collection from dried-droplets of two Wcontaining
proteins (c = 0.0625 mg.ml-1
to 1 mg.ml-1
) deposited as 100 nl fractions.
Linear correlation coefficent (R2
) was 0.998 for lysozyme and 0.997 for carbonic
anhydrase.
Nevertheless, the rigidness of the entire system has been tweaked up resulting in an
ability to compete with on-column detectors by means of limits of detection and
repeatability. Linear dynamic range of W-containing proteins lysozyme (6W) and
carbonic anhydrase (7W) deposited on a stainless-steel target has covered 2 orders of
magnitude as seen in Fig. 10. The irregularities in a peak shape presumably relate
with the way the deposition capillary moved downwards to create a droplet. Detection
limits (3σ) of W-containing proteins were 2 ng of lysozyme and 3 ng of carbonic
anhydrase in a 100-nl dried-droplet; the deposition repeatability of the midpoint
concentration (10 replicate RSD) was 12 and 13 %, respectively.
4.1.3. Time-resolved laser-induced native fluorescence (VI, IX)
In addition to steady-state fluorescence experiments, the time-resolved
fluorescence collection using a digital oscilloscope has been studied. By adding
0
0,02
0,04
0,06
0,08
0,1
0,12
600 800 1000 1200 1400 1600
spot number
F(a.u.)
lysozyme
carbonic anhydrase
1 mg.ml
-1
0.5 mg.ml
-
0.25
0.125 mg.ml
-
0.0625 mg.ml
-1
0
0,01
0,02
0,03
0,04
0,05
0,06
1110 1115 1120 1125 1130 1135 1140 1145 1150
spot number
IF(a.u.)
43
another dimension into data acquisition, the entire platform setup has become, on one
hand, more complex with larger data sets, on the other hand, the selectivity of the
system increased. The fluorescence collected as intensity and its decay in time could
have improved the short-lifetime scatter light elimination. 100-nl droplets of
anthranilic acid (10 µM) and lysozyme (0.5 mg.ml-1
) solution were deposited onto a
sample target in ten replicates, and time-resolved fluorescence was collected, as seen
in Fig. 11. Acquisition conditions were similar to those of steady-state fluorescence
collection, i.e. linear stage scanning rate along the deposited fractions was 1-mm.s-1
,
and ten points per fraction were obtained. Each point consisted of an average from
500 decay spectra.
44
Fig. 11: Ten 100-nl droplets from 0.5 mg.ml-1
solution of A) anthranilic acid (τ = 8.3
ns), B) lysozyme (τ = 3.4 ns) deposited onto a target plate.
4.1.4. Matrix-assisted laser desorption/ionization MS (III, X)
Although MALDI mass spectrometry is a commonly used technique for
analysis of polar biopolymers with detection limits down to amol levels, ionization of
non-polar compounds, such as sterols, seems to be very challenging, especially when
regular MALDI matrices are employed. Use of silver nanoparticles as a cationizing
agent has shown to be valuable when testing MALDI matrices for such compounds.44
A HPLC separation (C8, 150x4.6 mm, 3 μm) of model sterols (stigmasterol, βsitosterol
and cholesterol) was recorded onto a MALDI target at atmospheric pressure
45
after splitting 100:1. The collected 500 nl fractions were analyzed by MALDI MS; the
signal of separated sterols was approximately 3 orders of magnitude above MALDI
MS detection limits of individual sterols.
4.1.5. Substrate-assisted laser desorption inductively-coupled
plasma MS (I, II, III, V, VIII, IX)
Substrate-assisted laser desorption is based on the ability of the employed
substrate material to enhance the dried-droplet desorption. The study of the
enhancement mechanism should not be a basis of this thesis; nevertheless, the project
leading to SALD publishing18
was coordinated by the multidetection platform setup.
The initial idea was studied on a model system of chromium species separation.
A simple electrophoresis of Cr3+
species in a formic acid electrolyte, from
chromium (VI) existing as Cr2O7
2,
CrO4
2,
HCrO4
or
H2CrO4 cannot be
accomplished. The mobility of highly charged species caused the loss of the CrIII
signal in the electropherogram. EDTA was used as a complexing agent to create a
negatively charged species CrIII
(EDTA),
which could be well separated from CrVI
under the same conditions.45
CE with UV detection (214 nm) was used to optimize the
Cr species separation prior to deposition on a target. The 100-µM solution of
chromium species in water prepared from CrIII
(EDTA)and
CrVI
was used for
hydrodynamic injection; approximately 200 femtomoles of each species were
injected. The UV and SALD ICP MS detection of CE separation is shown in Fig. 12.
Detection limits were comparable to conventional ICP MS, taking into
consideration that low sample volumes were used for analysis in this case. The
separation efficiency has been conserved while the rapid analysis time has remained.
Contrary to the use of nebulizers, low flow rate in the separation columns is
advantageous as it leads to small sample spots, high sensitivity and short analysis
time.
46
Fig. 12: CE separation of CrIII
/CrVI
species with A) UV (214 nm) and B) ICP MS (53
a.m.u.) detection. Injection from 100 µM CrIII
(EDTA)and
100 µM CrO4
2-
solution;
injected amount was ~200 fmol.
0.0
0.2
0.4
0.6
0.8
1.0
0 30 60 90 120 150
time (s)
integratedICPMSsignal(a.u.)×106
0.0
0.5
1.0
1.5
2.0
2.5
0 30 60 90 120 150
time (s)
A(a.u.)
CrO4
2-
CrIII
(EDTA)-
B)
A)
CrO4
2-
CrIII
(EDTA)-
47
4.2. Operational software development
Effluent deposition and steady-state fluorescence collection were controlled by
a NI-DAQ board 6035E via virtual laboratory software LabViewTM
(National
Instruments, Austin, TX). In order to fully exploit maximum the experimental setup,
we have made a decision to write an own program code. The laboratory-written code
is based on event-driven programming and was used to concurrently control both
deposition and fluorescence collection operation modes. The program allowed a
manual and/or automatic control of the xy-stage with various loadable serpentine-like
trajectories, time-stable movement of the z-probe for deposition, and steady-state
fluorescence signal collection with data imaging and exporting included.
Fig. 13: The main window for controlling both deposition and fluorescence detection.
Deposition was viable on any addressable target position, detection enabled stepped
control scanning along dried-droplet fractions.
In the case of time-resolved fluorescence acquisition, the xy-stage was
controlled by the LabView code while data collection was performed by an
independently operating digital oscilloscope. MALDI MS and SALD ICP MS were
48
commercial instruments supplied with original software allowing raster acquisition,
serpentine-like trail stage movement, and data acquisition and processing.
49
5. Conclusion
The off-line coupling of microcolumn separations and various detection
modes, which can include on-target reactions, has been re-designed, tested, and
employed in related areas of knowledge. Present configuration allowed a stable and
reliable CE or LC separation recording, steady-state or time-resolved fluorescence
collection and both soft and hard mass spectrometric detection of deposited
microfractions. The dried droplet size was essentially influenced by the microfraction
volume and target surface hydrophobicity. Both steady-state and time-resolved
fluorescence detection was tested on model samples deposited on a stainless-steel
MALDI target and optimized for future use in protein separations. Application of
MALDI and SALD ICP MS is rewarding on samples, which require detection by two
or more detection techniques. This includes, for instance, metallomics where both
metal and bio-ligand are expected to be determined altogether. Applications
employable in the platform setup included LC separation of the plant sterols, SALD
ICP MS of element species determination, or preparation of single-use targets
intended for element calibration in LA/LD ICP MS.
A review with comprehensive and detailed characteristics on microcolumn
separation techniques off-line coupled to mass spectrometry brought out a
classification of junctions between the separation column and the deposition needle,
as well as the process by which the liquid is transferred on the target. Moreover,
commercially available devices have been compared in terms of their potential
utilization in analytical chemistry with a summarization of applications used over the
past few years.
50
6. List of publications and presentations
6.1. Full papers
I. Peš, O.; Jungová, P.; Vyhnánek, R.; Vaculovič, T.; Kanický, V.; Preisler,
J., Off-line coupling of capillary electrophoresis to substrate-assisted laser
desorption inductively coupled plasma mass spectrometry. Analytical
Chemistry 2008, 80, (22), 8725-8732.
II. Jungová, P.; Navrátilová, J.; Peš, O.; Vaculovič, T.; Kanický, V.; Šmarda,
J.; Preisler, J., Substrate-assisted laser desorption inductively-coupled
plasma mass spectrometry for determination of copper in myeloid
leukemia cells. Journal of Analytical Atomic Spectrometry 2010, 25, (5),
662-668.
III. Peš, O.; Preisler, J., Off-line coupling of microcolumn separations to
desorption mass spectrometry. Journal of Chromatography A 2010, 1217,
(25), 3966-3977.
6.2. Extended abstracts
IV. Peš, O.; Vyhnánek, R.; Krásenský, P.; Vrábel, P.; Preisler, J, On-target
laser-induced native fluorescence detection of proteins. 13th International
Symposium on Separation Sciences 2007, High Tatras, Slovakia.
6.3. Oral presentation
V. Peš, O.; Foltynová, P.; Vyhnánek, R.; Vaculovič, T.; Kanický, V.; Preisler,
J., Off-line capillary electrophoresis laser ablation inductively coupled
plasma mass spectrometry, Mass Spec Forum, Vienna, Austria, 2008.
6.4. Poster presentations
VI. Peš, O.; Přikryl, J.; Vyhnánek, R.; Preisler, J., On-target (time-resolved)
laser induced fluorescence detection, 4th
international interdisciplinary
meeting on bioanalysis (CECE 2007), Brno, Czech Republic, 2007.
51
VII. Peš, O.; Vyhnánek, R.; Krásenský, P.; Vrábel, P.; Preisler, J., On-target
laser induced native fluorescence detection of proteins, 13th
International
Symposium on Separation Sciences, Štrbské Pleso, High Tatras, Slovakia,
2007.
VIII. Peš, O.; Jungová, P.; Vyhnánek, R.; Vaculovič, T.; Kanický, V.; Preisler,
J., Off-line capillary electrophoresis laser ablation inductively coupled
plasma mass spectrometry for elemental speciation, XIXth Slovak - Czech
Spectroscopic Conference, Častá - Papiernička, Slovakia, 2008.
IX. Preisler, J.; Peš, O.; Jungová, P.; Vaculovič, T.; Krásenský, P.; Kanický,
V.; Vyhnánek, R., A multidetection platform for proteome analysis, 8th
Csaba Horváth Medal Award Symposium, Innsbruck, Austria, 2008.
X. Hégrová, B.; Peš, O.; Yeung, E. S.; Preisler, J., RP-HPLC with off-line
MALDI MS detection of selected sterols, 25th International Symposium
on MicroScale Bioseparations, Prague, Czech Republic, 2010.
6.5. Contributions not enclosed within the thesis
XI. Preisler, J.; Vrábel, P.; Peš, O.; Řehulková, H.; Havel, J., Capillary
electrophoresis combined with matrix-assisted laser desorption/ionization
mass spectrometry and native fluorescence detection for protein analysis,
53rd
ASMS Conference on Mass Spectrometry and Allied topics, San
Antonio, TX, 2005.
XII. Preisler, J.; Krásenský, P.; Vrábel, P.; Peš, O.; Vaculovič, T.; Foltynová,
P.; Vyhnánek, R., Multidetection platform for microcolumn separation of
proteins and peptides, 13th
International Symposium on Separation
Sciences, Štrbské Pleso, High Tatras, Slovakia, 2007.
XIII. Ryvolová, M.; Peš, O.; Fohlerová, R.; Hejátko, J.; Preisler, J., Laser diode
(405 nm) as an excitation source in capillary electrophoresis with laserinduced
fluorescence detection analysis of GFP-tagged proteins, 4th
international interdisciplinary meeting on bioanalysis (CECE 2007), Brno,
Czech Republic, 2007.
XIV. Jungová, P.; Peš, O.; Vyhnánek, R.; Vaculovič, T.; Kanický, V.; Preisler,
J., Off-line coupling of capillary electrophoresis to laser desorption
52
inductively coupled plasma mass spectrometry, 51 Zjazd Polskiego
Towarzystwa Chemicznego i Stowa, Opole, Poland, 2008.
XV. Preisler, J.; Peš, O.; Jungová, P.; Kanický, V.; Krásenský, P.; Vaculovič,
T.; Vyhnánek, R., Off-line coupling of capillary electrophoresis to
inductively coupled plasma mass spectrometry for elemental speciation,
Vitamins 2008 Nutrition and Diagnostics, Zlín, Czech Republic, 2008.
XVI. Preisler, J.; Peš, O.; Foltynová, P.; Vyhnánek, R.; Vaculovič, T.; Kanický,
V., Off-line coupling of capillary electrophoresis to laser ablation
inductively coupled plasma mass spectrometry for elemental speciation,
56th
ASMS Conference on Mass Spectrometry and Allied topics, Denver,
CO, 2008.
XVII. Preisler, J.; Peš, O.; Jungová, P.; Kanický, V.; Krásenský, P.; Vaculovič,
T.; Vyhnánek, R., Off-line coupling of capillary electrophoresis to
inductively coupled plasma mass spectrometry for elemental speciation,
Vitamins 2008 Nutrition and Diagnostics, Zlín, Czech Republic, 2008.
XVIII. Preisler, J.; Jungová, P.; Peš, O.; Vaculovič, T.; Navrátilová, J.; Šmarda, J.;
Kanický, V., Substrate-assited laser desorption inductively-coupled plasma
mass spectrometry for elemental detection in microcolumn effluent and
biological submicroliter samples, 23rd
International Symposium on
MicroScale Bioseparations, Boston, MA, 2009.
XIX. Jungová, P.; Peš, O.; Vaculovič, T.; Navrátilová, J.; Šmarda, J.; Kanický,
V.; Preisler, J., Preparation of microsamples for elemental analysis of
biological samples, 25th
International Symposium on MicroScale
Bioseparations, Prague, Czech Republic, 2010.
XX. Preisler, J.; Jungová, P.; Peš, O.; Kanický, V.; Navrátilová, J.; Šmarda, J.;
Krásenský, P.; Vaculovič, T., A multidetection platform for microcolumn
separations, 25th
International Symposium on MicroScale Bioseparations,
Prague, Czech Republic, 2010.
XXI. Vaculovič, T.; Jungová, P.; Peš, O.; Preisler, J.; Kanický, V.; Šmarda, J.;
Navrátilová, J., Substrate-assisted laser desorption inductively coupled
plasma mass spectrometry, Winter Conference on Plasma
Spectrochemistry, Fort Myers, FL, 2010.
53
7. Selected publications and presentations
54
I.
Peš Ondřej, Jungová Pavla, Vyhnánek Radek, Vaculovič Tomáš, Kanický
Viktor and Preisler Jan
Off-line coupling of capillary electrophoresis to substrate-assisted
laser desorption inductively coupled plasma mass spectrometry.
Analytical Chemistry 80, (22), 8725-8732
2008
55
DOI: 10.1021/ac801036x
56
II.
Jungová Pavla, Navrátilová Jarmila
Peš Ondřej, Vaculovič Tomáš, Kanický Viktor, Šmarda Jan and Preisler
Jan
Substrate-assisted laser desorption inductively-coupled plasma mass
spectrometry for determination of copper in myeloid leukemia cells.
Journal of Analytical Atomic Spectrometry 25, (5), 662-668
2010
57
DOI: 10.1039/b919811c
58
III.
Peš Ondřej and Preisler Jan
Off-line coupling of microcolumn separations to desorption mass
spectrometry.
Journal of Chromatography A 1217, (25), 3966-3977.
2010
59
DOI: 10.1016/j.chroma.2010.02.058
60
IV.
Peš Ondřej, Vyhnánek Radek, Krásenský Pavel, Vrábel Patrik and
Preisler Jan
On-target laser-induced native fluorescence detection of proteins.
13th International Symposium on Separation Sciences, High Tatras,
Slovakia
2007
61
ON-TARGET LASER-INDUCED NATIVE FLUORESCENCE DETECTION
OF PROTEINS
O. Peš, R. Vyhnánek, P. Krásenský, P. Vrábel, J. Preisler
Department of Chemistry, Faculty of Science, Masaryk University
Kotlářská 2, 61137 Brno, Czech Republic
Key words: capillary electrophoresis, laser-induced fluorescence, MALDI MS, protein
identification
Protein detection and identification is commonly performed by gel electrophoresis in
combination with enzyme digestion and MALDI MS. Alternative approach utilizes
microcolumn separation techniques, such as capillary electrophoresis (CE) or nanoLC,
in on-line coupling with spray ionization techniques. In this contribution, coupling
CE with off-line laser-induced native fluorescence detection (LINF), respectively
MALDI MS, is presented as an additional outlook for proteins study. This off-line
coupling is established by a MALDI MS target acting as a common platform for
purposes of on-target LINF, MALDI MS and/or enzymatic digestion.
CE separation fraction collection is carried out by linking a separation capillary via
liquid junction to an infusion capillary, which directs the eluent onto a MALDI target
sealed in a laboratory-built chamber. The flow inside the infusion capillary is induced
by subatmosheric pressure inside the chamber, which is about 20 kPa.
Next, the protein bands may be located either by LINF or MALDI MS after prior
MALDI matrix addition. The LINF detection has been engaged due to protein
identification by peptide mass fingerprinting, whereas the trypsin digestion prefers no
MALDI matrix addition. Once the protein bands are located by LINF, regular MALDI
MS, respectively peptide mass fingerprinting, can be performed.
Generally, native fluorescence of proteins is based on fluorescence of three amino
acids, tryptophan, tyrosine and phenylalanine, which are excited by a light source
emitting in UV region. Contribution of these amino acids to the fluorescence yields is
not evenly balanced and, as indicated by chemical structures, tryptophan provides
the highest fluorescence yield. Thus, the total amount of tryptophans in the protein
molecule is the crucial aspect of appropriate native fluorescence detection, see Fig.
1.
62
concave
mirror
266 nm
laser
PMT
filter
xystage
MALDI
target
reflective objective
extension tube
slit, filter
1
In this case, a commonly available solid-state Nd:YAG laser (frequency quadrupled)
emitting 266 nm light has been used. The instrumental setup of the optical system is
shown in
Fig. 2.
1
P. Pekárková, Bachelor Thesis, Masaryk University, 2005.
0,00
0,40
0,80
1,20
1,60
2,00
2,40
2,80
3,20
270 290 310 330 350 370 390 410 430 450 470
λ [nm]
I
[a.u.]
W
Y
F
Fig. 1: Emission spectra of tryptophan, tyrosine and phenylalanine1
,
c(W,Y) = 10-5
mol.l-1
, c(F) = 5 × 10-5
mol.l-1
, pH = 2.5, excitation wavelength 266 nm
Fig. 2: On-target laser-induced native fluorescence detection system setup
63
Utilization of native fluorescence of biomolecules brings several challenges that
originate from the requirement of a laser emitting in UV region where the great
energy may excite various organic compounds. Furthermore, eluent fractions from
CE separation are solid due to rapid evaporation in the subatmospheric chamber and
the native fluorescence yields of proteins in solid-state and in native environment can
principally differ.
Therefore, major attention has been paid to the MALDI target cleaning techniques
involving hydrogen peroxide cleaning procedure for complete oxidation of organic
residues; selection of proper buffers, which can recompense the irregularities of the
MALDI target and tuning the optical system by suitable filters and slits. Moreover, the
robustness of the entire system has been tweaked up resulting in ability to compete
with on-column detectors by means of limits of detection and repeatability.
Subpicomolar quantities of tryptophan containing proteins have been detected, see
Fig. 3, and forwarded for further analysis, e.g. MALDI MS or peptide mass
fingerprinting. As a final point, the off-line CE – LINF system has been tested using a
model protein mixture.
R
2
= 0,986
-0,02
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 0,09 0,1
c [mg.ml-1
]
I[a.u.]
lysozyme
Fig. 3: Linear dynamic range of laser-induced native fluorescence of lysozyme
64
V.
Peš Ondřej, Přikryl Jan, Vyhnánek Radek, and Preisler Jan
On-target (time-resolved) laser induced fluorescence detection.
4th
international interdisciplinary meeting on bioanalysis (CECE 2007),
Brno, Czech Republic
2007
65
66
VI.
Peš Ondřej, Vyhnánek Radek, Krásenský Pavel, Vrábel Patrik and
Preisler Jan
On-target laser-induced native fluorescence detection of proteins.
13th International Symposium on Separation Sciences, High Tatras,
Slovakia
2007
67
68
VII.
Peš Ondřej, Jungová Pavla, Vyhnánek Radek, Vaculovič Tomáš, Kanický
Viktor and Preisler Jan
Off-line capillary electrophoresis laser ablation inductively coupled
plasma mass spectrometry for elemental speciation.
XIXth Slovak - Czech Spectroscopic Conference, Častá - Papiernička,
Slovakia
2008
69
70
VIII.
Preisler Jan, Peš Ondřej, Jungová Pavla, Vaculovič Tomáš, Krásenský
Pavel Kanický Viktor and Vyhnánek Radek
A multidetection platform for proteome analysis.
8th Csaba Horváth Medal Award Symposium, Innsbruck, Austria
2008
71
72
IX.
Hégrová Blanka, Peš Ondřej, Yeung S. Edward and Preisler Jan
RP-HPLC with off-line MALDI MS detection of selected sterols.
25th International Symposium on MicroScale Bioseparations, Prague,
Czech Republic
2010
73
74
8. Attachments
8.1. Curriculum Vitae
Personal information
Family name: Peš
First name: Ondřej
Date of birth: April 22, 1982
Address: Křenová 57
60200 Brno
Czech Republic
Citizenship: Czech Republic
Academic information
2006 - present Ph.D. study, Department of Analytical Chemistry, Faculty of Science,
Masaryk University, Brno; focus on mass spectrometry
2001 – 2006 M.Sc., Department of Analytical Chemistry, Faculty of Science,
Masaryk University, Brno;
Diploma Thesis: On-target enzymatic digestion PMF MALDI MS
2002 – 2004 B.Sc., Department of Analytical Chemistry, Faculty of Science,
Masaryk University, Brno; Bachelor Thesis: Fluorescence dye study for laser 532 nm
induced fluorescence detection
Languages
Czech, English
Interests
steady-state and time-resolved luminescence, mass spectrometry, capillary
electrophoresis, hyphenated techniques, peptide mass fingerprinting protein
identification, G- language programming
Grant projects
75
Investigator of MALDI Mass Spectrometry of proteins and peptides (No. 746/2008,
Ministry of Education, Youth and Sports of the Czech Republic, 2008).
Educational experience
High school project supervisor: Kristýna Šimoníková, Ivana Vojtová
76
9. List of abbreviations
APCI Atmospheric-Pressure Chemical Ionization
CCD Charge-Coupled Device
CE Capillary Electrophoresis
CFR Curve-Field Reflectron
CHC α-cyano-4-hydroxycinnamic acid
DAQ Data AQuisition
DESI Desorption ElectroSpray Ionization
DHB 2,5-dihydroxybenzoic acid
DIOS Desorption/Ionization On Silicon
EDTA EthyleneDiamineTetracetic Acid
ESI ElectroSpray Ionization
F Phenylalanine
FTICR Fourier Transform Ion Cyclotron Resonance
FTIR Fourier Transform InfraRed
GE Gel Electrophoresis
GFP Green-Fluorescent Protein
HPLC High Performance Liquid Chromatography
ICP Inductively-Coupled Plasma
IR InfraRed
LA Laser Ablation
LC Liquid Chromatography
LDI Laser Desorption/Ionization
LIF Laser-Induced Fluorescence
LINF Laser-Induced Native Fluorescence
LOD Limit Of Detection
MALDI Matrix-Assisted Laser Desorption/Ionization
MELDI Material-Enhanced Laser Desorption/Ionization
MS Mass Spectrometry
NALDI Nano-Assisted Laser Desorption/Ionization
NI National Instruments
77
NMR Nuclear Magnetic Resonance
PETG PolyEthylene Terephthalate Glycol
PMF Peptide Mass Fingerprinting
PMT PhotoMultiplier Tube
RP Reversed Phase
RSD Relative Standard Deviation
SA 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid)
SALD Substrate-Assisted Laser Desorption
TCSPC Time-Correlated Single Photon Counting
TOF Time-Of-Flight
TR Time-Resolved
UV Ultra Violet
Vis Visible
W Tryptophan
Y Tyrosine
YAG Yttrium Aluminium Garnet
78
10. References
(1) Hirschfeld, T. Anal. Chem. 1980, 52, 297-313.
(2) Elipe, M. V. S. Anal. Chim. Acta 2003, 497, 1-25.
(3) Hu, L. H.; Ye, M. L.; Jiang, X. G.; Feng, S.; Zou, H. F. Anal. Chim. Acta
2007, 598, 193-204.
(4) Schmitt-Kopplin, P.; Frommberger, M. Electrophoresis 2003, 24, 3837-3867.
(5) Wilkins, C. L. Science 1983, 222, 291-296.
(6) Gaspar, A.; Englmann, M.; Fekete, A.; Harir, M.; Schmitt-Kopplin, P.
Electrophoresis 2008, 29, 66-79.
(7) Edwards, E.; Thomas-Oates, J. Analyst 2005, 130, 13-17.
(8) Huck, C. W.; Bakry, R.; Huber, L. A.; Bonn, G. K. Electrophoresis 2006, 27,
2063-2074.
(9) Staub, A.; Schappler, J.; Rudaz, S.; Veuthey, J. L. Electrophoresis 2009, 30,
1610-1623.
(10) Ahmed, F. E. J. Chromatogr. B 2009, 877, 1963-1981.
(11) Haselberg, R.; de Jong, G. J.; Somsen, G. W. J. Chromatogr. A 2007, 1159,
81-109.
(12) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301.
(13) Shen, Z. X.; Thomas, J. J.; Averbuj, C.; Broo, K. M.; Engelhard, M.; Crowell,
J. E.; Finn, M. G.; Siuzdak, G. Anal. Chem. 2001, 73, 612-619.
(14) Feuerstein, I.; Rainer, M.; Bernardo, K.; Stecher, G.; Huck, C. W.; Kofler, K.;
Pelzer, A.; Horninger, W.; Klocker, H.; Bartsch, G.; Bonn, G. K. J. Prot. Res. 2005, 4,
2320-2326.
(15) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306,
471-473.
(16) Daniels, R. H.; Dikler, S.; Li, E.; Stacey, C. J. Assoc. Lab. Automat. 2008, 13,
314-321.
(17) Gray, A. L. Analyst 1985, 110, 551-556.
(18) Pes, O.; Jungova, P.; Vyhnanek, R.; Vaculovic, T.; Kanicky, V.; Preisler, J.
Anal. Chem. 2008, 80, 8725-8732.
79
(19) Graber, A.; Juhasz, P. S.; Khainovski, N.; Parker, K. C.; Patterson, D. H.;
Martin, S. A. Proteomics 2004, 4, 474-489.
(20) Hasegawa, T.; Wakita, Y.; Zhu, Y. B.; Matsuura, H.; Haraguchi, H.;
Umemura, T. Bull. Chem. Soc. Jpn. 2007, 80, 503-506.
(21) Pes, O.; Preisler, J. J. Chromatogr. A 2010, 1217, 3966-3977.
(22) Ardrey, E. R. Liquid chromatography - mass spectrometry: an introduction;
John Wiley & Sons: Chichester, 2003.
(23) Craig, L. C. J. Biol. Chem. 1944, 155, 519-534.
(24) Martin, A. J. P.; Synge, R. L. M. Biochem. J. 1941, 35, 1358–1368.
(25) Harvey, D. Modern Analytical Chemistry; McGraw-Hill: Boston, 2000.
(26) http://www.idex-hs.com/, 2010
(27) http://www.richardcarbonaro.com, 2010
(28) Lindon, J.; Tranter, E. G.; Koppenaal, D. Encyclopedia of spectroscopy and
spectrometry; Academic Press: Toronto, 2010.
(29) Snyder, R. L.; Kirkland, J. J.; Dolan, W. J. Introduction to modern liquid
chromatography; John Wiley & Sons: New York, 2010.
(30) Lakowicz, R. J. Principles of fluorescence spectroscopy; Springer Science:
New York, 2006.
(31) http://www.olympusmicro.com, 2010
(32) Táborský, P. Dissertation Thesis, Masaryk University, 2005.
(33) Ryvolová, M. Dissertation Thesis, Masaryk University, 2009.
(34) Thomson, J. J. In Royal Society 1913; Vol. A-89, p 1-20.
(35) Karas, M.; Bachmann, D.; Hillenkamp, F. Anal. Chem. 1985, 57, 2935-2939.
(36) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion
Processes 1987, 78, 53-68.
(37) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T.; Matsuo, T.
Rapid Commun. Mass Spectrom. 1988, 2, 151-153.
(38) Li, L. MALDI mass spectrometry for synthetic polymer analysis; John Wiley
& Sons: New Jersey, 2010.
(39) http://biomikro.vscht.cz, 2010
(40) Herbert, C. G.; Johnstone, R. A. W. Mass spectrometry basics; CRC Press:
New York, 2003.
(41) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science
1989, 246, 64-71.
80
(42) Preisler, J.; Hu, P.; Rejtar, T.; Moskovets, E.; Karger, B. L. Anal. Chem. 2002,
74, 17-25.
(43) Pes, O. Diploma Thesis, Masaryk University, 2006.
(44) Sluszny, C.; Yeung, E. S.; Nikolau, B. J. J. Am. Soc. Mass Spectrom. 2005, 16,
107-115.
(45) Jung, G. Y.; Kim, Y. S.; Lim, H. B. Anal. Sci. 1997, 13, 463-467.