Schweizerische Gruppe für Massenspektrometrie
Gruppo svizzero di spettrometria di massa
| Swiss
Group for Mass Spectrometry Schweizerische Gruppe für Massenspektrometrie |
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Groupe
suisse de spectrométrie de masse Gruppo svizzero di spettrometria di massa |
| The annual meeting of the SGMS took place on November 2 / 3, 2006, again at the Mercure Hotel Beatenberg, high above Lake Thun in the Bernese Oberland, with a scenic view of the Swiss Alps! |
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Biological morphology tries to elucidate
body plans (“bauplans”) of plants and animals. It is mainly comparative
morphology (organography), dealing with concepts in order to compare the organs
of different organisms. — There are various groups not fitting the bauplan
of typical flowering plants. Such morphological “misfits” deviate
considerably from the classical root-stem-leaf (or root-shoot) model known from
most ferns and seed plants. The presentation will focus on river-weeds (family
Podostemaceae) as morphological misfits among dicotyledonous flowering plants.
They mainly occur in tropical waterfalls and river rapids, e.g. in Cameroon,
Ghana, India and Mexico. Analyses of the genome (DNA) indicate that the river-weeds
(podostemads) are closely related to St. John's wort (Hypericum) that also occurs
in Europe! The podostemads are enigmatic for people interested in developmental
genetics and evolution, for botanists studying their morphology and ecology:
Which adaptations allow the river-weeds to survive in their extreme habitats?
Most podostemads transformed their “roots” into green ribbons or
disk-like crusts that are fixed to rocks inside the torrents. The example of
the river-weeds exemplifies how botanists progress in their biodiversity research:
(i) analysis of the plant forms; (ii) identification of the genera and species;
(iii) listing the molecular and morphological characters into a data matrix;
and (iv) phylogenetic reconstruction using computer programs. There are complementary
views (perspectives) serving as heuristic devices for the elucidation of evolutionary
and developmental aspects of plant forms, e.g. the holographic paradigm anticipated
by AGNES ARBER (1879 – 1960), the continuum model and process morphology
proposed by ROLF SATTLER. — The talk will finish with artificial life,
i.e. virtual plants that only grow and flower inside computers.
Sehen und Denken in der modernen Pflanzenmorphologie, beleuchtet am Beispiel morphologischer „Misfits“ tropischer Wasserfälle. Biologische Morphologie befasst sich mit der Erforschung der Baupläne von Pflanzen und Tieren. Es handelt sich dabei um vergleichende Morphologie (Organographie), da sie mit Begriffen arbeitet, die Organe verschiedener Organismen miteinander vergleicht. — Es gibt Blütenpflanzengruppen, die sich nicht an den üblichen Bauplan halten. Diese morphologischen „Misfits“ weichen vom Wurzel-Stängel-Blatt-Schema ab, das wir von den meisten Farn- und Samenpflanzen kennen. Im Vortrag werden die Blütentange (Familie Podostemaceae) als morphologische „Misfits“ innerhalb der zweikeimblättrigen Blütenpflanzen vorgestellt. Sie gedeihen hauptsächlich in tropischen Wasserfällen und Stromschnellen, z.B. in Ghana, Kamerun, Indien und Mexiko. Analysen der Erbsubstanz (DNA) weisen darauf hin, dass die Blütentange nahe mit dem auch in Europa heimischen Johanniskraut (Hypericum) verwandt sind! Die Blütentange sind für den entwicklungsgenetisch, morphologisch und ökologisch interessierten Betrachter noch voller Rätsel: Welche Spezialisierungen erlauben ihnen das Überleben im tosenden Wasser? Zahlreiche Blütentange haben ihre „Wurzeln“ zu grünen, am überspülten Fels haftenden Bändern und Krusten umgestaltet. Am Beispiel der Blütentange wird gezeigt, wie Botaniker heute die Formenvielfalt von Blütenpflanzen analysieren, einen Katalog mit molekularen und morphologischen Merkmalen erstellen, und daraus digital die Phylogenie (den Stammbaum) rekonstruieren. Am Beispiel der Blütentange wird auch klar, dass komplementäre Betrachtungsweisen für ein besseres Verständnis von Pflanzenformen und ihrer Evolution hilfreich sind, so z.B. das bereits von AGNES ARBER (1879 – 1960) vorweggenommene holographische Paradigma und das von ROLF SATTLER vorgeschlagene Kontinuumsmodell (Prozessmorphologie). — Zum Ausklang wird im Vortrag auf virtuelle Pflanzen hingewiesen, die nur im Computer wachsen und blühen.
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The mechanism of electron capture
dissociation is a matter of substantial debate at the moment. The original,
non-ergodic mechanism implied one electron captured equals one backbone cleavage.
This was cleanly disproved in an experiment that performed ECD on doubly charged
cyclic peptides, leading to the proposal that ECD initiates a free radical reaction
cascade. Since then, a series of experiments have been performed to test this
free radical cascade mechanism.
First, if the free radical cascade is true, then it implies that the radical can move through the molecule prior to cleavage. Since the best site for a radical in a peptide is on the alpha carbon of glycine, peptides were synthesized with deuteroglycine. ECD of these peptides showed that the deuterium atoms moved, which is observable as a mass shift. Clearly the radicals migrate during ECD.
Second, radical migration is presumed to occur on a >1 microsecond timescale, suggesting that ECD proceeds through a long lived radical intermediate. A fairly simple, double resonant experiment can probe lifetimes on the >10-100 microsecond timescale, and this was applied to a series of peptides to see if some of the fragments observed were products of a long lived intermediate that exists for at least that long. New results will be presented to explore this, but in general, it appears that many or most (but not all) of the ECD fragments appear on a >1 msec timescale. Clearly both fast and slow mechanisms exist, but the fastest reactions that can currently be probed this way are slower than 10 microseconds.
Third, if radical propagation occurs on a slow timescale, then it can be modulated by adding groups to a peptide that contain large, distributed pi-bonded systems which should act as radical traps by resonantly stabilizing the radical. Coumarin can be easily tagged onto the molecule, providing such a system. ECD of a peptide with such a radical trap indicates that backbone fragments are suppressed, but side chain fragments are increased. Furthermore, double resonance shows that most of the side chain fragments are very slow, on the millisecond timescale.
Thus, the free radical cascade mechanism has withstood challenge by a series of experiments designed to test it. While the theory cannot be proven, it has not yet been disproved, and further experiments are planned to test it further.
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Miniaturization strategies for protein sample preparation prior to mass spectrometry readout has clear benefits and is currently a focus area within the microchip and microfluidics field. The strategies along this line as pursued by the Lund Nanobiotechnology group will be reviewed. Piezodispensing as a means for precise sample deposition and on-spot enrichment as well as chip integrated solid phase extraction linked to MALDI-TOF MS readout will be discussed. A new approach to high capacity on-MALDI-target sample preparation - (ISET Integrated Sample Enrichment Target) - will also be presented.
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In recent years, liquid chromatography/mass spectrometry (LC/MS) has become one of the most powerful analytical techniques for qualitative and quantitative analysis. This is mainly due to the invention of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). With these interfaces, polar compounds are typically detected by protonation in the positive ion mode and by deprotonation in the negative ion mode.
Non-polar analytes, however, which are particularly well separated in reversed-phase HPLC, are either detected with unsatisfactory results or not at all under these conditions. Therefore, strategies to improve the ionization for these compounds have to be developed. Some respective approaches are shown within this presentation.
One promising approach is the electrochemical conversion of the analytes to more polar or even charged products, which are then readily accessible by ESI or APCI. We have developed an on-line LC/electrochemistry/MS method with post-column oxidation of the analytes occurring in a commercial "coulometric" cell, which provides for up to quantitative conversion of the analytes prior to the mass spectrometer. Examples from different applications areas will be presented.
Another approach is based on an electron-capture effect, which is observed for selected nitroaromatic compounds when using the APCI interface as a source of low-energy electrons in the negative ion mode. Depending on the structure of the compound to be analysed, either a dissociative or a non-dissociative electron capture effect is observed. The electron capture effect competes with the classical deprotonation in LC/MS and provides for additional structural information on the analytes.
A third approach has been directed to the coordination of inorganic ions to analytes with limited polarity under formation of charged adducts. This has been explored with respect to the addition of Ag+ ions after separation of rubber vulcanization accelerators.
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2D-LC-MS/MS based functional proteomics approach for the analysis of stress response in aquatic model organisms
Marc J-F Suter and Victor J Nesatyy
Eawag, Swiss Federal Institute of Aquatic Science and Technology
CH-8600 Dübendorf
suter@eawag.ch
A current, powerful and frequently
used method for the assessment of integrative responses on the molecular level
is the analysis of whole organism gene expression profiles, called transcriptomics.
Inside the cells in organisms, the RNA molecules (transcripts) are subsequently
translated into proteins, each protein having its specific physiological functions.
Protein expression profiles can be assessed as well, in what today is called
proteomics. In this approach, the protein expression of exposed organisms is
compared to a control. If a certain protein is observed at higher concentrations
in the exposed organism, then it is part of the cellular stress response. Proteomics
is by now an established and sensitive tool for identifying protein markers
for (multiple) stress that can provide insights into the underlying modes of
action and can be used to assess exposure. Usually a 2D separation technique
is needed to reduce the complexity of a protein extract. This has traditionally
been 2D gel electrophoresis, but more recently stacked columns have allowed
2D LC separation culminating in the multidimensional protein identification
technology (mudPIT) (Washburn et al, 2001). More importantly, the data produced
needs to be matched with in silico digested proteins derived from the genome
of the organism in question. Commercial as well as open-source programs are
available that match proteins identified from MS data with proteins in the database.
Using this technique we could demonstrate that zebrafish eggs show different
induction patterns when comparing eggs exposed to cadmium or 17ß-estradiol.
Our most recent results will be presented.
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Taxonomic characterization of halophilic archaea by mass spectrometry
Laurent Bigler (1), Urs Stalder (1),
Fatemeh Ghorbani (1), Huda Al-Ajmi (2), and Heiko Patzelt (2)
(1) Universtity of Zurich,
Institute of Organic Chemistry, CH-8057 Zurich
(2) Department of Chemistry, Sultan Qaboos University, Al-Khod 123, Sultanate
of Oman
lbigler@oci.unizh.ch
Increasing numbers of extremely halophilic (= salt-loving) microorganisms - both archaea and bacteria - are isolated from salt lakes and saline marshes in many parts of the world. These organisms, which grow well at NaCl concentration up to 4M, appear to play a vital ecological role in their habitats and show great promise for applications in environmental biotechnology [1]. The de-replication and ultimately the taxonomic characterization of halophilic cells from natural isolates, however, still remains laborious and expensive. We present here a rapid and facile identification method, based on the fingerprint comparison of whole-cell MALDI mass spectra.
Archaea, isolated from oil-contaminated sites in the saline deserts of the Sultanate of Oman [2], were cultivated in halophilic standard media (15-30% NaCl) and were shown to efficiently degrade aliphatic hydrocarbons. The cells were harvested and – without any extraction steps - applied onto the sample plates of the MALDI mass spectrometer (“cell smear method”) [3]. Spectra were recorded both in the positive and negative detection mode, leading to reproducible sets of data for the polar membrane lipid fractions in the negative and for the short peptide/protein domain [4] in the positive mode. Combined, the two sets of mass data constitute an unambiguous fingerprint for a particular cell type. Control experiments, where membrane lipids were extracted and analyzed by LC-MS methods, confirmed the MALDI results but were much more time-consuming. The direct MALDI analysis of whole cells is thus the method of choice for the rapid characterization of the archaeal species in complex natural isolates.
[1] R Margesin, F Schinner, Extremophiles
5 (2001) 73-83.
[2] H Patzelt, Hydrocarbon Degradation under Hypersaline Conditions, in: N Gunde-Cimerman,
A Oren, A Plemenitas (eds) Adaptation to life at high salt concentrations in
Archaea, Bacteria, and Eukarya, Springer, Berlin, 2005, pp 105-122.
[3] P Krader, D Emerson, Extremophiles 8 (2004) 259-268.
[4] C Fenselau, P Demirev, Mass Spectrom Rev 20 (2001) 157-171.
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Phosphorylation of the mediator complex: is Cdk8 the sole operating kinase?
Manuel Tzouros, Albert Heck and Jeroen
Krijgsveld
Biomolecular Mass Spectrometry, Utrecht University
Sorbonnelaan 16, 3584 CA, Utrecht, The Netherlands
The yeast Mediator complex is an
assembly composed of 21 proteins that acts as a transcriptional regulator of
RNA polymerase II (pol II) [1]. For instance, Mediator operates as a repressive
regulator of gene transcription when an additional module composed of four proteins
— the Srb8-11 module — is associated to it. The cyclin-dependent
kinase Cdk8 (or Srb10), part of the Srb8-11 module, is responsible for the phosphorylation
of several Mediator subunits and is thus a key player in modulating the function
of the complex. Some recent investigations have shed light on the presence of
extra kinases capable of modifying Mediator.
We have chosen a phospho-proteomics approach to map the modification sites due
to Cdk8. We used metabolic labeling (15N) to compare the Mediator subunits expressed
by a wild-type and a CDK8-deleted yeast strain. The complex was selectively
isolated from the yeast lysates by tandem-affinity purification and submitted
to an in-solution trypsin digestion. The resulting digest was fractionated by
off-line strong cation exchange (SCX) chromatography and phosphopeptides were
enriched using titanium dioxide (TiO2). Identifications were performed by nanoLC-MS/MS
using the LTQ-FT or -Orbitrap operating in the MS2/MS3 or multistage activation
mode. The corresponding signals for the “light” (14N) and “heavy”
(15N) phosphopeptides originating from both yeast strains were integrated and
the ratios obtained were used to evidence the contribution of Cdk8. The data
clearly support that Cdk8 is not the sole kinase responsible for the phosphorylation
of Mediator subunits and further investigations need to be undertaken to determine
their identities.
[1] S Björklund, CM Gustafsson, Trends Biochem Sci 30 (2005) 240.
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Opportunities and limits of the combination of linear ion trap with Orbitrap analyzer to detect and identify contaminants in environmental water samples
Juliane Hollender, Heinz Singer,
Kathrin Fenner
Eawag, Swiss Federal Institute of Aquatic Science and Technology
CH-8600 Dübendorf
juliane.hollender@eawag.ch
The challenges in analysing polar
organic chemicals and their transformation products in environmental samples
by LC-MS are twofold. First, the generally low but nevertheless potentially
toxicologically relevant concentrations, which usually lie in the ng/L range,
require enrichment, separation of the matrix and sensitive detection. The second
challenge is the identification of unknown peaks in LC-MS, where the interpretation
of fragmentation pattern without large spectra libraries as those available
for GC-MS is more difficult. In recent years the combination of LC-TOF (providing
accurate mass measurements to generate elemental compositions of ions) with
LC ion trap (providing structural information from fragmentation studies) has
a few times been applied in non-target-screening of environmental samples [1-3].
The limitation of TOF instruments in comparison to quadrupoles is their lower
sensitivity, which hampers the detection and identification of analytes at low
concentrations [2].
The scope of our ongoing study is to determine the potential of the new hybrid
system linear ion trap and Orbitrap analyzer to detect and identify polar organic
contaminants like pesticides and their metabolites in environmental water samples
without using reference standards. Surface and groundwater samples were enriched
and analyzed by LTQ Orbitrap using automatic data dependent scanning, enabling
the simultaneous acquisition of high resolution MS spectra and several MS/MS
scans of the most abundant mass peaks. Screening for stable transformation products
was focused by intensive literature surveys as well as the use of chemical fate
models and biodegradation pathway prediction tools to produce a list of transformation
products that are likely to occur. We managed to identify several pesticides
and their transformation products in the lower ng/L concentration range by this
method, but it is still an important issue to achieve sufficient detection sensitivity.
Upon further refinement, the approach presented opens up an avenue to more realistically
assess water quality with regard to emerging contaminants and their transformation
products and, at the same time, to refine risk assessment methods to include
transformation products.
[1] EM Thurman, I Ferrer, JA Zweigenbaum,
JF García-Reyes, M Woodman, AR Fernández-Alba J Chromatogr A 1082
(2005) 71-80.
[2] F Hernández, ÓJ Pozo, JV Sancho, FJ López, JM Marín,
M Ibánez TrAC 24 (2005) 596-612.
[3] F Hernández, M Ibánez, JV Sancho, OJ Pozo, Anal Chem 76 (2004)
4349-4357.
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Significantly improving quantitative LC-MS/MS analysis for large number of analytes (>500) with scheduled-MRM
Yves LeBlanc
MDS Sciex, Concord, Canada
The selectivity and sensitivity of triple quadrupole systems have made them the instrument of choice for quantitative LC-MSMS analysis. The main reasons for this acceptance is their high duty-cycle when operated in multiple reaction monitoring (MRM) mode. However, as the number of analytes monitored increases, the accuracy and precision of peak areas decreases significantly due to an effective decrease of the duty-cycle and sensitivity of the instrument. The present work report on the concept of Scheduled-MRM to improve the data collection per unit time. Using retention time (RT) information, only the required MRM transitions are monitored over a given time window. This approach enables the ability to monitor a significantly larger number of MRM’s (>500) during an LC analysis while sustaining appropriate quantitation characteristics (CV and accuracy).
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New MS/MS experiments in the c-trap (curved trap) of an Orbitrap mass spectrometer
Paul-Gerhard Lassahn
Spectronex AG, Hochstrasse
48, CH - 4002 Basel
Injection of ions into the Orbitrap mass analyzer is not an easy task. Ions packets must enter the trap in a very short pulse, they must get highly focused and at the same time the ion beam must not suffer from space charge effects. The c-trap seems to be an ideal interface for fulfilling all these needs.
At the same time the c-trap expands and improves the capabilities of the Orbitrap itself in several ways. The c-trap is an ideal storage device for calibrants selected from known background signal and ensures mass accuracies in the Orbitrap of 1 ppm or better on a routine basis. In addition the c-trap is a collision cell of its own and is able to yield complementary information to MS/MS obtained in the linear ion trap of the Orbitrap mass spectrometer. Due to the dynamics of the collision process the spectra are similar to MS/MS obtained in a triple quad mass spectrometer and in addition they do not suffer from any low mass cut-off.
The talk highlights some important characteristics of the curved trap and discusses recent results obtained in the different MS/MS modes on the Orbitrap in our laboratory.
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Extractive electrospray ionization mass spectrometry: concept, instrumentation and applications
Huanwen Chen (1,2), Andre Vetor (2),
Graham Cooks (2), Renato Zenobi (1)
(1) Chemistry Department and
Applied Bioscience, ETH Zurich, 8093 Switzerland
(2) Chemistry Department, Purdue University, West Lafayette, IN 47907 USA
Conventionally, extraction is performed offline before sampling and ionization in mass spectrometry. In a novel extractive electrospray ionization source (EESI) (schematically shown in Figure 1), on-line droplet-droplet extraction occurs when a sample spray intersects a reagent electrospray; this makes online extraction and ionization happen simultaneously, and allows continuous mass spectrometric direct analysis of trace amounts of compounds in complex matrices for extended periods of time.
Figure 1 Schematic diagram of extractive electrospray ionization showing ionizing
and sample sprays (the distance a, b and the angles alpha, beta are adjustable).
Initially, EESI was demonstrated by using a homemade EESI source coupled to
a LTQ mass spectrometer. Analytical performance has been shown with an ion trap
mass analyzer. Recently, EESI has been implemented with a commercial ESI-QTOF-MS
instrument without any hardware modification, making EESI available to a more
users with ease.
Intrinsically, flexible configuration of the EESI source allows it to tolerate
complex matrices and require no sample pretreatment for real time monitoring
of various samples. Trace components present in raw urine, milk, serum, drug
powder, E. coli strands and breath aerosol samples have been successfully detected.
Atmospheric pressure ion/molecule reactions and ion-ion reactions have also
been demonstrated in EESI-MS. Potential applications include but not limited
to proteomics, biomedical, clinical diagnosis, metabolomics, microbiological
applications, pharmaceutical products and process monitoring, food analysis,
environmental analysis, explosives detection, homeland security and forensics
science.
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The separation of protein ion charge states and their associated fragments using a travelling wave IMS device.
Jennifer Burgess (1), Steven D Pringle
(1), Kevin Giles (1), Iain Campuzano (1), Stormy L Koeniger (2), Stephen J Valentine
(3), Robert H Bateman (1), Sam Merrenbloom (2), David E Clemmer (2), Georges
Froidevaux (4), Robert Funck (4) and Jan Claereboudt (5)
(1) Waters MS Technologies Centre,
Manchester, UK
(2) Department of Chemistry, Indiana University, Bloomington, IN
(3) Predictive Physiology and Medicine, Bloomington, IN
(4) Waters AG Switzerland, Rupperswil, Switzerland
(5) Waters Central Europe, HRMS Division, Zellik, Belgium
Electrospray mass spectrometry is a firmly established tool for the identification of proteins, via the analysis of complex tryptic peptide mixtures. It has also proven to be an extremely powerful technique for determining protein structure by mass analysis at the intact protein level. Often the complexity of the associated mass spectrum limits the information content that can be obtained form the data and additional stages of separation prior to analysis by mass spectrometry are desirable. The potential of using ion mobility spectrometry adds another, orthogonal, dimension of separation to the MS experiment, providing separation of species by their associated mobility, or drift time, a factor which is dependant upon ion size, shape and charge. Consequently, it is possible to separate co-eluting isobaric species which exhibit different drift times. We have combined a travelling wave ion mobility (TWIMS) device within a quadrupole orthogonal acceleration time-of-flight mass spectrometer, enabling ion mobility separations to be combined with electrospray mass spectrometry at high sensitivity.
We have investigated the potential of this configuration for the separation and subsequent mass analysis of multiply charged protein ions and to separate fragments derived from these multiply charged species, following CID. If mobility separation of fragment ions was efficient, this would reduce mass spectral complexity and facilitate detection and subsequent identification.
In this study, two new ion mobility spectrometry (IMS) techniques have been used to separate the protein ion fragments. These techniques will be compared and contrasted, and the potential of IMS for biological applications will be discussed.
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Comparison of dissociation constant of calmodulin-melittin complex determined using surface plasmon resonance, circular dichroism and mass spectrometry.
Sonal Mathur (1), Michael Scott (2),
Renato Zenobi (1)
(1) Department of Organic Chemistry
and Applied Biosciences, Wolfgang-Pauli-Strasse-10, ETH Zurich, CH-8093 Zurich,
Switzerland
(2) Functional Genomics Center, UNI ETH Zurich, Winterthurerstrasse 190, CH-8057
Zurich, Switzerland
Mass spectrometric methods have been used for studying biomolecular interactions since decades [1]. In particular, electrospray ionization (ESI) is a promising tool for the determination of binding affinities of noncovalent complexes. This work is focused on the comparison of standard (surface plasmon resonance and circular dichroism) and mass spectrometric techniques (ESI-MS and MALDI-SUPREX) for the determination of binding strength of these noncovalent interactions. Calmodulin-melittin complex was selected as model system, which shows a high binding affinity of 3 nM with 1:1 stoichiometry in the presence of Ca2+ [2].
The results obtained in this study show a good agreement between ESI-MS (Kd
= 5 +/- 4 nM) and surface plasmon resonance (Kd = 6 +/- 3 nM) data. An ESI spectrum
showing multiply charged peaks of the free protein and the complex (bold letters)
is shown above. Preliminary experiments with circular dichroism give high affinity
in low nanomolar range. However, the SUPREX analysis indicates a lower affinity
in micromolar range. Instrumental and experimental conditions for each technique
were also optimized during the course of this study. As a conclusion, this investigation
clearly demonstrates the potential of mass spectrometric methods for the quantitative
determination of noncovalent interactions.
[1] JM Daniel, SD Friess, S Rajagopalan,
S Wendt, R Zenobi, Int J Mass Spectrom 216 (2002) 1.
[2] M Comte, Y Maulet, JA Cox, Biochem J 209 (1983) 269.
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Therapeutic drug monitoring of antiretroviral drugs using LC-MS.
Katharina M Rentsch, Ursula Gutteck-Amsler,
Arnold von Eckardstein
Institute for Clinical Chemistry, University Hospital Zürich, Rämistrasse
100, 8091 Zürich, Rentsch@ikc.unizh.ch
Prospective and retrospective studies
have provided some evidence of the clinical and virological benefit of incorporating
therapeutic drug monitoring (TDM) into routine patient care. The bases for using
TDM with today’s knowledge are data demonstrating considerable inter-individual
variability in the concentration of antiretroviral drugs among patients and
data demonstrating relationships between drug concentrations and responses,
either virological or toxicological. Because antiretroviral therapy consists
always of a combination of different drugs, analysis can be simplified if different
drugs are measured at the same time. Therefore, LC-MS or LC-MS/MS is nowadays
the analytical method of choice. Due to the high specificity of the mass spectrometric
detection the times of analysis can be shortened and the risk of interferences
can be minimized as compared to UV detection.
In 2003 we have published an LC-MS method [1] for the quantification of amprenavir,
efavirenz, indinavir, lopinavir, nelfinavir, nevirapine, ritonavir and saquinavir
after solid-phase extraction. In the meantime atazanavir and tipranavir have
been introduced into the marked. In the process of including them in our analytical
procedure we have reduced the sample volume, simplified sample preparation and
shortened the chromatographic run time. The result of this optimisation process
will be presented.
Sample preparation consisted in the addition of a solution of the internal standard
(proteinase inhibitor analogue) in acetonitrile to 100 µl serum which
resulted in protein precipitation. After centrifugation the supernatant was
diluted with buffer before injection into the HPLC system. The different drugs
were analyzed by reversed-phase chromatography and detected by negative or positive
atmospheric pressure chemical ionization (APCI) mass spectrometry.
Depending on the target concentrations in patients, the calibration curves of
the new method were linear in the range of 0.01 – 30.0 mg/l. The limit
of quantification was accordingly between 0.01 and 0.3 mg/l. The imprecision
was < 10% and the accuracy 92 – 108%. The performance data of the new
and simplified method are comparable or even better to the published numbers
and demonstrate that this method allows the quantification of 10 different proteinase
inhibitors or non-nucleoside reverse transcriptase inhibitors in patients with
HIV infection.
[1] KM Rentsch. Sensitive and specific determination of eight antiretroviral agents in plasma by high-performance liquid chromatography-mass spectrometry J Chromatogr B Analyt Technol Biomed Life Sci 788 (2003) 339-350.
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Analysis of oligosaccharides by capillary-scale high-performance anion-exchange chromatography with pulsed amperometric detection (CHPAEC-PAD) and on-line electrospray-ionization ion-trap mass spectrometry (CHPAEC-ITMS).
C Bruggink (1,3), CAM Koeleman (1),
V Barreto (2), Y Lui (2), C Pohl (2), A Ingendoh (4), M Wuhrer (1), CH Hokke
(1), AM Deelder (1)
(1) Department of Parasitology, Leiden University Medical Center, Leiden, The
Netherlands.
(2) Dionex Corporation, Sunnyvale, California, USA
(3) Dionex Benelux, Breda, The Netherlands
(4) Bruker Daltonik GmbH, Bremen, Germany
High-pH anion-exchange chromatography
with pulsed amperometric detection (HPAEC-PAD) is an established technique for
the selective separation and analysis of underivatized carbohydrates. The miniaturization
of chromatographic techniques by means of capillary columns, and on-line coupling
to mass spectrometry are critical to the further development of glycan analysis
methods, which are compatible with the current requirements in clinical settings.
A prototype system has been developed based on a Dionex BioLC equipped with
a microbore gradient pump and a PEEK flow splitter, a FAMOS micro autosampler,
a modified electrochemical cell for on-line capillary PAD and a capillary column
(380 ?m i.d.) packed with a new type of anion-exchange resin. This system operates
with sensitivity in the low fmol range. In addition, an on-line capillary desalter
has been developed to allow direct coupling to a Bruker Esquire 3000 ion-trap
mass spectrometer with an electrospray ionisation interface (ESI-IT-MS). Both
systems have been evaluated using standard oligosaccharides as well as urine
from children with various lysosomal oligosaccharide storage diseases. Our data
indicate that the robust and selective anion-exchange system in combination
with ESI-IT-MS for structure confirmation and analysis provides a powerful platform
which is complementary to existing nano-/capillary LC-MS methods for analytical
investigations of oligosaccharides from biological samples.
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Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) : New Developments in Toxicological and Pharmaceutical Analysis
LA Leuthold, E Varesio, G Hopfgartner
Life Sciences Mass Spectrometry, EPGL, Université de Genève
20 Bd d’Yvoy, 1211 Genève 4, Switzerland
Desorption Electrospray Ionization
(DESI) is an ambient ionization technique introduced in 2004, allowing the sampling
of surfaces under ambient conditions, without the use of any matrix. Jet propelled
droplets generated by an electrospray hit the surface to be analyzed, generating
analyte ions sampled by the mass spectrometer.
From then on, numerous examples have been published, usually for the detection
of relatively high amounts of material (micrograms) on simple or relatively
complex matrices, such as drug tablets, biological tissues or plant material;
or low amounts of material (down to picograms) on simple matrices as clean surfaces
or paper. Detection of relatively low amounts of analytes in complex matrices
such as biological fluids or tissues still needs to be investigated and is of
great challenge, due to sensitivity and selectivity issues.
Following our report on the analysis of commercial pharmaceutical and Ecstasy
tablets [1] and our work on the analysis of compounds of toxicological interest
in Dried Blood Spots (DBS) [2], new results will be shown on DESI-MS/MS in toxicological
and pharmaceutical analysis, including direct analysis of LSD on paper strips
and current work on DBS. The sensitivity issue and reasonable applications will
be assessed.
[1] LA Leuthold, J-F Mandscheff, M Fathi, C Giroud, M Augsburger, E Varesio,
G Hopfgartner. Rapid Commun Mass Spectrom 20 (2006) 103-110.
[2] LA Leuthold, J-F Mandscheff,
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