2007 SGMS Meeting - celebrating our 25th anniversary!

The 25th anniversary meeting of the SGMS was held at the
Dorint Resort Blüemlisalp Beatenberg (new name, same place)
October 24 - 26, 2007, high above Lake Thun in the Bernese Oberland, with a scenic view of the Swiss Alps!

Plenary Lectures

Molecular Profiling and Imaging of Tissues by Mass Spectrometry: Applications to Clinical and Biological Research

Richard M Caprioli
Vanderbilt University
School of Medicine
Nashville TN
USA

a copy of the talk is available HERE (8.8 MB)

Imaging Mass Spectrometry (IMS) is a molecular discovery technology that takes advantage of the methodology and instrumentation of MALDI mass spectrometry. It can be used to locate specific molecules such as drugs, lipids, peptides and proteins directly from the surface of fresh frozen tissue sections. Frozen tissues specimens are cut in very thin (~10 ?m) sections and thaw-mounted on flat metallic target plates. Matrix can be manually or automatically deposited on the sections. Molecular profiles recovered upon analysis typically contain over 500 or more distinct signals in the m/z range up to 200,000. When imaging from a tissue section, matrix is deposited in a homogeneous manner minimizing the lateral dispersion of molecules of interest. This can be achieved by automatically printing arrays of small droplets. Each micro spot is then automatically analyzed generating a mass spectrum. When monitoring the intensity of a signal within the data array, a two-dimensional ion density map (or image) can be reconstructed giving information on the location and relative abundance of a given analyte. From the analysis of a single section, images at virtually any molecular weight may be obtained.
IMS is an effective discovery tool for the qualitative and quantitative analysis of molecular differences unhealthy versus normal tissues and in helping identifying potential protein markers in lesions and in various stages of disease progression. In this regard, histology directed profiling permits higher sample throughput and reproducibility. The visual specificity of histology is combined with the positioning accuracy of the robotic micro-dispenser to direct placement of matrix drops onto specific cells with high placement accuracy. Processing digital images of the spotted plate provides relative locations of each matrix spot. These coordinates are transferred and registered to the mass spectrometer for automated data acquisition. Thousands of molecular profiles can now be acquired from large sample sets in very short periods of time, improving analysis statistics. The margins of lesions can be further imaged to define the extent of the molecular advances in surrounding healthy tissues. We have applied this technology for the creation of 3-D protein images of substructures of mouse brain. Finally, we have successfully applied IMS to drug targeting and metabolic studies and the measurement of concomitant protein changes in specific tissues after systemic drug administration. Identification of statistically significant protein markers can be identified in high throughput mode by mass spectrometry based proteomic approaches.

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Ambient Ionization and Miniature Mass Spectrometers

R Graham Cooks
Department of Chemistry
Purdue University
West Lafayette, IN 47907
USA

a copy of the talk is available HERE (4.7 MB)

Ambient ionization using desorption electrospray ionization (DESI) is presented with emphasis on the range of applications and the underlying ionization mechanisms. Applications to metabolomics include direct analysis of biological fluids. Lipid analysis directly from untreated biological tissue is discussed in the context of tissue imaging for disease diagnosis and tumor margin determination. Rapid bacterial typing is shown. Laser doppler spectroscopy is used in characterizing the DESI mechanism. Phase transfer occurs from the condensed phase analyte to the solution phase droplet. Modification of the spray solvent is shown to optimize reactions with particular compounds (reactive DESI) and the value of this capability in selective recognition of particular functional groups is demonstrated. A new version of the DESI ion source in which the geometry of the inlet and outlet tubes is fixed is shown to be more rugged and easily used than the earlier devices.

The recent combination of DESI with a miniature mass spectrometer is discussed. Miniature rectilinear ion trap (RIT) mass analyzers have been developed for applications to trace organic analysis in air and in aqueous solutions. There applications include public safety and forensics. A shoebox sized, 10 kg handheld miniature mass spectrometer, Mini 10, based on a rectilinear ion trap mass analyzer has been built and characterized. More recently, the even smaller Mini 11 instrument, weighing just 4 kg for the whole system has been shown to give unit resolution (to m/z 600) mass and MS/MS spectra. Data for protein analysis will be shown.

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The Role of Mass Spectrometry in Comprehensive Two-dimensional Gas Chromatography: It is Not Just About Speed

Philip Marriott
Australian Centre for Research on Separation Science
RMIT University
GPO Box
2476V Melbourne, Australia

In the decade or so that research surrounding comprehensive two-dimensional gas chromatography (GC×GC) has been progressing, there has been almost a fascination with what MS can – or should – be able to deliver to this field of analysis. The fascination is largely to do with such basic questions as ’How do we do it?’ Since GC×GC generates peaks as narrow as 100 ms or narrower, then the conventional rule-of-thumb for quantitative analysis might be that we require 10+ data points per peak in order to measure peak area. This translates as 100 Hz data acquisition. Only one MS can deliver this speed – the time-of-flight system. However, this does not mean a laboratory shouldn’t try GC×GC with a quadrupole MS (qMS), to try to obtain identification, although parallel use of a fast detector such as FID can then provide for quantification) and once the method is proved then move to a TOFMS system.
We have used qMS with GC×GC to accomplish what we consider to be very competent identifications. A qMS scanning at maybe 20-30 Hz might only give 4 spectral scans per second dimension peak, with attendant concerns about spectral scan bias reducing spectral quality. However, the fact is that good library matches can be obtained. With qMS, we do push the system for speed. We reduce the scan range to a minimum; we might also decide to take only the higher or lower mass scan region (eg 100 u range). We find that SIM operation does not provide adequate speed when more than a few ions are selected. We have also used the GC×GC-qMS system in a vacuum 2D column operational mode to improve resolution and column efficiency. With these lengths taken to achieve GCxGC-qMS, one might now believe that the TOFMS system solves all the speed problems. It can scan at up to 500 Hz – though 125 or 150 Hz seems to be the maximum acquisition used practically. TOFMS sampling is also believed to offer the best deconvolution of overlapping spectra (notwithstanding that GC×GC should reduce overall peak overlap).
The above suggests that speed is all-important. But brute force of speed needs to be finessed by data processing that harnesses the considerable data that are produced. No qMS manufacturer provides software dedicated to GC×GC. So the conventional GCMS user will find that ‘running a report’ cannot be done in the same way in GC×GC-qMS, as for GC-MS. The one commercial GC×GC-TOFMS supplier offers a data system that must undergo continual refinement as processing speed and system capability are extended. Hands-on operation and manipulation is still a necessity.
But within the scope of MS hyphenation with GC×GC, accurate mass MS has been proposed as an exceptional, ultimate identification tool, for volatile chemical analysis. This can be considered along with ‘comprehensive GCxMS’, where soft ionisation MS (eg FIMS) is almost akin to a separation technique (now in the mass domain) similar to that of GC (in the time domain). Here a 2D plot of retention vs mass provides exquisite sample characterisation. Still further MS methods that have found specific application niches in conventional GCMS, but demand improved separation, require investigation with GC×GC, provided the acquisition and ‘system’ speeds can be matched. Clearly the challenges that GC×GC has laid at the feet of MS are many, and most are still to be met.

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Airborne Cell Chemistry & Nanoparticle based CEC

Staffan Nilsson
Applied Biochemistry, Lund Institute of Technology
Lund University, P.O.box 124
S-221 00 Lund, Sweden

Fig.1 Instrumental set-up.

Cell-containing 100-500 nL drops are levitated in a specially designed ultrasonic field. Cells and reagents are added to the drop using flow-through dispensers, and the cell reactions are monitored using fluorescence imaging detection. The cells are subsequently lysed and extractions performed in the levitated drops. The drop or part of its contents is transferred into capillaries, after which the contents are separated by CE/CEC and detected using nano-ESI-MS. We have previously shown that the levitated method can be used to follow the lipolysis in primary adipocytes and cell-cell communication between adipocytes and B-cells. The cell response from a single cell has been successfully detected. Among other things, this technique can be used to determine insulin resistence in connection with Type 2 diabetes and obesity. After exposure of the cells in the levitated drop to drugs, activators or inhibitors, the cell response (or lack of response) is monitored using fluorescence imaging detection or other non-invasive detection methods. We have also shown that micro extractions can be performed in the levitated drops. In solid phase and liquid extractions, mixing and phase separation is achieved by adjusting the ultrasonic field. The phase containing the molecules of interest is kept in the levitator and the other phase removed using a micropipette.

The drop or part of its contents is easily transferred into capillaries, after which the contents are separated by CE/CEC and detected using nano-spray ESI-MS. To detect molecules at the single cell level: We are currently developing a mass spectrometer interface with the possibility to take out aliquots in Real-Time of the levitated droplet for ESI or MALDI-MS analysis.

Fig. 2. Levitated two-phase drop just before complete phase separation. Right photo shows how one of the phases is discarded from the drop with a micropipette.

We are currently developing a mass spectrometer interface with the possibility to take out aliquots in Real-Time of the levitated droplet for ESI or MALDI-MS analysis.

Nanoparticle based CEC will be discussed as well. An alternative way to perform CEC, compared to traditional formats, is to use a pseudostationary phase (PSP). Dextran-coated nanoparticles have been used as PSP for highly efficient CEC separations (plate numbers up to 700 000 /m) of neutral analytes. The dextran coated nanoparticles role to suppress non-coated capillary wall interactions.

Molecular Imaging of Biochemical Functions Using (Small animal) PET

P August Schubiger
Center for Radiopharmaceutical Science of ETH, PSI and USZ
PET-Animal Imaging Center HCI IPW H433
ETH Hönggerberg D-CHAB
Wolfgang-Pauli Str. 10
8093 Zürich, Switzerland

a copy of the talk is available HERE (3 MB)

Molecular Imaging has become a very popular term in medicine. In the literature and at scientific meetings images are presented under the term ‘molecular’ – irrespective of the imaging method (CT, US, MRI, BLI or PET) and the information gained from the imaging method. However some methods will lead to e.g. structural images, whereas molecular imaging methods make molecular processes visible, quantifiable and trackable over time in a live animal or human. Understanding biology at the molecular levels needs molecules (molecular imaging probes), which are part of the biological processes underlying normal or diseased states. The choice of a certain imaging modality depends primarily on the specific question to be addressed. Answering those questions requires methods with specific properties on spatial resolution, sensitivity and specificity.

It is obvious, that only PET has the sensitivity needed to visualize most interaction between physiological targets and ligands as e.g. neurotransmitter and brain receptors. Therefore, if the question concerns monitoring drug distribution, pharmacokinetics and pharmacodynamics for most organs PET is the only choice as a nuclear imaging technique. Two examples are given, the first concerns potential PET-ligands for the metabotropic glutamatergic receptor subtype 5 (mGluR5). The only compound which is selective and shows high affinity is C-ABP688. It’s the first known PET-ligand displaying an in vivo distribution pattern consistent with the known regional density of mGlur5.
The second example is about the uptake of 18F-FECNT (2ß-carbomethoxy-3ß-/4-chlorophenyl)-8-(2-fluoroethyl) nortropane), a dopamintransporter ligand in the striatum of mice. Parkinson’s disease (PD) is characterized by a progressive degeneration of nigrostriatal neurons and depletion of dopamine in the striatum. This striatal degeneration can be analyzed non-invasively by small animal PET imaging using the DAT tracer [18F]FECNT in a mouse model of PD.

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Short Communications

Miniature Mass Spectrometers for Planetary Research

P Wurz (1), JA Whitby (1), M Managdze (2)
(1) Physics Institute, University of Bern, Switzerland,
(2) Space Research Institute (IKI),Moscow, Russia

Knowing the chemical, elemental, and isotopic composition of planetary objects allows the study of their origin and evolution within the context of our solar system. Exploration plans in planetary research of several space agencies consider landing spacecraft for future missions. Although there have been landers in the past, more landers are foreseen for Mars and its moons, Venus, the jovian moons, and asteroids. Furthermore, a mass spectrometer on a landed spacecraft can assist in the sample selection in a sample-return mission and provide mineralogical context, or identify possible toxic soils on Mars for manned Mars exploration. Given the resources available on landed spacecraft mass spectrometers, as well as any other instrument, have to be highly miniaturised. We will present our instrumentation developed for the Mercury and Phobos-Grunt landers.

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Does a Mass Spectrum Really Mirror the Sample composition? – Gas-Phase Reduction in the API Source as the Origin of Artifacts.

S Eichenberger, S Bienz, L Bigler
University of Zurich, Institute of Organic Chemistry, CH-8057 Zurich

In the last two decades, electrospray (ESI) and atmospheric pressure chemical ionization (APCI) have been established as two of the most important ionization techniques for mass spectrometry, in particular when the mass spectrometer is on-line coupled with liquid chromatography (LC/MS). Typically, ESI and APCI produce quasimolecular ions of the analytes without fragmentation, thus providing directly information about the composition of a sample. This is not the case, however, when fragmentation or decomposition reactions take place prior to, during or after the ionization. It is therefore of great importance to be aware of potential side-reactions that might lead to artifacts and could provoke wrong conclusions about the constitution of a sample.
Such a side reaction, occurring during the ionization process, was found in connection with our mass spectrometric investigations of spider venoms. In the APCI- but not in the ESI-source, N-hydroxylated polyamine derivatives were partially reduced to the respective amines.
To gain a better insight in this unexpected gas-phase reaction, which is of particular importance for the study of natural product and drug metabolism, the APCI and ESI behaviors of several synthetic N-hydroxylated compounds were investigated.

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Structure Elucidation of Neoefrapeptins, Insecticidal Peptides with Rare Cyclic Amino Acids

A Fredenhagen*, G Laue*,
Louis-Pierre Molleyres & Bettina Böhlendorf
Syngenta Crop Protection Research, 4002 Basel, Switzerland
* present address: Novartis Inst for BioMed Research, Basel, Switzerland

Neoefrapeptins are linear peptides which display insecticidal activity. Their amino acid composition and sequence showed a close similarity to efrapeptin. However, all neoefrapeptins contain the very rare amino acids 1-amino-cyclopropane-carboxylic acid (Acc) and, in some cases, (2S,3S)-3-methylproline. It’s the first time, that these amino acids were found as building blocks in linear peptides. They were identified by comparison of silylated hydrolyzate to reference material by GC/MS (EI-mode).
The sequence of neoefrapeptins was elucidated using mass spectrometry (ESI+ mode). Full scan spectra show two fragments in a high yield, even under mild ionization conditions. MS/MS of these two fragments yielded fragment rich spectra from which the sequence was determined almost completely.
The proteolytic cleavage with the proteinase papain yielded products that allowed to prove the rest of the sequence and the identity of the C-terminus to efrapeptin. The proteolytic cleavage products allowed furthermore to determine the position of the isobaric amino acids, pipecolic acid and 3-methylproline in neoefrapeptin F, as, well as the location of R-isovaline and S-isovaline. Papain digestion was such established as a tool for structure elucidation of peptides rich in a,a-dialkylated amino acids. CD spectra suggested a 310 helical structure for neoefrapeptins A and F.

Reference: J Antibiot 2006, 59, 267-280

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Intact Estrogen Receptor Complexes Measured by Soft Ionization Mass Spectrometry: an Approach to Identify and Classify Endocrine Disruptors

C Bovet 1, A Wortmann 1, M Ruff 2, S Eiler 2, F Granger 2, A Nazabal 1,3, R Wenzel 1,3, B Gerrits 4, D Moras 2, and R Zenobi 1
1 Dept of Chemistry and Applied Biosciences, ETH Zurich, Switzerland;
2 Inst Génétique et de Biologie Mol et Cell, CNRS, 67404 Illkirch, France;
3 CovalX AG, Technoparkstrasse 1, 8005 Zurich, Switzerland;
4 Functional Genomics Center, UZH | ETH Zurich, 8057 Zurich, Switzerland

Estrogens, a group of steroid hormones, regulate the differentiation and maintenance of a variety of tissues by binding noncovalently through the estrogen receptor (ER). ER binds not only to the natural hormone, but also to a wide repertoire of non-steroidal compounds such as pharmaceutical drugs and environmental contaminants also known as endocrine disruptors. The increase of endocrine-related abnormalities in humans and wildlife in response to endocrine disruptors and the optimization of new drugs to prevent endocrine-related cancers require an efficient method to identify and classify these estrogenic compounds. For this purpose, we have developed a new approach based on nondenaturing nanoelectrospray mass spectrometry (nanoESI-MS) and high mass matrix-assisted laser desorption ionization MS (MALDI-MS) combined with chemical cross-linking. Cross-linking chemistry was used to prevent noncovalent complex dissociation induced by standard MALDI protocols.
Using proper experimental conditions, nanoESI-MS allowed the detection of specific ligand interactions with a native triple mutant human ER ? ligand-binding domain (hER? LBD). The best approach to evaluate relative solution-phase binding affinity by nanoESI-MS was to perform competitive binding experiments with 17?-estradiol (E2) as a reference ligand. Among the ligands tested, the relative binding affinity for hER? LBD measured by nanoESI-MS was 4-hydroxtamoxifen ˜ diethylstilbestrol > E2 >> genistein >> bisphenol A, consistent with the order of binding affinities in solution. Furthermore, we measured with high mass MALDI-MS an increase of the homodimer abundance after incubating the receptor with a ligand. This ligand regulation of the dimerization allows also using MALDI for the identification of suspected endocrine disruptors.
hER? LBD samples were then incubated with a peptide containing the binding sequence of coactivator proteins (CAP). It is known from structural studies that the conformational rearrangement induced by antagonist ligands blocks the hER? LBD binding site of CAP. As expected, intact hER? LBD-CAP complexes were detected by nanoESI and high mass MALDI only in the presence of an agonist ligand. The ligand character identified by MS for the tested ligands was in good agreement with the one reported in biological studies. Therefore, the results clearly demonstrate that both MS methods are a suitable tool to identify estrogenic compounds.

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Mass Spectrometry for Lipidomics: Tracking Potential Lipid Biomarker (e.g. PAFs, lysoGPCs or eicosanoids)

R Geyer
Applera Europe BV, Rotkreuz, Switzerland

The rapid recovery and analysis of regulatory lipids in biological (or environmental) samples could provide a means of monitoring critical processes in responses to a multitude of factors like induction of inflammatory stress.

Rapid extraction together with utilizing reversed-phase chromatography and ESI mass spectrometry enables simultaneously quantification of a multitude of regulatory lipids like platelet-activating factors (PAFs), the metabolic successor lyso-glycerophosphatidyl-cholins (lysoGPC), or eicosanoids in serum samples or cell culture supernatants at trace level sensitivity.

Although the ESI in the positive mode is very sensitive it can not distinguish between isobaric PAF und lysoGPC species (e.g. PAF16:0 and stearoyl-lyso-GPC) which coalesced into the same peak at standard normal phase and reversed phase chromatography. The results show significant benefits of analysis of lyso-GPC (and related lipids) performed in the negative ion mode. Using a rather unusual modifier the applied method enables the formation of [M-H]- ion at higher abundance than the [M-CH3]- ions without the need for a high declustering potential normally needed to obtain charged PC molecules. The fragmentation of [M-H]- in the collision cell of a triple quadrupole MS yields fair amounts of substantially different product ions at m/z 59 from PAF and m/z 283 (stearate) from stearoyl-lyso-GPC making the compounds easily distinguishable. The selective detection of PAF enables the monitoring of critical processes in responses induced stress. However, even the detection of increased levels of the lysoGPC, which is very abundant in human tissue samples, serum or cell lines, in the positive ESI mode can indicate stress response as shown for serum samples obtained at different time points after treatment with LPS. Principle-component-analysis (PCA) of the mass spectrometry raw data, integrated in a software tool (MarkerView), makes differences between sample sets and evaluation of potential biomarkers easily accessible. The approach can also be applied to targeted multi compound analysis (e.g. profiling of eicosanoids).

1 White, DC, Geyer, R, Cantu, J, Jo, S-C, Mani, S, Jett, M, Moss, OR, 2005, Assessment of Regulatory Lipids in Breath Condensate as Potential Presymptomatic Harbingers of Pulmonary Pathobiology, J Microbiol Methods, 62:293-302
2 Lechner, U, Brodkorb, D, Geyer, R, et al, 2007, Aquincola tertiaricarbonis gen. nov., sp. nov., a tertiary butyl moiety-degrading bacterium, IJSEM, 57: 1295–1303
3 Curtis, PD, Geyer, R, White, DC, Shimkets, LJ, 2006, Novel Lipids in Myxococcus xanthus and Their Role in Chemotaxis. Environmental Microbiology, 8(11): 1935-1949

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Challenges for Sample Preparation and Data Validation in Imaging MALDI

M Macht 3, S-O Deininger 3, M Schuerenberg 3, C Luebbert 3, A Fuetterer 3, M Ebert 2, C Roecken 1
1 Department of Medicine II, Technical University of Munich, Germany
2 Institute of Pathology, Charité University Hospital, Berlin, Germany
3 Bruker Daltonik GmbH, Bremen, Germany;

MALDI imaging is a technique with increasing importance for marker discovery and in clinical research. The sample preparation, especially the application of matrix onto the sample is of utmost importance on the quality of the results. The main parameters in judging the results are the achievable spatial resolution and the spectra quality -unfortunately these parameters seem to be negatively correlated. Here, various preparation methods, such as pneumatic spray, robotic and manual spotting were evaluated. The images are usually analysed visualizing the spatial resolution of selected peaks on an image. The data however contain more complex information, such as overall changes in the proteomic pattern. Here we discuss how such information can be tapped.
Human tissue sections of different types of cancer were prepared on a microtome, transferred onto electrically conductive glass slides were used for the experiments. Images were acquired with a MALDI-TOF/TOF instrument equipped with a 200 Hz laser with changeable focus size (~120 µm to 10 µm). Data evaluation was done using dedicated software packages.
The data quality is directly correlated with the time of solvent exposure and the amount of matrix. To achieve good quality of spectra at maximal spatial resolution it proved necessary to optimize preparation parameters such as the number of repeated matrix applications, solvent composition and matrix concentration.
When tissues were compared that contained only homogenous tumor or non-tumor regions the simple presence or absence of a peak was not sufficient to classify the tissue because of the lack of an “internal control”. Therefore the data evaluation of the several thousands of spectra was performed by chemometric, multivariate statistical models to distinguish between various tissue areas and to locate specific outlier compounds.
It was not only possible to distinguish tumor from non-tumor areas, but also between tumors of different types. Moreover, specific known biomarkers could be allocated which may determine the further patient therapy.

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MS Imaging in Drug Discovery

B Prideaux, D Staab, M Stoeckli
Novartis Institutes for BioMedical Research, Basel, Switzerland

Imaging MALDI MS has been demonstrated to be a suitable technique in biomedical research for providing information of the distribution of drugs and metabolites within biological tissue sections. In our labs, we have been developing and applying this technology to a number of current compounds, gaining relevant information for our drug discovery process. In this presentation we will show technical improvements which allow routine usage and applications thereof.
The development of suitable sample preparation methodology is essential in the acquisition of high quality, reproducible MALDI MSI images on a routine basis. The application of the matrix to the tissue section is arguably the most important step in sample preparation for direct imaging MALDI MSI. The methodology and instrumentation utilised for this purpose has a great effect on the properties of the matrix coating (such as homogeneity over the tissue surface) and is of paramount importance for the production of a high quality MALDI MSI image.
To optimise matrix deposition for MALDI MSI, a variety of manual and automated deposition methods and instrumentation have been investigated for the application of matrix (in our case typically alpha-cyano-4-hydroxycinnamic acid) to tissue sections. Direct comparisons between automated matrix deposition instrumentation (including commercial sprayers and in house developments) and manual airspray for coating whole-rat sections for analysis of a compound and its metabolites has been conducted. Mass spectrometric images were acquired using a 4700 Proteomics analyzer, Voyager DE-STR and Q-Star Elite mass spectrometers (Applied Biosystems). The highest sensitivity in the low mass range was achieved using a manual spray with a Q-Star instrument. However, we decided that there is not one single solution optimal for all MS imaging tasks, but that one has to carefully select the components matching to the experimental task at hand.
As well as whole body sections, tissues from specific organs have been studied at higher resolution using the optimised matrix application methods. Rat lung sections have been analyzed to show the distribution of a compound for the current drug discovery pipeline throughout the lung over time. The compound was observed to remain in the alveoli for longer than 168 hours before being completely excreted. MALDI MSI images will be presented showing the localization of a compound in the skin following topical application. The compound was observed to penetrate through the stratum corneum and epidermis with the highest concentration of the drug located in the epidermal skin layers.

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Selective Isolation and Massspectrometric Analyses of Phosphopeptides :
Optimisation with LC MALDI-MS Coupling

RA Brunisholz 1, R Türk 2, Y Auchli 1, T Wallimann 2, D Neumann 2
1 Functional Genomics Center and
2 Institute for Cell Biology, ETH Zürich

Reversible phosphorylation of proteins plays an important role in many cellular processes. To investigate the functions of kinases and their substrates in vivo and in vitro e.g. production of phospho-specific antibodies or generation of phospho-site mutants, the precise identification of phosphorylation sites are mandatory.
A rapid and efficient workflow has been elaborated using prespotted 384 (HCCA) MALDI targets as fractionation unit which are ideally suited to visualize 32P labelled phosphopeptides.
For these purposes, upstream and downstream1) targeting of AMP-activated protein kinase (AMPK) -an important regulator of cellular and whole-body energy balance - was investigated. e.g. ingel-tryptic digests of phosphorylated AMPK (either by PKB or autophosphorylation in the presence of 32P-ATP) were separated on an Agilent 1100 capillary LC-system coupled to a microfractionation unit. The tryptic peptides were deposited in 1 ul portions onto a PAC-MALDI-target with 384 prespotted matrix preparations as well as calibrant spots. In order to visualize 32P labelled peptides the PAC-plate was then exposed to a Kodak MR autoradiography film. Accordingly, the 32P positive spots were screened for phosphorylation sites performing MS and MSMS with the Ultraflex II TOF/TOF.
This new workflow for phosphorylation site identification evades tedious manual handling of digested samples thus reduces the needed initial sample material dramatically. Furthermore, we observe that the separated peptides can be stored on the PAC plate for several weeks without any significant loss of resolution and signal intensity.

1 Tuerk RD, Thali RF, Auchli Y, Rechsteiner H, Brunisholz RA, Schlattner U, Wallimann T, Neumann D., New target candidates of AMP-activated protein kinse in murine brain revealed by a novel mulitdimensional substrate-screen for protein kinases (MuDSeeK). J Proteome Res 2007 (in press).

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UPLC-TOF-MS and CAP-NMR a powerful metabolomic platform for studying stress biomarkers in Arabidopsis thaliana

E Grata 1,2 , G Glauser 1,2, J Boccard 2,3, D Guillarme 2, PA Carrupt 3, S Rudaz 2, JL Wolfender 1
1 LPP, 2 LCAP, 3 LCT, Ecole de Pharmacie Genève-Lausanne, Section des Sciences Pharmaceutiques, Université de Genève, Université de Lausanne, 1211 Genève, Suisse

Recent developments in analytical methods and data mining have permitted metabolomics to evolve from an ambitious concept to a valuable technology which provides a global picture of molecular organisation at the metabolite level. With this approach, various plant physiology issues such as those in relation with stress induction phenomena can be tackled under new perspectives.
In this work, a metabolomic strategy was developed for the detection, isolation and identification of stress-induced metabolites produced in Arabidopsis thaliana after wounding the leaves, which mimics the herbivore attack. Although several defence signalling compounds are well known, e.g. oxylipins and jasmonates, the expression of some of the defence genes is probably dependent on other compounds which still need to be characterized. Therefore, the structure determination of these wound biomarkers represents an important analytical challenge since they are only found in minute amounts in plants, occur as closely related isomers and are convoluted with major constitutive plant secondary metabolites.
The developped approach was based on these sequential steps. 1. High throughput metabolite fingerprinting involving rapid UPLC-TOF-MS gradients on numerous wounded and unwounded leaf samples. 2. Data mining for group discrimination and determination of peaks (m/z , RT) responsible for the main metabolome variations in relation with wounding. 3. High resolution metabolite profiling of selected pool samples on high peak capacity UPLC columns after efficient gradient transfer for the localisation and deconvolution of the putative biomarkers. 4. Targeted LC-MS triggered microfractionation of the biomarkers at the semi-preparative level based on computed LC conditions from UPLC gradients. 5. Complete structural determination of the unknown biomarkers based on at-line capillary-NMR (CAP-NMR) experiments at the microgram level.
Thanks to this strategy a broad survey of wound-biomarkers with various physicochemical properties was obtained in the leaf extracts and, besides known signalling molecules, original oxylipins and related products were identified. The approach enables both a rapid estimation of the significant wound metabolome variations and the precise identification of biomarkers involved in these changes. The biological activity of these products in relation with their defence gene expression potential is evaluated based on DNA microarray experiments.

The Swiss National Science Foundation (grant n° 205320-116274/1 to J.-L Wolfender and S. Rudaz) is thanked for supporting this work.

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Mass Spectrometry in Structural Biology -
Characterizing Protein: DNA Interactions in Mismatch Repair

KB Tomer 1, JM Cutalo 1, A Schorzman 1, L Pedersen 1, TA Kunkel 1,2
1 Laboratory of Structural Biology, 2 Laboratory of Molecular Genetics
NIEHS, Research Triangle Park, NC

Maintaining genome stability that is critical to survival depends on a variety of biological processes. One is DNA mismatch repair (MMR), which corrects replication errors. Although MMR has been extensively investigated, there remain many unanswered questions regarding the roles of the large number of proteins required. One of these proteins (in yeast) is post meiotic segregation protein 1 (PMS1). PMS1 forms a heterodimer with the protein MLH1 (mutator L homolog). This complex has several functions in MMR, one of which is binding to DNA. The PMS1-MLH1 complex, and the N-terminal domains (NTD) of PMS1 and MLH1 can bind both dsDNA and ssDNA, and the intact heterodimer can bind cooperatively to long DNA molecules and simultaneously interact with two different regions of dsDNA. Understanding the DNA binding properties and other functions of these proteins is important because mutations that result in loss of MMR activity in human result in genome instability, cancer and infertility. Thus, we are using mass spectrometry to probe how these proteins recognize and bind to DNA. We began by probing the DNA recognition surface on the N-terminal domain of PMS1 using limited proteolysis. We compared the rate of proteolysis at specific sites in the unliganded PMS1-NTD with the PMS1-NTD bound to either ssDNA or dsDNA. Proteolysis involves an exposed polypeptide chain that is sufficiently flexible to adapt to the proteases active site. If the polypeptide chain becomes less exposed or less flexible when it is part of a complex, the rate of proteolysis can be significantly decreased. In addition to native PMS1 NTD, we also probed several mutant proteins. Using Lys-C and Arg-C with mass spectrometric analysis, we identified basic residues that, when changed, result in strongly reduced proteolysis. We are also using selective surface modification of lysine and arginine residues to further define the role of specific residues in DNA binding by PMS1. These data, along with the crystal structure on the NTD, have allowed use to formulate a model of the PMS1-NTD bound to DNA.

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A novel Method for Identification of Compounds related to Clinical and Forensic Toxicology and Doping Control in Urine and Serum Samples using a GC/MS System with a Heart Cutting Device

B Rothweiler
Agilent Technologies, Waldbronn, Germany

It is always very difficult to identify a number of target compounds in a high matrix background like urine and serum. The matrix may influence the ion ratios of the target compounds or even hide them. Quadrupole GC/MS has the required sensitivity to confirm toxicological relevant compounds but lacks often the selectivity over matrix interferences. Therefore techniques like GC/MS/MS and LCM/MS are often required for secure confirmation. The higher level of selectivity via tandem mass spectrometry is used to overcome interference problems.
The use of a two-dimensional (heart cutting) GC together with a standard quadrupole MS can be a simpler and less expensive alternative. New improved technologies like precise electronic pressure and flow control together with small and very inert capillary flow devices do increase the confidence and usability of the “Deans switch” technique drastically. The system used here involves as a first column a nonpolar DB-1 MS and uses a polar DB-17MS as 2nd. Upon injection, the analytes separate on the first column. The Deans switch is time programmed to heart cut the elution time range of the analytes from the first column onto the second column. The analytes are further separated on the second column from the matrix compounds that co-eluted with them on the first column. The 2-D GC separation is used instead of a secondary mass spectrometric operation. At the end of the analyte elution, the carrier gas in the first column can be reversed to backflush the non interesting heavy sample components out of the split vent from the inlet. This saves analysis time, increases column life time and reduces maintenance requirements.
The extremely high chromatographic resolution afforded by the 2-D approach resolves most matrix interferences from the compounds of interest. This results in detection levels comparable to MS/MS techniques.
It has been proven by former experiments that anabolic steroids and beta agonists showing a considerably increased selectivity by operating in positive chemical ionization (PCI) compared to standard electron impact (EI) ionization. The described application for the determination of anabolic agents in human urine involves additional positive chemical ionization with ammonia as reactant gas to further optimize the selectivity for this compound class. Comparison of a standard analyzed in PCI mode with methane and ammonia as reactant gas shows a clear advantage in the resulting sensitivity for ammonia (~7 x more sensitive than methane).

By combining the separation power of 2-D GC with the increased selectivity of positive chemical ionization with ammonia, detection of anabolic steroids and beta agonists in urine samples was possible down to the 1 ng/ml level.

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Multi-residue Analysis of Pesticides in Food using GC/MS/MS with the TSQ Quantum GC. 1

R Stoop
Brechbühler AG, Steinwiesenstrasse 3, 8952 Schlieren

Food safety concerns are on the rise amongst consumers worldwide. There are numerous types of pesticides regularly used in industry, including insecticides, fungicides, herbicides, and growth regulators. Because each type has different physicochemical properties, there are limitations on simultaneous analysis. GC/MS/MS can analyze approximately 300 compounds. The superior selectivity of this technique allows interference-free quantification, even with peak oelution, and provides positive confirmation of various pesticides in a single analytical run.

To monitor pesticide residues in routine measurement, a high throughput multi-residue screening method that can quantitate a large number of compound in a single analytical run is needed.

We will present a total of 103 pesticides which were analyzed on a TSQ QUANTUM GC. Results obtained indicated excellent sensitivity (0.1 ppb), reproducibility (10% at 5 ppb) and linearity (R2 > 0.995) in the range of 0.1-100 ppb. No cross-talk was observed for the analysis of closely eluting multi-component mixtures. Using H-SRM, interferences from the sample matrix background were substantially reduced, leading to improved LOQs. In addition, QED provided MS/MS structural confirmation of the analytes undergoing quantification.
The SRM transitions, the optimum collision energy, a summary of the calibration range, linearity, and the reproducibility are available. Selected example will be presented.

1 K Sugitate; M Kanai; M Okihashi, D Ghosh. Multi-residue Analysis of Pesticides in Food using GC/MS/MS with the TSQ Quantum GC. Application Note 387, june 2007

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Development and validation of a library-assisted toxicological screening method in urine by LC-MS2

D Müller, KM Rentsch
Institute for Clinical Chemistry, University Hospital Zürich 100, Zürich,

In clinical toxicology a fast and specific method is necessary for the screening for different drug classes. In former time this has been done by GC-MS or HPLC with UV or diode-array detection, in recent years the development of LC-MS instruments and especially software tools enabled the use of LC-MS in this respect.
In order to confirm immunological screening assays for drugs of abuse in urine and to test for the presence of drugs often present in intoxications, a library-assisted toxicological screening method has been developed and validated. The drugs or drug classes which have been considered are amphetamines, antidepressants, beta-blockers, benzodiazepines, cocaine, dextromethorphan, methadone, neuroleptics, opiates and psilocin.
After solid-phase extraction of 2 ml urine, the different compounds were separated using HPLC with mobile phases consisting of acetonitrile, methanol, ammonium formiate buffer (pH 3.0), ammonium acetate buffer (pH 4.0) or ammonium carbonate buffer (9.3). After atmospheric pressure chemical ionization, the analytes have been detected using data-dependent acquisition (DDA). The library was assembled by injecting all compounds directly into the MS with different collision energies and the precursor spectra as well as the product ion spectra have been recorded. In order to estimate the sensitivity for the toxicological screening, the limits of detection were compared to estimated concentration in urine (ECU) after therapeutic use of the drug.
The library which has been established contains 136 different substances out of the above mentioned drug classes. Due to the different ionization properties of the different compounds and the limited number of DDAs which can be performed simultaneously on our 10 years old instrument, 6 different analytical methods have been developed which differ in the use of the buffers in the mobile phase and the gradients applied. Of the 136 substances which have been included in the library, 20% could be identified in a concentration in urine which corresponds to the ECU, about 70% even in a sometimes much lower concentration.
In addition, we compared > 100 patient urines which have been screened by HPLC and UV detection and/or GC-MS with the newly established library-assisted LC-MS method. More than 95% of all compounds which have been identified before, have been reconfirmed by the new screening approach. Sometimes in addition new drugs have been identified. As the amount of patient urine is restricted, occasionally less than 2 ml urine has been available for this comparison, leading to the conclusion that the rate of falsely negative results will be less if the amount of sample is sufficient.
In conclusion the newly developed and validated library-assisted toxicological screening method allows a fast and specific identification of the 136 substances which have been included in the library until now.

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Negative-ion chemical ionization tandem mass spectrometry in forensic toxicology: Application to analysis of cannabinoids in biological matrices.

A Thomas, K Vuignier, C Staub
Institute of Forensic Medecine, University of Geneva, Geneva

Negative ion chemical ionization coupled with tandem mass spectrometry (NCI-MS/MS) is an interesting tool in forensic toxicology. With its properties of soft ionization, it offered a better selectivity and sensitivity than electronic impact (EI), allowing the use of a simpler sample pre-treatment, even with complex matrices like blood and hair.
Cannabis is considered to be the most widely abused illicit drug in Europe. Such large consumption levels necessitate fast, sensitive, and reliable methods of analysis to be devised by forensic laboratories. In humans, tetrahydrocannabinol (THC) is extensively metabolized in its two main metabolites: 11-hydroxy-tetrahydrocannabinol (THCOH) and 11-nor-tetrahydrocannabinol-9-carboxylic acid (THCCOOH). Knowledge of THC and its two metabolites concentrations becomes interesting with the application of mathematical models, which can predict the time when marijuana was consumed and also in estimating the user’s driving capacity. The detection of cannabinoids in hair is a great analytical challenge, since the concentration of analytes (particularly THCCOOH) to be detected is very low. From the forensic point of view, the detection of one or two metabolites in hair is of crucial interest to establish cannabis use.
The purpose of our work was first, to show the power of NCI-MS/MS in analyses of toxicological compounds, and second, to develop and establish the validity of routinely applicable methods that allow quantification of cannabinoids in blood and hair samples.
The cannabinoids were extracted from 500 ul of whole blood or from 50 mg of hair by a simple liquid-liquid extraction and then derivatized by using trifluoroacetic anhydride (TFAA) and hexafluoro-2-propanol (HFIP) as fluorinated agents. Mass spectrometric detection of analytes was performed in the selected reaction monitoring mode on a triple quadrupole instrument after negative-ion chemical ionization. The following quantitation transitions were used: 410.3 > 313.3 or 410.3 > 245.4 for THC, 422.3 > 361.2 for THCCOOH and 409.2 > 339.2 for THCOH.
The assay in blood was found to be linear in the concentration range of 0.5-20 ng/ml for THC and THCOH, and of 2.5-100 ng/ml for THCCOOH and the assay in hair was found to be linear in the range of 20 to 2000 pg/mg for THC and of 1 to 50 pg/mg for THCCOOH.
Under standard gas chromatographic conditions the run cycle time would have been 15 minutes. By using fast chromatographic separation conditions, the assay analysis time could be reduced to 5 minutes. Our developed procedures were also used to determine the concentration levels off more than a hundred real forensic samples. Some relevant cases will be presented and discussed.
NCI-MS/MS constitutes a true force for toxicological analysis. With its properties of soft ionization, it offers a better selectivity than EI and a higher sensibility than both EI and positive ion chemical ionization (PICI), allowing use of a simpler sample pre-treatment with complex matrices like blood and hair.

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Ambient Ion Mobility Spectrometry with Time-of-Flight Mass Spectrometry

M Gonin 1, K Fuhrer 1, S Graf 1, C Tanner 1, P Dwivedi 2, HH Hill, Jr 2*
1 TOFWERK AG. Feuerwerkerstrasse 39, CH-3602 Thun, Switzerland
2 Dept of Chemistry, Washington State University, Pullman, WA, USA

Electrospray ionization (ESI) and secondary electrospray ionization (SESI) coupled with an ambient ion mobility spectrometer was interfaced to a reflectron time-of-flight mass spectrometer with a mass resolving power of 2000. The ion mobility spectrometer was constructed from lead-glass technology to produce a smooth electric field improving IMS resolving power from segmented IMS designs. A segmented quadrupole ion guide was used as the interface between the IMS (ambient pressure) and the MS (vacuum) for high ion transmission without significant loss in IMS resolving power.
Ambient IMS has advantages over low pressure IMS in size, resolving power, separation selectivity and cost. Potential applications of this powerful IMS instrument will be discussed and demonstrated, including combinations with both LC and GC separation methods. Application to isomer separations in complex samples will be presented. IMS coupled with MS has a number of advantages for real applications. We have applied this technology to metabolomics, proteomics, explosive detection, gas phase chiral separations, glycomics, drug detection. Selected applications will be presented to demonstrate the advantages of this adding high resolution IMS to mass spectrometry.
A description of the instrument design and construction strategies will be presented along with initial evaluation of the instrument’s sensitivity, resolving power and reproducibility. Results of parametric investigations will be presented such as the effects of IMS temperature, electric field, drift gas flow rate, electrospray flow rate and the position of the electrospray needle on ion chemistry, ion counts and IMS resolving power will be presented. New 4D software developed for data acquisition and visualization will be described.

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The Application of Novel UPLC-Ion Mobility–TOF Mass Spectrometry Technology for Analysis of Xenobiotic Metabolites.

JP Shockcor, J Castro-Perez, K Yu, E Marsden-Edwards, A Davies
Waters Corporation Milford, MA USA

The novel aspects of this technology are based on ; an extra dimension in mass spectrometry separation with drift time and an information rich approach for metabolite detection and identification with multiple stages of fragmentation.
One of the typical problems when running in-vivo samples is that without the use of radiolabel compounds there are no reference points to look for xenobiotics. Therefore, in the vast majority of cases the analyst relies heavily on personal experience and analytical strategies to detect and identify low-level metabolites. In principle, the problems described above could be reduced through use of an additional stage of separation which is orthogonal to the LC and mass spectrometric separations and occurs on a timescale that is intermediate between the two. A technique that potentially has this capability is ion mobility spectrometry (IMS). IMS is the separation of ionic species as they drift through a gas under the influence of an electric field. The rate of drift depends on the particular mobility of an ion species in the gas and is dependent on factors such as the mass of the ion, its particular charge state and the interaction cross-section of the ion with the gas. Consequently it is possible to separate species of nominally the same m/z ratio if they have different charges or different interaction cross-sections. Ion mobility separations are generally on the millisecond timescale and so many can be acquired over the timescale of peaks eluting from the LC. Previously, a factor that has prevented IMS from becoming a mainstream separation approach in conjunction with mass spectrometry has been the low sensitivity of conventional DC-only ion mobility spectrometers as a result of poor duty cycle and radial diffusive losses. However, developments in sub-ambient pressure ion mobility instrumentation have improved this situation with ion trapping prior to mobility separation improving duty cycle, the use of RF ion guides to minimize diffusive losses either during mobility separation or post-mobility separation and the use of periodic focusing in a DC-only system to minimize diffusive loss. The combination of ion trapping prior to mobility separation coupled with minimal diffusive loss provides a system with sufficient sensitivity to be useable for sample analysis at analytically significant levels .In this study we investigate the capabilities of a hybrid quadrupole/Travelling Wave IMS/ oa-TOF instrument coupled to an UPLC inlet for drug metabolite analysis. In addition to the orthogonal separation afforded by ion mobility, this instrument has the capability for both pre-IMS and post-IMS ion fragmentation which can be selectively used to provide high sensitivity MS3 information. Typically, one of the main problems with metabolite id samples are the biological matrices analyzed. These samples may contain a very large number of endogenous compounds which may interfere with the detection of the drug-related component or may obscure its detection. This is true for example in the case of bile or any other complex biological matrix where there is a high content of endogenous compounds and sometimes these co-elute with the putative drug metabolites. In this paper we have utilized a Travelling Wave-IMS-TOF to detect and separate endogenous metabolites from xenobiotics. The main advantage of this configuration is the fact that we can separate isobaric interferences if they have different interaction cross sections. Another interesting feature of this device is the fragment separation for metabolites of interest in the IMS by different drift times (Time Aligned Parallel fragmentation – TAP) which can be used very selectively for multiple stages fragmentation experiments.

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