2003 SGMS Meeting

Date and Location

The next meeting of the SGMS will be held on 30 and 31 October 2003, again at the Hotel Dorint on the Beatenberg ... high above Lake Thun in the Bernese Oberland, with a scenic view of the Swiss Alps! Further details will be published as soon as they are available.

BioAnalytical Applications of ICP-MS

Vladimir I. Baranov, Scott D Tanner, Zoë A. Quinn and Dmitry R. Bandura
MDS-SCIEX
71 Four Valley Drive, Concord, Ontario L4K 4V8 Canada

The particular characteristics of Inductively Coupled Plasma Mass Spectrometry (ICP-MS) that address perceived shortcomings of other mass spectrometric approaches to bioanalysis will be discussed. These benefits include: exceptional sensitivity (to elements) that is independent of the chemical form in the sample, relative insensitivity (tolerance) of concomitant materials in the sample, large (9 orders of magnitude) linear dynamic range, capability for absolute quantification. The deficiency of ICP-MS for this application is that the ionization source totally destroys molecular (speciation) information, other than that which is gained through prior sample manipulation (such as chromatography or immunoassay). This deficiency, however, is turned to advantage for the determination of the state-of-phosphorylation of proteins through measurement of the total P concentration, especially when normalized to another element that can be taken as characteristic of the total protein content of the sample. In many instances, S is a convenient normalizing element, especially where homologous proteins or samples are assayed. Recent phosphorylation assays will be presented, including the discrimination of cancerous whole cell lysates.
A novel application of ICP-MS is to multiplex immunoassays where the various protein complexes may be distinguished and quantified by the use of distinguishable elemental tags. In many instances, the presence of an elemental tag (or multiple copies thereof) does not affect the efficiency of the affinity binding; in fact, many commercial antibodies specifically employ lanthanides as fluorescence enhancers. In the instance that many copies of an isotopic tag (for example, Nanogold™) are incorporated, the sensitivity and detection limit are directly enhanced. Because there are more than 50 elements that are suitable to the purpose, and more than a hundred isotopes of those elements that can be obtained in enriched form, the potential for multiplex analysis is evident. Data that demonstrates the advantages anticipated will be given, and the conditions that allow for multiplex analysis will be discussed. Current results for the simultaneous determination of multiple biomarkers are presented.

ATime-Of-Flight mass spectrometer: a new horizon in GC separations

René JJ Vreuls
Vrije Universiteit
Department of Analytical Chemistry and Applied Spectroscopy
de Boelelaan 1083, 1081 HV Amsterdam
The Netherlands

Time-of-flight mass spectrometry (TOF-MS) is a very powerful technique with limits of detection in the low pg range. Furthermore, the spectrum storage rate can be as high as 500 spectra per second. Both advantages have been exploited using a faster GC mode than is commonly used in the GC laboratory. In this mode columns are short and narrow, while temperature gradients are steep (complete volatility range in 3-10 min). In combination with TOF-MS, detection limits in the low pg-range have been obtained, viz. 1-6 pg for organophosphorus pesticides, 4-60 pg for triazine herbicides and 0.3-6 pg for polycyclic aromatic hydrocarbons. Linearity of response (2 pg-10 ng range) and RSD values at adequate levels were good. Due to the high acquisition rate, automated spectral peak deconvolution could be used. This highly powerful software option enabled calculation of spectra from overlapping peaks. In actual practice this meant that peaks had to be separated only by 3 spectra (e.g. 0.15 s at a spectrum storage rate of 20 Hz). This was especially valuable in case of complex samples. GC-TOF-MS was applied to the determination of the above compound classes in various aqueous and solid extracts (by means of miniaturised extraction procedures). An aliquot was injected into the GC system using splitless or large-volume injection. The technique has meanwhile been accepted and applied to various analyte groups, e.g. doping in urine, VOCs in drinking water, explosives in waste water and many more.
Comprehensive GC (GCxGC) is a relatively new technique, which has proven to be very powerful for analysis of very complex samples. The compounds eluting from the first-dimension column are continuously trapped and reinjected into a very fast second-dimension column by means of a timed modulator. In the early stage of this technique, it has mainly been demonstrated in the field of oil industry, where various distillate fractions showed up containing thousands of ordered peaks. In the past few years many other applications have been described in the literature. Due to the narrow peaks in the second dimension, detection has to be in the 50-200 Hz range. Flame ionisation and electron capture detection have successfully been used. Identification is usually based on retention time determination in both dimensions using standards. Expertise knowledge and chemical intuition are also often used. The combination with TOF-MS resulted in a true three-dimensional separation technique. The high spectrum storage rate guaranteed full recording of the fast eluting peaks (150-250 ms at the base). Applications in the field of fruit (pesticides; volatile flavours), food (contaminants, flavours) and environmental analysis (pesticides; polycyclic aromatic hydrocarbons, PCBs and dioxins) will be demonstrated. Analytes of interest were isolated using newly developed miniaturised extraction procedures.

MS and structural studies of protein interactions

Mass spectrometry has long been known for its utility in peptide and protein identification, especially in the field of proteomics. The biological activity of molecules is dependent on its structure, on modifications, such as phosphorylation and glycosylation, and on its interactions with other biomolecules. The utility of mass spectrometry for characterization of these more complex structural interactions has been less recognized.
Over the past several years, we have been developing and applying mass spectrometry-based techniques to probe protein structural problems and protein:protein interactions. In these studies, we have used protection assays, differential chemical modification studies of surface-accessible amino acids of complexed and non-complexed proteins, and cross-linking agents to obtain information about the interacting surface of the proteins. A variety of separation techniques, such as nanoscale capillary LC and affinity chromatography, combined with mass spectrometry have been used to determine the results of these experiments. Molecular modeling based on these results has enabled us to determine structural parameters.
The major biological focus of these studies has been the structural characterization of HIV proteins and their interactions with other proteins that are relevant to HIV infection. Specifically, we have been characterizing glycan heterogeneity at glycosylation sites on HIV gp120, mapping epitopes on HIV proteins recognized by antibodies and have been probing interactions between the HIV surface glycoprotein, gp120, and its cellular receptors. In this talk, our approaches to these problems will be outlined and several examples of their application will be presented.

Quantitative and qualitative analysis of small molecules
by mass spectrometry: with or without liquid chromatography ?

Gérard Hopfgartner
University of Geneva, School of Pharmacy
Life Sciences Mass Spectrometry
20, Bd d'Yvoy, 1211 Geneva, Switzerland

For the analysis of pharmaceutical compounds in biological fluids liquid chromatography combined with mass spectrometric detection is the method of choice either as a concentration, purification or/and a separation step in the analytical procedure. High throughput quantitative analysis is achieved with high-flow or parallel LC using generic sample preparation approaches, such as direct plasma injection.
Triple quadrupole mass analysers are largely used for the quantification of small molecules, while ion trap mass spectrometers are more suited for drug metabolism studies. Both mass spectrometers are complementary and ideally one would like to have all features of both instruments in one system. The situation becomes even more complex with hybrid instruments such as quadrupole time of flight mass spectrometer, where medium resolution and accurate mass of the precursor ions and the fragment ions can be obtained with high sensitivity. Accurate mass MS/MS spectra can also be recorded on triple quadrupoles with enhanced resolution. Recently, hybrid instruments using linear ion traps have become available opening completely new possibilities in qualitative and quantitative analysis.
Quantitative bioanalysis on an disposable ESI Chip has been demonstrated without chromatographic separation. With the high selectivity of mass spectrometric detection how much chromatography do we really need ?
A chromatographic peak elutes within 10 and 30 seconds. For quantitative analysis this is sufficient while for qualitative analysis several injections of the same sample are required to perform all relevant experiments. Fraction collection prior MS detection and infusion of the relevant peaks by ESI Chip is one approach to overcome the time constrain. With off-line analysis various MS experiments can be performed for structure elucidation by minimising analysis time and sample consumption.
Selected examples in drug metabolism and quantitative analysis will be presented with the emphasis fish out needles in a haystack and quantify them.

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