Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Final Activity Report Summary - CHIEFTAIN (Chromatography with heteroelement specific detection for protein profiling in complex matrices)

From about the middle of 90's there has been a missing link in Se-speciation. That is, the well-established and widespread use of ICP-MS as a Se-specific detector could not provide unambiguous structural information of the detected Se-species that could be only obtained that time from direct infusion ESI-MS experiments of ultimately purified fractions. Especially the minor, non-peptide Se-species have been difficult to identify as most of the predicted or already known Se-species have not - still - been available as commercialised standards. Similar situation has been the landmark of selenoproteomics as well, where the genome of the targeted samples in Se-related studies is usually unknown (except for the yeast Saccharomyces cerevisiae). Therefore, one of our objectives was to develop a protocol that could almost simultaneously provide elemental (ICP-MS) and organic mass spectrometry (ESI-MS) derived information.

In 2004, it was clear that ESI-MS could work on its highest potential if hyphenated to low flow, especially nano-flow HPLC techniques; this coupling was unrealisable before for ICP-MS. This situation was changed by the development of a 'nanonebuliser', patented by the host laboratory before my contract started, which made the coupling of nano HPLC and ICP-MS possible. Therefore, one of my work package was intended to develop the possibility of parallel elemental and molecular mass spectrometric (nanoLC-ICP-MS and nanoLC-ESI-MS, respectively) analyses in the fields of Se-speciation and selenoproteomic research. By exploiting the capacity of this new hyphenation technique at low-flow HPLC conditions, several trend-setting achievements were made such as a) the first successful selenoproteomic analysis dealing with a naturally Se-containing (not artificially Se-enriched) food sample, Brazil nut, by identifying and quantifying 15 Se-containing peptides; b) identification and quantification of previously undiscovered adenosyl-derived Se-metabolites from the most widespread Se-supplement, selenised yeast, by complete structural and accurate mass analyses; c) description of artefact formation during Se-speciation on the level of selenised peptides; d) the first molecular mass spectrometry based proof of the presence of selenocystathionine, the Se-analogue of cystathionine, and its γ-glutamylated derivatives in a natural sample, Lecythis minor.

The correct quantification of sulphur with the standard (not high-resolution) quadrupole ICP-MS instruments is practically impossible because of spectral interferences hampering the targeting of the most abundant 32S isotope. This problem is highly referred in the field of proteomics as well, from another point of view, i.e., usual proteomic ESI-MS applications are not able to correctly quantify the tryptic peptides arising from a complex sample. The common links between the two research areas are on one hand the S-containing amino acids, Met and Cys (or in other applications, phosphorylated residues), and on the other hand, the low-flow HPLC techniques that can be hyphenated to ICP-MS and ESI-MS as well for the chromatographic separation of tryptic peptides.

We succeeded in developing a dedicated ICP-MS collisional cell based technique that applies xenon as collision gas to be able to measure the 32S isotope. This achievement, backed up with a dedicated S-free tryptic digestion technique and a modified isotope dilution analysis proved to be viable for the quantification of Met- and Cys-carrying tryptic peptides and their originating proteins.

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CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
TALENCE
France
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