17th International Mass Spectrometry Conference :: Prague, 2006
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|Presentation date:||Thu, Aug 31, 2006|
|Presentation time:||09:50 – 11:20|
Stefan Pieper1, Robert Ahrends1, Andreas Kuehn1, Christian Scheler2, Michael Linscheid11 Humboldt Universitaet zu Berlin, Berlin, Germany
Correspondence address: Stefan Pieper, Humboldt Universitaet zu Berlin, Department of Chemistry, Brook-Taylor-Str., 2, Berlin, 12489 Germany.
Keywords: Labeling, Isotope; Mass Spectrometry, Inductively Coupled Plasma; Protein; Quantitative Analysis.
Novel aspect: Analysis of proteins and peptides using LC/ICP-MS.
MeCAT (Metal Coded Affinity Tags) represents a new powerful approach for the analysis of complex mixtures of proteins. This alternative protein-coding technique based on labelling with different metal chelates was developed to quantify relative and absolute amounts of proteins and peptides relatively as well as absolutely. An affinity-tag allows the separation of coded target-peptides out of a complex mixture. In this contribution, as a proof of principle, we coded and analyzed peptides and proteins with MeCAT labelling.
Modified proteins were modified via the attachment of the tag to the cysteins. Afterwards, MeCAT-coded peptides and proteins were quantified by Micro-LC/ICP-MS. To increase the sensitivity, we have chosen the chelates of the monoisotopic metals of lanthanides. Data of different metals will be presented here to simulate the analysis of several proteomic states. The ICP-MS analysis provides not only a relative, but absolute quantification, since calibration and internal standardization is straight forward. In addition, this technique allows a screening of peptides and proteins without further purification in an unsurpassed sensitivity; the selected metals can be detected with fmol sensitivities using LC/ICP-MS or flow injection. Finally, ESI MS/MS may be used to identify unknown tagged peptides.
We are able to detect four different metals (Holmium, Lutetium, Thulium, Terbium) down to the low fmol range, and therefore an amount of substance of protein in the amol range. With this method it is possible to analyze intact modified proteins purified by analytical SDS-PAGE using silver staining and also a labeled digest of the protein. A Labeling of proteins and peptides with MeCAT reagents offers a new alternative to all common quantification methods (ICAT, ITRAQ and others). Last mentioned methods need concentrations in the high fmol range. Furthermore, these approaches allow only relative quantification, while the new method facilitates an absolute determination.
The next steps required for LC/ICP-MS measurements are analysis of labeled proteins and peptides out of complex mixtures.